IN VITRO MODELS FOR INVESTIGATING KERATINOCYTE RESPONSES...
Transcript of IN VITRO MODELS FOR INVESTIGATING KERATINOCYTE RESPONSES...
IN VITRO MODELS FOR INVESTIGATING
KERATINOCYTE RESPONSES TO
ULTRAVIOLET B RADIATION
Tara L. Fernandez
Bachelor of Biomedical Science (Honours)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Faculty of Health
School of Biomedical Sciences
Institute of Health and Biomedical Innovation
Queensland University of Technology
2013
i Keywords
This thesis is dedicated in loving memory of:
Dr. Shila C. Fernandez
i Keywords
Keywords
Photobiology, keratinocytes, dermal fibroblasts, in vitro skin culture models,
epidermal regeneration, UVB radiation, epidermal-dermal interactions, insulin-like
growth factors, skin cancer, photodamage.
iii Abstract
Abstract
Australia experiences extreme levels of solar ultraviolet radiation (UVR), as
well as increasing rates of skin cancer incidence among the Australian population.
Strong associations between chronic unprotected sun exposure (in particular the
UVB radiation wavelength) and photocarcinogenesis have been established.
However, major knowledge gaps still exist in our understanding of the biology of the
cutaneous photoresponse, in particular the complex cellular and molecular mediators
involved in resisting and repairing UVB-induced damage. Due to the intricate nature
of human skin, the lack of complete, accessible and biologically-relevant
experimental models to investigate the human photoresponse has limited in vitro
investigations into these processes.
Keratinocytes are the primary cell type of the epidermis, which functions as a
barrier against environmental hazards. Due to their frequent exposure to genotoxic
insults like UVB radiation, keratinocytes are equipped with complex protective and
repair strategies against photodamage, including the formation of DNA photolesions.
Intercellular interactions between keratinocytes and dermal fibroblasts and the role of
this dermal-epidermal crosstalk in modulating keratinocyte responses to UVB
radiation have been relatively uncharacterised. These fibroblasts are known to
synthesize various factors with potent effects on basal keratinocytes, such as
stimulating proliferation, regulating epidermal regeneration and homeostasis.
Importantly, the association of these fibroblast-secreted factors with the acute
keratinocyte UVB radiation response and potentially their role in epidermal
photocarcinogenesis need to be further investigated.
The overriding aim of this thesis was therefore to characterise and develop 2-
dimensional (2D) and 3D in vitro skin models as platforms for investigating the
responses of human keratinocytes to UVB radiation. A tissue-engineered human skin
equivalent (HSE) model consisting of primary human keratinocytes cultured on a de-
epidermised dermal (DED) scaffold has been established in our laboratory as an
organotypic model to study aspects of skin biology. Importantly, this HSE-KC skin
model (comprised of a stratified epidermal layer with primary keratinocytes on a
iv Abstract
DED scaffold) has been previously demonstrated to retain several structural and
functional properties of the native epidermis. Hence, in this thesis, the
characterisation and validation of the HSE-KC skin model as a tool to study
keratinocyte responses to UVB radiation experimentally are reported. Significantly,
the HSE-KC was shown to possess several biological parallels to ex vivo skin, such
as the expression of several epidermal protein markers. Furthermore, histological and
immunohistochemical analysis of irradiated HSE-KC constructs showed the
generation of key markers of the epidermal photoresponse, including the formation
of DNA photolesions, apoptotic sunburn cells, increased synthesis of inflammatory
cytokines and shifts in epidermal proliferation and differentiation patterns.
To study the paracrine interactions between dermal fibroblasts and
keratinocytes, the novel application of a cell co-culture system to study the influence
of fibroblasts on modulating keratinocyte responses to UVB radiation is described
herein. Using this previously undescribed technique, I report the enhancement of
keratinocyte viability, an inhibition of UVB radiation-induced apoptosis and the
enhancement of DNA repair in cells co-cultured with fibroblasts prior to irradiation.
Next, this HSE-KC model was further developed and characterised as a model of
epidermal-dermal interactions during the acute UVB irradiation response via the
incorporation of human dermal fibroblasts into the DED component, termed the
HSE-KCF. This construct was utilised as a novel in vitro 3D model for studying the
influence of epidermal-dermal crosstalk on photodamage and repair in keratinocytes
post-irradiation. The protective effect of fibroblasts previously described in the
abovementioned 2D co-culture model was also observed in the epidermis of these
HSE-KCF models. Hence, the molecular mechanisms surrounding this fibroblast-
induced keratinocyte protection during the UVB irradiation response were further
analysed. Importantly, the presence of fibroblasts in the HSE-KCF skin constructs
modulated the expression and activation of elements of the UVB radiation-induced
apoptotic cascade including Bcl-2-associated death promoter (Bad) and caspase-3,
but not of cleaved poly (ADP-ribose) polymerase (PARP).
To further investigate this fibroblast-mediated protection of UVB-irradiated
keratinocytes, the effect of insulin-like growth factor-I (IGF-I) on cellular
photoresponses was also analysed. The insulin-like growth factor (IGF) system plays
a central role in dermal-epidermal interaction during the homeostatic maintenance of
v Abstract
the epidermis and during wound healing. In the studies presented in this thesis, the
modulation of keratinocyte photoresponses to UVB radiation by IGF-I-mediated
activation of the insulin-like growth factor receptor type 1 (IGF-IR) and the resulting
downstream pathways were reported. Significantly, IGF-IR activation: 1) enhanced
keratinocyte viability; 2) suppressed UVB radiation-induced apoptosis; 3)
accelerated the rate of UVR-induced photolesion removal; 4) increased the activation
of p53; 4) modulated cell cycle progression; and 5) enhanced the activation of DNA
damage response pathways. Notably, these results elucidate several mechanisms
through which the IGF-I system may regulate appropriate epidermal responses to
UVB radiation exposure, through the use of a novel in vitro photobiology culture
system. These findings have implications not only for broadening our understanding
of the involvement of fibroblast-keratinocyte interactions in the initiation of NMSCs,
but also as a potential therapeutic or protective strategy against cutaneous
photodamage and photocarcinogenesis.
In summary, this project has characterised and validated previously
undescribed primary cell-based in vitro platforms for photobiology research. The 2D
Transwell® and 3D organotypic HSE-KC and HSE-KCF described herein have
numerous possible applications in studying the biology of the cutaneous
photoresponse. Significantly, the impact of fibroblast-keratinocyte interactions, in
particular via the IGF-I system as reported in this thesis, has highlighted potential
targets for the possible development of novel therapeutic interventions against the
harmful effects of UVB radiation exposure.
vii
Table of Contents
KEYWORDS I
ABSTRACT III
TABLE OF CONTENTS .................................................................................................................. VII
LIST OF FIGURES ........................................................................................................................ XIII
LIST OF TABLES ............................................................................................................................ XV
LIST OF ABBREVIATIONS ........................................................................................................ XVII
STATEMENT OF ORIGINAL AUTHORSHIP ......................................................................... XXV
ACKNOWLEDGEMENTS ........................................................................................................ XXVII
PUBLICATIONS AND PRESENTATIONS ............................................................................... XXX
CHAPTER 1: LITERATURE REVIEW ........................................................................................... 1
1.1 Introduction .................................................................................................................................. 1
1.2 Ultraviolet radiation and skin cancer ........................................................................................... 1
1.3 UVR and the acute effects of its exposure in skin ....................................................................... 4
1.3.1 UVR-induced DNA photodamage .................................................................................... 6
1.3.2 Cellular UVB radiation-induced DNA damage response ................................................. 8
1.3.3 DNA photodamage repair mechanisms .......................................................................... 10
1.3.4 UVR-induced cell cycle arrest and apoptosis ................................................................. 11
1.3.5 The role of p53 in the cellular UV-response ................................................................... 13
1.3.6 UVR-induced inflammation and tanning ........................................................................ 13
1.3.7 UVR-induced immunosuppression ................................................................................. 14
1.4 Fibroblast-keratinocyte interactions during the UVR-response ................................................. 15
1.4.1 Epithelial-mesenchymal interactions in skin .................................................................. 15
1.4.2 Growth factors mediate epithelial-mesenchymal cross-talk ........................................... 16
1.5 The insulin-like growth factor system........................................................................................ 17
1.5.1 IGF-IR signalling pathways ............................................................................................ 19
1.5.2 IGF-I and its role in influencing keratinocyte responses to UVR ................................... 20
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1.5.3 A potential role of the IGF system in photocarcinogenesis ............................................ 22
1.6 Experimental models of photobiology ....................................................................................... 23
1.6.1 Cell culture systems ........................................................................................................ 24
1.6.2 Animal models ................................................................................................................ 26
1.6.3 In vivo clinical studies .................................................................................................... 28
1.6.4 In vitro skin models ........................................................................................................ 29
1.6.5 HSEs as in vitro photobiology models............................................................................ 31
1.6.6 HSE-DED as an organotypic model ............................................................................... 32
1.7 Summary and significance ......................................................................................................... 27
1.8 Project Hypotheses..................................................................................................................... 28
1.9 Project aims ................................................................................................................................ 28
CHAPTER 2: MATERIALS AND METHODS .............................................................................. 29
2.1 Introduction ................................................................................................................................ 29
2.2 Experimental procedures............................................................................................................ 29
2.2.1 Ex vivo skin tissue collection .......................................................................................... 29
2.2.2 Primary cell culture......................................................................................................... 30
2.2.3 Construction of human skin equivalent .......................................................................... 32
2.2.4 Co-culture of keratinocytes and dermal fibroblasts using a Transwell® cell
culture system ................................................................................................................. 34
2.2.5 UVB irradiation protocols .............................................................................................. 35
2.2.6 Histological analysis ....................................................................................................... 37
2.2.7 Immunohistochemical analysis ....................................................................................... 38
2.2.8 Immunofluorescent analysis ........................................................................................... 38
2.2.9 Image analysis ................................................................................................................ 39
2.2.10 Immunofluorescent analysis of Transwell® insert membranes ...................................... 39
2.2.11 TUNEL Assay for the detection of apoptotic cells ......................................................... 40
2.2.12 Assessment of cellular viability ...................................................................................... 40
2.2.13 Detection and quantification of secreted cytokines in 2D and 3D cultures .................... 41
2.2.14 Analysis of cell cycle phase by fluorescent-labelling of DNA content .......................... 42
2.2.15 Extraction of total RNA from HSE composites .............................................................. 43
2.2.16 Purification of total RNA from cell lysates .................................................................... 43
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2.2.17 Synthesis of double-stranded cDNA .............................................................................. 44
2.2.18 Primer design .................................................................................................................. 44
2.2.19 Analysis of differential gene expression using qRT-PCR .............................................. 44
2.2.20 Extraction of protein from HSE epidermal cell lysates .................................................. 45
2.2.21 Extraction of protein from Transwell® cell monolayers ................................................ 45
2.2.22 Protein separation of cell lysates using SDS-PAGE ....................................................... 46
2.2.23 Western immunoblotting ................................................................................................ 46
2.2.24 Statistical analysis .......................................................................................................... 47
CHAPTER 3: VALIDATION OF THE HSE-KC PHOTOBIOLOGY MODEL ......................... 49
3.1 Introduction................................................................................................................................ 49
3.2 Materials and methods ............................................................................................................... 53
3.2.1 Fixation of native skin samples ...................................................................................... 53
3.2.2 Construction and culture of the HSE-KC ....................................................................... 53
3.2.3 Irradiation of the HSE-KC .............................................................................................. 53
3.2.4 Histology ........................................................................................................................ 53
3.2.5 MTT assay of cell viability ............................................................................................. 53
3.2.6 Immunohistochemistry ................................................................................................... 53
3.2.7 Detection and quantification of secreted cytokines in 2D and 3D cultures .................... 54
3.2.8 TUNEL assay ................................................................................................................. 54
3.3 Results ....................................................................................................................................... 55
3.3.1 Immunohistochemical characterisation of the HSE-KC epidermis ................................ 55
3.3.2 Optimisation of UVB-irradiation protocol ..................................................................... 57
3.3.3 Assessment of cell viability in irradiated HSE-KC ........................................................ 58
3.3.4 Formation of CPDs in irradiated HSE-KC composites .................................................. 60
3.3.5 Formation of apoptotic keratinocytes in the irradiated HSE-KC .................................... 60
3.3.6 UVB-induced expression of p53 in the HSE-KC ........................................................... 61
3.3.7 Secretion of inflammatory cytokines by irradiated HSE-KC constructs ........................ 63
3.3.8 Removal of CPDs in HSE-KC post-irradiation .............................................................. 65
3.3.9 Expression of Ki-67 in irradiated HSE-KC .................................................................... 68
3.3.10 Keratinocyte differentiation ............................................................................................ 71
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3.4 Discussion .................................................................................................................................. 75
3.5 Conclusion ................................................................................................................................. 85
CHAPTER 4: FIBROBLAST-KERATINOCYTE INTERACTIONS AND THE ACUTE UVB
RESPONSE 87
4.1 Introduction ................................................................................................................................ 87
4.2 Materials and methods ............................................................................................................... 92
4.2.1 Primary human cell isolation and culture ....................................................................... 92
4.2.2 Transwell® fibroblast and keratinocyte co-cultures ....................................................... 92
4.2.3 Analysis of cellular viability ........................................................................................... 92
4.2.4 TUNEL assay for the detection of apoptosis .................................................................. 92
4.2.5 Detection and quantification of secreted cytokines in 2D and 3D cultures .................... 92
4.2.6 HSE-KC construction and culture .................................................................................. 92
4.2.7 Irradiation of keratinocyte monolayers on Transwell® inserts ....................................... 92
4.2.8 Optimisation of HSE-KCF culture protocol ................................................................... 93
4.2.9 Irradiation of the HSE-DED composites ........................................................................ 93
4.2.10 Histological analysis of tissues ....................................................................................... 94
4.2.11 Immunofluorescent analysis ........................................................................................... 94
4.2.12 Immunohistochemical analysis ....................................................................................... 94
4.2.13 Analysis of apoptotic pathways using ELISA ................................................................ 95
4.2.14 qRT-PCR analysis of gene expression ............................................................................ 95
4.2.15 Western immunoblotting analysis .................................................................................. 95
4.3 Results ........................................................................................................................................ 97
4.3.1 Optimisation of a Transwell® co-culture system photobiology model .......................... 97
4.3.2 Effects of fibroblast co-culture on cell viability in irradiated keratinocytes ................... 98
4.3.3 Effects of fibroblast co-culture on cell death in irradiated keratinocytes........................ 99
4.3.4 Fibroblast co-culture and the removal of UVB-induced DNA dimers in irradiated
keratinocytes ................................................................................................................. 105
4.3.5 Development and characterisation of the HSE-KCF skin model .................................. 109
4.3.6 Characterisation of the UVB response in HSE-KCF .................................................... 115
4.3.7 Mechanism of fibroblast-induced keratinocyte survival post-irradiation ..................... 125
4.3.8 Fibroblast-induced regulation of p53 in irradiated keratinocytes ................................. 130
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4.4 Discussion ................................................................................................................................ 135
CHAPTER 5: INVESTIGATING THE ROLE OF IGF-I IN THE KERATINOCYTE UVB
RESPONSE 147
5.1 Introduction.............................................................................................................................. 147
5.2 Materials and methods ............................................................................................................. 151
5.2.1 Isolation and culture of primary human skin cells ........................................................ 151
5.2.2 Transwell® co-culture of keratinocytes and fibroblasts ............................................... 151
5.2.3 Irradiation of keratinocyte monolayers ......................................................................... 151
5.2.4 Quantification of IGF-I in cell culture supernatants ..................................................... 151
5.2.5 Testing the effects of exogenous IGF-I on irradiated keratinocytes ............................. 151
5.2.6 Testing the effects of fibroblast conditioned medium on irradiated keratinocytes ....... 152
5.2.7 Analysis of keratinocyte viability ................................................................................. 152
5.2.8 Analysis of IGF-IR-mediated cell signalling pathways using Western
immunoblotting ............................................................................................................ 153
5.2.9 Cell cycle analysis by flow cytometry .......................................................................... 153
5.2.10 Analysis of apoptosis in irradiated keratinocytes ......................................................... 153
5.2.11 Analysis of the formation of UVB-induced CPD photolesions in irradiated
keratinocytes ................................................................................................................. 154
5.2.12 Blocking IGF-IR function using a neutralisation antibody ........................................... 154
5.2.13 Analysis of differential gene expression using qRT-PCR ............................................ 154
5.2.14 UVB radiation-induced DNA damage response antibody array ................................... 154
5.3 Results ..................................................................................................................................... 157
5.3.1 In vitro concentration of IGF-I in 2D and 3D cultures ................................................. 157
5.3.2 Effect of IGF-I on irradiated keratinocyte viability ...................................................... 158
5.3.3 IGF-IR signalling in irradiated keratinocytes ............................................................... 160
5.3.4 Effect of IGF-I on UVB-induced apoptosis .................................................................. 162
5.3.5 Effects of the blocking of IGF-IR activation on cell death in irradiated
keratinocytes ................................................................................................................. 163
5.3.6 Effect of IGF-I on the repair of UVB-induced DNA damage ...................................... 166
5.3.7 Repair of UVB-induced CPDs with IGF-IR neutralisation in irradiated
keratinocytes ................................................................................................................. 167
5.3.8 Effect of IGF-I on p53 expression in irradiated keratinocytes ...................................... 169
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5.3.9 Effect of IGF-I on cell cycle phase ............................................................................... 171
5.3.10 DNA damage response proteins ................................................................................... 172
5.4 Discussion ................................................................................................................................ 176
CHAPTER 6: GENERAL DISCUSSION ...................................................................................... 187
BIBLIOGRAPHY ............................................................................................................................. 203
xiii List of figures
List of Figures
Figure 1.1. The structure of human skin. ................................................................................................ 2
Figure 1.2. UVR-induced skin cancers ................................................................................................... 4
Figure 1.3. Penetration of UVR wavelengths into human skin. .............................................................. 5
Figure 1.4. Effects of UVB irradiation on skin cells. .............................................................................. 6
Figure 1.5. Cell cycle checkpoint pathways initiated by UVB radiation-induced DNA damage ............ 9
Figure 1.6. Signalling cascades associated with IGF-IR activation ...................................................... 20
Figure 1.7. Downstream cell survival effects from IGF-IR activation. ................................................. 21
Figure 1.8. A potential role of the IGF system in photocarcinogenesis ................................................ 23
Figure 1.9. HSE-DED composites in culture ........................................................................................ 33
Figure 1.10. Comparison of histology between native skin and HSE-DED sections. ........................... 34
Figure 2.1. Timelines of 2D and 3D keratinocyte in vitro culture methods .......................................... 34
Figure 2.2. Transwell® co-culture of primary human keratinocytes and fibroblasts ............................ 35
Figure 2.3. UVB irradiation of cells and skin tissue samples ................................................................ 37
Figure 3.1. Comparison of cutaneous markers in HSE-KC and native skin ......................................... 56
Figure 3.2. Dose-response of HSE-KC to UVB radiation. .................................................................... 58
Figure 3.3. Epidermal viability following UVB-irradiation in HSE ..................................................... 59
Figure 3.4. UVB exposure induces the formation of CPDs in HSE-KC ............................................... 60
Figure 3.5. Apoptotic keratinocytes in irradiated HSE-KC ................................................................... 61
Figure 3.6. UVB induces p53 expression in irradiated HSEs................................................................ 62
Figure 3.7. Quantification of IL-6 and IL-8 concentrations in HSE-KC culture supernatants .............. 64
Figure 3.8. Detection of CPDs in irradiated HSE-KC over time........................................................... 66
Figure 3.9. Repair of CPDs in irradiated HSE-KC ............................................................................... 67
Figure 3.10. Detection of Ki-67 in irradiated HSE-KC ......................................................................... 69
Figure 3.11. Quantification of Ki-67 expression in irradiated and non-irradiated HSE-KC ................. 70
Figure 3.12. Time-course analysis of keratin 1/10/11 expression in irradiated and control HSE-
KC ........................................................................................................................................ 72
Figure 3.13. Quantification of keratin 1/10/11 expression in HSE-KC over time ................................ 73
Figure 3.14. Validation of the HSE-KC as a photobiology model ........................................................ 86
Figure 4.1. Irradiation of keratinocyte monolayers cultured in different co-culture conditions ............ 93
Figure 4.2. Cell viability of keratinocytes in UVB-irradiated Transwells®.......................................... 98
Figure 4.3. UVB-induced keratinocyte apoptosis in a 2D co-culture model ....................................... 101
Figure 4.4. Fibroblast co-culture inhibits apoptosis in irradiated keratinocyte monolayers ................ 102
Figure 4.5. Detection of cleaved caspase-3 in irradiated keratinocyte monolayers ............................. 104
xiv List of figures
Figure 4.6. Repair of CPD in irradiated keratinocyte monolayers in different culture conditions ...... 107
Figure 4.7. Fibroblasts enhance repair in irradiated keratinocyte monolayers .................................... 108
Figure 4.8. Optimisation of fibroblast seeding in the construction of HSE-KCF ............................... 110
Figure 4.9. Collagen IV expression in HSE-KCF and native skin ...................................................... 112
Figure 4.10. Expression of vimentin in native skin and HSE composites ........................................... 113
Figure 4.11. Histology of HSE-KCF in culture................................................................................... 114
Figure 4.12. Histology of irradiated HSE-KCF .................................................................................. 116
Figure 4.13. Quantification of IL-6 and IL-8 concentrations in HSE-KCF culture supernatants........ 117
Figure 4.14. UVB-induced formation of CPDs in HSE-KCF ............................................................. 119
Figure 4.15. Rate of CPD repair in irradiated HSE-KC and HSE-KCF .............................................. 120
Figure 4.16. Effect of UVB on keratinocyte proliferation in the HSE-KCF ....................................... 122
Figure 4.17. Keratinocyte apoptosis in irradiated HSE-KC and HSE-KCF following UVB
irradiation. .......................................................................................................................... 124
Figure 4.18. Relative expression of Bad and phosphorylated-Bad in irradiated HSE-KC and
HSE-KCF ........................................................................................................................... 126
Figure 4.19. Relative expressions of cleaved PARP in irradiated HSE-KC and HSE-KCF ............... 128
Figure 4.20. Relative expressions of cleaved caspase-3 in irradiated HSE-KC and HSE-KCF .......... 129
Figure 4.21. Relative expression of p53 in HSE-KC and HSE-KCF after UVB irradiation. .............. 131
Figure 4.22. Relative levels of p53 protein in irradiated HSE-KC and HSE-KCF composites .......... 133
Figure 5.1. Experimental set up for testing the effects of IGF-I on irradiated keratinocytes .............. 152
Figure 5.2. Experimental set up for testing the effects of fibroblast conditioned medium on
irradiated keratinocytes ...................................................................................................... 152
Figure 5.3. Quantification of IGF-I concentration in cell culture supernatants ................................... 158
Figure 5.4. Effects of IGF-I treatment on the viability of irradiated keratinocyte monolayers ........... 159
Figure 5.5. Activation of IGF-IR-mediated signalling pathways in irradiated keratinocytes ............. 161
Figure 5.6. Effect of IGF-I on UVB-induced apoptosis in human keratinocytes ................................ 163
Figure 5.7. Effect of IGF-IR blocking antibody treatment on UVB-induced apoptosis in
irradiated keratinocytes ...................................................................................................... 165
Figure 5.8. Effect of IGF-I on the repair of UVB-induced CPDs in irradiated keratinocytes ............. 167
Figure 5.9. Effect of IGF-IR blocking antibody treatment on the formation of UVB-induced
DNA dimers in irradiated keratinocytes ............................................................................. 168
Figure 5.10. Relative gene and protein expressions of p53 in irradiated keratinocytes ...................... 170
Figure 5.11. Effect of growth factor pre-treatment on cell cycle phases in irradiated
keratinocytes ...................................................................................................................... 172
Figure 5.12. Expression of DNA damage response proteins in IGF-I-treated irradiated
keratinocytes ...................................................................................................................... 173
xv List of tables
List of Tables
Table 1.1.1. Characteristics of commonly used in vitro and in vivo models for studying skin
cancer biology. ..................................................................................................................... 25
xvii List of abbreviations
List of Abbreviations
(6-4)PP (6-4) pyrimidone photoproducts
°C degrees Celsius
µg/µL micrograms per microlitre
µg/mL micrograms per millilitre
µm micrometer
µM micromolar
µmol micromoles
2D two-dimensional
2-∆∆C
T relative comparative threshold cycle method
3D three-dimensional
3T3 3T3 mouse fibroblast cell line
8-oxo-dG 8-oxo-2'-deoxyguanosine
25(OH)D 25-hydroxyvitamin D3
ABAM antibiotic/antimycotic solution
ACTH adrenocorticotropic hormone
AKT protein kinase B
ANOVA analysis of variance
Arg arginine
Asp aspartic acid
ATM Ataxia telangiectasia mutated
ATR Ataxia telangiectasia mutated and Rad3-related protein
ATRIP ATR-interacting protein
Bad Bcl-2-associated death promoter
Bax Bcl-2-associated X protein
xviii List of abbreviations
BCC basal cell carcinoma
Bcl-2 B-cell lymphoma 2
b-FGF basic fibroblast growth factor
BM basement membrane
BSA bovine serum albumin
CDK cyclin dependent kinase
cdk-1 cyclin dependent kinase-1
cDNA complementary DNA
cm centimetres
CM conditioned medium
CMM cutaneous malignant melanoma
CO2 carbon dioxide
CPD cyclobutane pyrimidine dimer
CT threshold cycle
DAPI 4',6-diamidino-2-phenylindole
DED de-epidermised dermis
DMBA 7,12-dimethylbenz[α]anthracene
DMEM Dulbecco’s modified Eagles medium
DMEM/F-12 Dulbecco’s modified Eagles medium/ Ham’s F-12
DNA deoxyribonucleic acid
DNase I deoxyribonuclease I
dUTP 2´-deoxyuridine, 5´-triphosphate
ECM extracellular matrix
EDTA ethylenediamine tetra-acetic acid
EGF epidermal growth factor
ELISA enzyme-linked immunosorbent assay
ELK-1 E-twenty-six (ETS)-like transcription factor 1
xix List of abbreviations
ERK 1/2 extracellular signal-related kinase-1 and 2
FCS foetal calf serum
FGM full Green’s medium
Fib fibroblast
FKH fork head
g grams
G0 Gap zero (resting) phase
G1 Gap one (post-mitotic) phase
G2 Gap two (pre-mitotic) phase
GAPDH glyceraldehyde 3-phosphate dehydrogenase
GM-CSF granulocyte-macrophage colony-stimulating factor
Gy gray
HGF hepatocyte growth factor
HGF/SF hepatocyte growth factor/scatter factor
HRP horseradish peroxidase
HSE human skin equivalent
HSE-DED human skin equivalent- de-epidermised dermis
HSE-KC human skin equivalent (keratinocyte only)
HSE-KCF human skin equivalent (keratinocyte and fibroblast-populated DED)
IGF-2R insulin-like growth factor type-2 receptor
IGFBP insulin-like growth factor binding protein
IGF-I insulin-like growth factor-I
IGF-II insulin-like growth factor type-2
IGF-IR insulin-like growth factor type-1 receptor
IGF-IRα insulin-like growth factor receptor α subunit
IGF-IRβ insulin-like growth factor receptor β subunit
IgG immunoglobulin G
xx List of abbreviations
IL interleukin
IRS-1 insulin-receptor substrate type-1
JNK c-Jun N-terminal kinase
kb kilobases
kDa kilo Dalton
KGF keratinocyte growth factor
Leu leucine
m metres
M molar
MAPK mitogen-activated protein kinase
MC1R human melanocortin 1 receptor
MDM2 mouse double minute 2
MED minimal erythemal dose
MEK mitogen-activated protein kinase
mins minutes
MITF microphthalmia-associated transcription factor
mJ millijoules
mL millilitre
mm millimetres
MRN MRE11-RAD50-NBS1
mRNA messenger ribonucleic acid
mTOR mammalian target of rapamycin
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium)
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
mw molecular weight
NER nuclear excision repair
xxi List of abbreviations
ng/µL nanogram per microlitre
nm nanometers
NMSC non-melanoma skin cancer
N-terminal amino-terminal
p p-value
p-AKT phosphorylated AKT
PARP poly (ADP-ribose) polymerase
PBS phosphate buffered saline
PDK-1 pyruvate dehydrogenase lipoamide kinase isozyme 1
PFA paraformaldehyde
PGE2 prostaglandin E2
pH H+ ion concentration
PI3K phosphoinositol 3-kinase
p-IGF-IR phosphorylated IGF-IR
POMC pro-opiomelanocortin
PTEN phosphatase and tensin homolog
PTH-rp parathyroid hormone-related protein
PTP protein tyrosine phosphatases
Px passage number x
qRT-PCR quantitative real time polymerase chain reaction
Raf-1 raf proto-oncogene serine/threonine protein kinase-1
RNA ribonucleic acid
ROS reactive oxygen species
RTK receptor tyrosine kinase
S-phase synthesis phase
SCC squamous cell carcinoma
SCF stem cell factor
xxii List of abbreviations
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEM standard error of the mean
Ser serine
SFM serum-free medium
ssDNA single-stranded DNA
T time (hours)
T3 triiodothyronine
Tm melting temperature
TAE tris-acetate/ethylenediaminetetraacetic acid
t-AKT total-AKT
TBS-T tris-buffered saline Tween-20
t-ERK total extracellular signal-related kinase
TGF-β transforming growth factor β
Thr threonine
t-IGF-IR total insulin-like growth factor type-1 receptor
t-IGF-IR total insulin-like growth factor receptor
TMB 3,3',5,5'-tetramethylbenzidine
TNF-α tumour necrosis factor-α
TPA 12-O-tetradecanoylphorbol-13-acetate
TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
Tyr tyrosine
TYR tyrosine kinase
U/mL units per millilitre
UVA ultraviolet radiation A
UVB ultraviolet radiation B
UVC ultraviolet radiation C
UVR ultraviolet radiation
xxiii List of abbreviations
V volts
XP Xeroderma pigmentosum
α alpha
α-MSH alpha-melanocyte-stimulating hormone
β beta
xxv
Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the
best of my knowledge and belief, the thesis contains no material previously
published or written by another person except where due reference is made.
Signature: _________________________
Date: _________________________ 17 July 2013
QUT Verified Signature
xxvii
Acknowledgements
Firstly, I would like to extend my heartfelt gratitude to the wonderful supervisory
team who supported me through my PhD journey. Firstly, thank you Zee for the
opportunity to be a part of the TRR team and for all your guidance and advice. Your
accomplishments have been a great inspiration and your encouragement and belief in my
abilities certainly mean a lot. I am also very appreciative of the chances I’ve been given
for international conference travel, which have definitely been highlights of this journey.
A great big thank you, Derek, for all the generosity you’ve shown with your time,
practical help, looking through my many drafts and posters, keeping me on track and for
always delivering it all with a huge smile. Thanks for celebrating even the smallest of
my accomplishments. It was a big motivation having your unwavering support from start
to finish.
To Michael, you have been a wonderful mentor. Thank you so much for being
always enthusiastic and optimistic throughout my project, while always injecting a
healthy dose of reality when needed! I am very grateful for being included as an
honorary member of CRESH and the AusSun group and for all your advice and support.
Thank you Rebecca for being the human Google for all things HSE/skin related,
for all the knowledge and techniques you’ve passed on to me, for being genuinely
excited about my work and for always showering me with praise along the way. Your
friendship and guidance have definitely helped me keep my head above water.
Finally, thank you David for your insightful comments and for your signature
post-presentation questioning, which has certainly prepared me for even the toughest of
audiences! Your input and assistance (and lunch-time humour) are very much
appreciated.
I would like to extend my gratitude to the brilliant members of the TRR Skin
Group. A special thank you goes out to Jacqui and Emily for assisting me with qRT-PCR
and immunohistochemistry. You have all always been incredibly supportive of me and
so generous with ideas and constructive criticism. The many, MANY hours spent in the
Primary Room were only bearable thanks to the never-ending supply of laughs and good
conversation.
xxviii
My wonderful friends from the TRR group, I thank you for all your support and
many fond memories. Thank you Gary for the supply of IGF-I and sarcasm, Ali: thanks
for being a great pal and always making me laugh, Tony for your encouragement and
chats over beers, Dayle: thesis writing was a little less painful with you in the same boat,
Svenja for letting me be a part of your PhD project (ouch!), and Arnulf, thanks for
always cheering me on and being a great friend!
I would like to thank those who have generously contributed their time to making
this project a success: Leo de Boer, for assistance with cell imaging and FACS, to Elke
Hacker for being a fantastic source of guidance and advice towards this project, Clay
Winterford and the QIMR Histology team for your assistance with
immunohistochemistry, Dod and David for their hard work behind the scenes, Nicky
Gillott, Sharyn McCormack and Stella Winn for all your wonderful support and help.
To my buddy Abhishek ‘Beebs’ Kashyap, you have been a continuous source of
entertainment with your crazy sense of humour, our many ideas on how we can become
millionaires, and of course being my human guinea pig for my study on protein
consumption and (lack of) muscle growth. This journey would have been unbearable
without you sitting right there behind me, and for that you have my eternal gratitude.
And to the other member of the Wolfpack, my beloved friend: Manas ‘Chubes’
Sivaramakrishnan. Thanks for being right beside me through all the funniest, best, worst,
craziest (and slightly fuzzy) memories. Your zest for life and obsession with antioxidants
has helped keep me sane and youthful. You are truly an amazing person and your
friendship means the world to me!
My heartfelt thanks go to my family for being my pillar of strength. To my
parents, Ivan and Shila Fernandez, for always making me believe I have limitless
potential and for providing countless opportunities to make my dreams a reality. To
Devin Fernandez, my best friend and incredibly talented brother: knowing how proud
you are of me is what keeps me going. To my aunt, Mema, thank you for the continuous
supply of love and prayers, which were great sources of strength. Claudine Michael, you
are my rock and I could not have achieved this without your blind faith in my abilities,
being right beside me through this rollercoaster ride and your constant iChats. Your
resilience and philosophies on life will always be a huge inspiration to me. Uncle
Lawrence & Mama Yvonne: thanks for all your encouragement and for always being so
proud of me. Aunty Gill, thank you for being so supportive and genuinely excited about
all my academic endeavours and achievements.
xxix List of abbreviations
To my wonderful friends who have always been behind me despite the miles
between us. Thank you to Vanessa Low for all the years of loyal friendship, for sharing
in all my joys and for being a ray of hope through the tough times. Thank you, Ganeshini
Sri Kanthan for being a wonderful sister and for believing in my biology skills since we
were kids. Janita Kumar, thanks for cheering me on, the good advice and for being ever
ready to celebrate my arrival. To Zarina Muhammad for over a decade of love and
laughs and for being the biggest fan of my scientific pursuits.
Last but not least, I would like to acknowledge all the generous skin donors for the
donation of their tissues, as well as the research and clinical staff members that facilitate
skin collection; without their contributions, this project would not be possible.
xxx Presentations and publications
Publications and presentations
Fernandez T.L., Dawson, R. A., Van Lonkhuyzen, D. R., Kimlin, M. G., Upton, Z.
(2012) A tan in a test tube: in vitro models for investigating ultraviolet radiation-
induced damage in skin. Experimental Dermatology 21:404-10.
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2012) Human dermal fibroblasts protect against UVB radiation-
induced keratinocyte damage in a human skin equivalent model. Society for
Investigative Dermatology Meeting, Raleigh, North Carolina, 8-12 May (Poster
presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2012) Human dermal fibroblasts protect against UVB radiation-
induced keratinocyte damage in a human skin equivalent model. 6th Australian
Health and Medical Research Congress, Adelaide, 25 - 28 Nov (Poster presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2012) Human dermal fibroblasts protect against UVB radiation-
induced keratinocyte damage in a human skin equivalent model. Australasian Wound
& Tissue Repair Society Meeting – ‘Repair and regeneration’, Sydney, Australia,
22-24 May (Oral presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2011) The human skin equivalent model: A tool for studying the
effects of UV radiation in skin. Molecular and experimental pathology society of
Australia Annual Conference, Brisbane, Australia 30 Nov – 2 Dec (Oral
presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2011) Investigating the effects of UVB radiation on human skin – a
human skin equivalent model. Centre for Research Excellencein Sun and Health
(CRESH) Annual Scientific Meeting, Brisbane, Australia, 30 Nov (Oral presentation)
xxxi Presentations and publications
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2011) The role of IGF-I in the responses of skin cells to UV radiation.
IGF-Oz – The Insulin-like Growth Factor System and Related Proteins in
Development and Disease, Parkville, Victoria, 27-28 Oct (Poster presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2011) Investigating the effects of ultraviolet radiation on skin – a
human skin equivalent model. Institute of Health and Biomedical Innovation Inspires
Postgraduate Conference, Brisbane, Australia 22-24 Nov (Oral presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2011) A human skin equivalent model for studying responses of skin
to ultraviolet radiation. AusBio GlaxoSmithKline Student Excellence State Finals,
Brisbane, Australia, 27 Sep (Oral presentation, State Finalist)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2011) A human skin equivalent model for studying responses of skin
to ultraviolet radiation. Australian Society for Medical Research Postgraduate
Student Conference, Brisbane, Australia, 30-31 May (Poster presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2011) Role of IGF-I in the responses of human keratinocytes to UVR.
Gordon Research Conference – ‘Insulin-like growth factors in physiology and
disease’, Ventura, California, USA, 27 Feb – 4 Mar (Poster presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. Human skin equivalents: Applications of an in vitro cell-based model.
Institute of Health and Biomedical Innovation Research Showcase, Brisbane,
Australia (Oral presentation)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2010) A human skin equivalent model for studying responses of skin
to ultraviolet radiation. AusBio GlaxoSmithKline Student Excellence State Finals,
Brisbane, Australia, 28 Sep (Oral presentation, State Finalist)
Fernandez T.L., Van Lonkhuyzen, D. R., Dawson, R. A., Leavesley, D., Kimlin, M.
G., Upton, Z. (2010) Investigating the effects of ultraviolet radiation on skin – a
human skin equivalent model. Institute of Health and Biomedical Innovation Inspires
Postgraduate Conference, Gold Coast, Australia 22-24 Nov (Poster presentation).
1 Literature review
Chapter 1: Literature review
1.1 INTRODUCTION
Skin is the largest organ in the human body and forms the frontline of defence
against environmental hazards, including ultraviolet radiation (UVR). Accordingly,
skin possesses sophisticated systems to resist and repair UVR-induced damage.
Chronic exposure to UVR, coupled with the failure of these regenerative processes,
has been associated with photodamage and the onset of skin cancers. The molecular
mechanisms underpinning UVR exposure and the resultant damage to skin have been
the subject of extensive research but, to date, have yet to be fully characterised. The
future of photocarcinogenesis research is therefore highly-dependent on deepening
our understanding of the impact of UVR on cutaneous biology.
1.2 ULTRAVIOLET RADIATION AND SKIN CANCER
Human skin encompasses an avascular epidermis comprised of distinct
differentiated layers and a characteristic polarized pattern of keratinocyte growth,
with actively proliferating cells situated along the basement membrane (BM) (Figure
1.1). Pigment-producing melanocytes, Merkel cells and Langerhans cells are also
found in this epidermal layer. Underlying the epidermis is the dermis, predominantly
made up of connective tissue and with dermal fibroblasts as the major cell type of
this region. In addition, hair follicles, blood vessels, sweat ducts, sebaceous glands
and nerve endings are also present in the dermis.
Predisposing risk factors for the development of skin cancers include skin
colour, gender and genetic factors, such as mutations in the melanocortin 1 receptor
(MCIR), p16 (CDKN2A) and glutathione S-transferase (GST) genes (Ramsay et al.,
2001; Matichard et al., 2004; Goldstein et al., 2007). Both epidemiological and
experimental studies have demonstrated that excessive and unprotected exposure to
UVR triggers a cascade of biological processes in the skin, which is the main risk
factor associated with the onset of skin cancers (Matsumura and Ananthaswamy,
2002). An individual’s cumulative lifetime exposure to UVR is thought to be the
leading risk factor for developing all types of skin cancers (Armstrong and Kricker,
2 Literature review
2001). Solar keratoses are among the first pre-malignant skin changes resulting from
chronic UVR exposure (Holmes et al., 2007). Histologically, solar keratoses exhibit
hyperproliferative keratinocytes with atypical cellular and nuclear morphology and
an abnormal organisation at the epidermal-dermal junction (Schwartz, 1996). Genetic
analysis of solar keratoses reveal that more than half of the keratinocytes in these
lesions posses genetic mutations, particularly in the tumour suppressor gene, p53
(Nelson et al., 1994). As a result of these and other similar oncogenic mutations,
solar keratoses possess the propensity to develop into squamous cell carcinomas
(SCC).
Figure 1.1. The structure of human skin.
The epidermis and dermis, separated by the basement membrane (BM), make up the two major components of
skin. Actively proliferating keratinocytes and melanocytes lie along the epidermal-dermal junction, with
keratinocytes at varying stages of terminal differentiation making up the distinct epidermal layers (stratum
corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale). The papillary region of
the dermis features rete ridges, seen histologically as areas of epidermal thickening. The reticular region is
primarily composed of dense irregular connective tissue. Dermal fibroblasts generate connective tissue,
extracellular matrix proteins and a variety of factors critical for the maintenance of the epidermis. Figure © T.
Fernandez, 2012.
3 Literature review
Australia has the highest rates of skin cancer in the world (Australian Institute
of Health and Welfare, 2007). Alarmingly, it is estimated that 450 000 Australians
are diagnosed with a form of skin cancer annually (Australian Institute of Health and
Welfare, 2008). As a result, this disease is the largest financial burden on the
Australian health care system as compared to other cancers (Australian Institute of
Health and Welfare, 2005). The main categories of skin cancers are melanomas, or
cancers involving malignant transformations in melanocytes, and those that result
from the transformation of keratinocytes, or non-melanoma skin cancers (NMSCs)
(Figure 1.2). Cutaneous malignant melanomas (CMM) account for about 4% of all
skin cancers (Boscoe and Schymura, 2006). Despite being relatively less common,
they are associated with the greatest mortality rates (Tsao et al., 2004). This is
correlated with an increased likelihood of melanoma metastasis in CMM patients
(Pollack et al., 2011). While it is believed that chronic UVR exposure leading to
oncogenic transformations in melanocytes forms the basis of CMM development, the
precise biological connection between UVR exposure and the risk of CMM has yet
to be established. For instance, it has yet to be determined whether the cumulative
effect of UVR exposure or childhood incidences of sunburn, play a greater role in the
development of CMMs (Zanetti et al., 1992).
Basal cell carcinomas (BCCs) and SCCs, on the other hand, are among the
most frequently diagnosed malignancies worldwide (Gallagher et al., 2007).
Epithelial in origin, these NMSCs occur as a result of neoplastic changes in
keratinocytes following chronic exposure to skin irritants, including UVR.
Histological and genetic analysis of NMSC tumours shows aberrant keratinocyte
proliferation, growth and keratinisation patterns (Ponec et al., 1988a; Lodygin et al.,
2003; Mancuso et al., 2004). These neoplasms commonly occur on sun-exposed
regions of the body such as the face, neck and head and represent the majority of skin
cancer incidences (Rass and Reichrath, 2008). Both BCCs and SCCs show strong
similarities in the genetic alterations of major oncogenes and tumour suppressor
genes in transformed keratinocytes (Wang et al., 2011).
4 Literature review
Figure 1.2. UVR-induced skin cancers
The photographs represent the clinical manifestations of (A) BCC, (B) SCC and (C) CMM in skin cancer
patients. Images were obtained from the Australasian College of Dermatologists website
(http://www.dermcoll.asn.au/public/a-z_of_skin-types_of_skin_cancers.asp).
1.3 UVR AND THE ACUTE EFFECTS OF ITS EXPOSURE IN SKIN
Sunlight is comprised of a continuous spectrum made up largely of visible and
infrared light, with about 10% consisting of UVR, radio, microwave and X-rays
(Holzle and Honigsmann, 2005). Solar-emitted UVR is predominantly long
wavelength ultraviolet A (UVA) (320 to 400 nm) radiation, followed by the UVB
(290 to 320 nm) and UVC wavelengths (254 nm). It is believed that both the
penetration of both the UVA and UVB wavelengths into skin during sun exposure
are responsible for the onset of short and long-term biological and clinical
consequences. These three UVR wavelengths penetrate into different depths of the
epidermis and dermis (Figure 1.3). The UVA wavelength permeates into dermis,
while UVB radiation mostly effects the epidermis through damage to the
keratinocytes at the dermal-epidermal junction (Bernerd et al., 2000). The UVC
wavelength, which only passes through the stratum corneum, is filtered by oxygen
and ozone in the atmosphere, and is not a major component of terrestrial solar UVR.
The major mode of action involved in cutaneous cell damage as a result of UVA
exposure is the generation of reactive oxygen species (ROS) (Tyrrell and Keyse,
1990). The damaging effects of UVB exposure are detailed in the following sections.
5 Literature review
Figure 1.3. Penetration of UVR wavelengths into human skin.
The UVB and UVA wavelengths, which make up the majority of UVR in sunlight are able to penetrate the skin,
affecting epidermal cells in the stratum granulosum, stratum spinosum, transitional zone and stratum basale. The
majority of the UVC wavelength is absorbed by atmospheric ozone, with artificial sources observed to only pass
through the stratum corneum. The image on the left shows a histological section of native human skin, with green
arrows on the right depicting the relative depths of UVR wavelength penetration. The scale bar represents 50 µm.
Figure © T. Fernandez, 2012.
The UVB radiation wavelength is readily absorbed by DNA, proteins and cell
membranes causing both harmful and beneficial effects. Exposure to UVB radiation
is responsible for the production of 25-hydroxyvitamin D3 (25(OH)D), a steroid
hormone crucial for normal bone and muscle function (Bogh et al., 2010). High
doses of UVR, however, have detrimental effects in the cell’s genetic material and
induce the generation of reactive oxygen species, the cross-linking of various cellular
proteins and the peroxidation of lipids (Setlow and Carrier, 1966). Consequently, this
triggers an inflammatory response in the form of erythema, or sunburn. The amount
of UVR that can be absorbed by the skin before the onset of erythema is known as
the minimal erythemal dose (MED), with this measurement being approximately 100
mJ/cm2
for UVB radiation (Poon et al., 2003). The cutaneous effects of UVB
irradiation on the skin are summarised in Figure 1.4.
6 Literature review
Figure 1.4. Effects of UVB irradiation on skin cells.
Exposure to UVB radiation results in the secretion of various inflammatory factors such as interleukins 1, 6, 8
(IL-1/6/8), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-
α) (Yoshizumi et al., 2008). Additionally, keratinocytes secrete melanocyte activating factors such as alpha-
melanocyte stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH) and prostaglandin E2 (PGE2)
(Gordon et al., 1989). Melanocytes respond by increasing the activity of tyrosinase (TYR) and the synthesis of
melanin-containing melanosomes (Yamaguchi et al., 2007). Dermal fibroblasts produce a variety of paracrine
factors which act in the orchestration and regulation of epidermal regeneration such as basic fibroblast growth
factor (bFGF) (Berking et al., 2001), stem cell factor (SCF) (Imokawa et al., 1998), hepatocyte growth factor
(HGF) (Imokawa et al., 1998), insulin-like growth factor (IGF) (Tavakkol et al., 1992) and keratinocyte growth
factor (KGF) (Kovacs et al., 2009). Damage to DNA in UVB radiation-exposed cells via the production of
DNA lesions and reactive oxygen species occurs. Severely damaged cells undergo apoptosis, with the
characteristic activation of caspases and DNA fragmentation. Alterations in the expression patterns of various
epidermal proteins are also observed in the period following UVB irradiation. Figure © T. Fernandez, 2012.
1.3.1 UVR-induced DNA photodamage
Damage to DNA as a result of UVR exposure is known to be one of the
initiating factors in photocarcinogenesis (Ley and Fourtanier, 1997). In irradiated
cells, DNA acts as a chromophore, directly absorbing photons of UVR, thus resulting
in the formation of mutagenic DNA lesions (Regan and Setlow, 1974). This occurs
when two adjacent pyrimidine bases on the same DNA strand form dimeric
photoproducts. The more common form of these pyrimidine dimers are cyclobutane
pyrimidine dimers (CPDs), as a result of carbon atoms at the C5 and C6 positions of
7 Literature review
thymine or cytosine bases forming covalent bonds and producing a four-membered
ring (Protic-Sabljic et al., 1986). Similarly, (6-4) pyrimidine-pyrimidone dimers ((6-
4)PPs) are formed from the covalent bonding of carbon atoms at the C4 and C6
positions, frequently between the TC and CC dinucleotides (Franklin et al., 1985).
This particular dimer can be converted to a third form of carcinogenic photoproduct,
the Dewar valence isomer, following photoisomerisation in the presence of UVA at
320 nm (Perdiz et al., 2000). The significance of these DNA insults as a result of
UVR exposure is the physical hindrances they create to replication and transcription
processes (Costa, 2003). Of these DNA lesions, (6-4)PPs are among the least
mutagenic and have been shown to be more efficiently repaired by the cells’ DNA
repair mechanisms, with up to 90% of these dimers being repaired three hours after
irradiation (Nakagawa, 1998).
In contrast, CPD photoproducts are thought to be a primary contributor to
mutations in skin cells, resulting in the transition of C to T and CC to TT
dinucleotide transitions, commonly known as the UV ‘signature’ mutations. Other
types of UVR-induced DNA damage include the formation of protein-DNA cross-
links, single strand DNA breaks and thymine glycol lesions (Ichihashi et al., 2003).
The hazards of UVA and UVB radiation exposure also lie in their ability to
form ROS, thus indirectly damaging DNA, proteins and lipids (Danpure, 1976).
Reactions with UVR and endogenous cellular porphyrins, riboflavin and quinines,
generate highly-reactive, DNA-damaging ROS molecules (Scharffetter-Kochanek et
al., 1997). These include superoxide anions, singlet oxygen and hydrogen peroxide,
which cause downstream single-strand breaks, DNA-proteins cross-links and altered
bases (Phillipson, 2002). One of the most frequently occurring altered base
phenomena is the oxidation of guanine to form 7,8-dihydro-8-oxoguanine (8-oxo-
dG), hence making its presence a ubiquitous marker of cellular oxidative stress
(Cadet, 2000). The presence of 8 oxo-dG alterations in DNA has been shown to be
premutagenic and is thought to contribute to the photocarcinogenic process (Kasai
and Nishimura, 1984). The other major DNA lesions generated from oxidative
damage include thymine glycol, pyrimidine hydrates and urea (Scharffetter-
Kochanek et al., 1997).
8 Literature review
1.3.2 Cellular UVB radiation-induced DNA damage response
Cells possess complex and multifaceted responses to sense and repair DNA
damage, in order to minimise the risk of propagating mutations during cellular
replication. Upon the detection of damaged DNA, the activation of cell cycle
checkpoints and arrest coupled with the initiation of DNA repair pathways or
apoptosis are orchestrated to preserve genomic integrity (Zhou and Elledge, 2000).
Cell cycle checkpoints serve as regulatory pathways which control the cell cycle by
establishing the proper completion of one phase before the transition to the next. The
cell cycle checkpoint pathways principally associated with UVB radiation-induced
DNA damage are depicted diagrammatically in Figure 1.5. Central mediators of
these checkpoint pathways include the ataxia telangiectasia mutated (ATM) and
ATM and Rad3-related (ATR) protein kinases, which respond to DNA damage via
their phosphorylation and subsequent activation (Morgan and Kastan, 1997). The
principle regulator in the photodamage response is ATR, which functions by
blocking DNA replication predominantly at the G1 checkpoint (Bender et al., 1997;
Zhou and Elledge, 2000). ATM and ATR activation result in the downstream
phosphorylation of the signal transducers Chk1 and Chk2, a process which is
required for the phosphorylation and proteosomal degradation of the cyclin
dependent kinase, cdc25C and the activation of p53 (discussed further in Section
1.3.5) (Su, 2006). Besides causing G1/S arrest, this process also activates elements
such as histone H2A.X, which within minutes of sensing damage, initiates chromatin
remodelling for DNA repair (Bao, 2011).
9 Literature review
Figure 1.5. Cell cycle checkpoint pathways initiated by UVB radiation-induced DNA damage
In response to UVB radiation-induced DNA damage, the phosphorylation of ATR and its interaction with ATRIP
initiate different pathways which aid in cell cycle arrest and the repair of DNA damage through such pathways as
the phosphorylation of the histone H2A.X (Kawasumi et al., 2011; Nam and Cortez, 2011). The G1 cell cycle
checkpoint prevents damaged DNA from being replicated and is largely triggered by the activation of p53.
Besides upregulating genes involved in the DNA damage response including MDM2, GADD45a and p21/Cip,
phospho-p53 causes the accumulation of p21 which suppresses Cdk2/Cyclin E kinase activity, thereby causing
cell cycle arrest at G1. Cellular entry into mitosis is also controlled by the activity of the cyclin dependent kinase
cdc25, which is regulated by the phosphorylation of kinase Chk-1. The phosphorylation of the dual specificity
phosphatase cdc25C creates a binding site for 14-3-3 proteins. The formation of the cdc25C/14-3-3 complex
maintains the Cdc2/Cyclin B1 in its inactive state, hence blocking cellular entry into mitosis at the G2 checkpoint
(Peng et al., 1997).
The synthesis phase (S-phase) checkpoint controls cell cycle progression
following DNA damage by suppressing the rate of DNA synthesis. Relatively little is
known of the precise mediators of this checkpoint, though the Chk1/2-regulated
phosphorylation of cdc25C is thought to inactivate the S-phase cyclin E/CDK2
14-3-3
UVB
DNA damage
Chk-1
P
ATR
P ATRIP
cdc25C
P
H2A.X
P
Cdc2/
Cyclin B1
p53
P
p21
Cdk2/
Cyclin E
cdc25C
P
DNA repair
G1 arrest G2 arrest
10 Literature review
complex, thus arresting the cell cycle (Hakem, 2008). The G2/M DNA damage
checkpoint suspends the cell cycle prior to chromosome segregation, thus preventing
mitotic entry of damaged cells. At this checkpoint, the Chk1/2 kinases phosphorylate
cdc25C at Serine 216 thus creating a binding site for 14-3-3 proteins (Peng et al.,
1997). This in turn results in the maintenance of the Cdc2/Cyclin B1 complex in its
inactive state, and the subsequent blockage of the cell cycle progression into mitosis
(O'Driscoll and Jeggo, 2006). Upon the completion of DNA repair, cells are able to
resume cell cycle progression and cellular functions. However, unsuccessful DNA
repair leads to cellular senescence or p53-mediated apoptosis in order to prevent the
replication of damaged cells (Chipuk and Green, 2006; Lewis, 2008; Handayaningsih
et al., 2012).
1.3.3 DNA photodamage repair mechanisms
Due to the deleterious effects of UVR on DNA, mammalian cells are equipped
with several sophisticated protective DNA repair mechanisms to eliminate this
damage. A combination of repair pathways including direct repair, mismatch repair,
double-stranded break repair, base excision repair and the nucleotide excision repair
(NER) systems are activated, depending on the type and extent of photodamage
(Sancar et al., 2004). The NER mechanism is central to the removal of both CPDs
and (6-4)PPs (Suzuki et al., 1978). This complex and versatile mechanism is able to
both sense and restore distortions in the DNA helix (de Laat, 1999). This is achieved
via either the more robust global genome NER, which can repair lesions over the
whole genome, or the faster transcription-coupled NER system which senses the
presence of lesions at a transcription level (Ogi et al., 2010). Both systems operate by
recruiting repair complexes to the site of the damage, excising about 10 to 20
nucleotides around the lesion, and then replacing the excised regions with the correct
sequence. The significance of these repair systems is made evident in patients
suffering from Xeroderma pigmentosum (XP), a disease resulting from genetic
defects in aspects of the NER complex, resulting in faulty UVR-induced DNA
damage repair. Studies on patients with this autosomal recessive genetic disorder
have demonstrated the connection between UVR exposure and the onset of skin
cancers (Kraemer et al., 1994; Tang et al., 2000; Lehmann, 2003). It has been found
that XP patients have an approximately 1000-fold increased risk of developing skin
cancer than those without the disease (Cleaver, 2000). In fact, UVR is known as a
11 Literature review
‘complete carcinogen’, as it directly induces mutations in DNA, and also triggers a
variety of cellular processes in skin such as the suppression of the natural anti-
tumour immune responses which creates a favourable environment for malignant cell
proliferation (de Gruijl, 2002).
Damage recognition systems are responsible for sensing and activating these
repair mechanisms, detecting even subtle alterations to DNA, proteins and lipids. For
example, the generation of reactive oxygen species causes the oxidation of certain
cellular proteins, thus altering their conformation and function. A well documented
example is the UVR-induced oxidation and inactivation of protein tyrosine
phosphatases (PTPs) which initiate several phosphotyrosine-dependent signal
transduction pathways (Gross, 1999). This PTP inactivation by UVR has been shown
to activate the epidermal growth factor (EGF) receptor, thus triggering various
proinflammatory and proliferative effects in keratinocytes (Knebel, 1996). Cellular
surveillance machinery for detecting protein damage, for instance, intracellular
pattern recognition receptors, may also aid in damage recognition and repair
induction (Meylan, 2006). The generation of damaged, single-stranded DNA
(ssDNA), has also been implicated as a mediator of cell cycle arrest and the
activation of DNA repair. The presence of ssDNA in the nucleus is usually detected
by sensor protein complexes such as MreII/Rad50/Nbs1 (MRN) and ataxia
telangiectasia mutated and Rad4-related interacting protein (ATRIP), which bind to
the damaged DNA activating and recruiting DNA repair complexes via multiple
signalling mechanisms (Zou, 2003).
1.3.4 UVR-induced cell cycle arrest and apoptosis
Although intricate cellular DNA repair mechanisms are usually able to restore
most UVR-induced damage, it is likely that high doses of radiation or the
accumulation of unrepaired damage can ultimately lead to the onset of skin
carcinogenesis (Van Steeg and Kraemer, 1999). Thus, to prevent potentially
oncogenic mutations from being propagated to daughter cells, damaged cells activate
cell cycle arrest or cell death responses (Cox and Lane, 1995). Growth arrest allows
for DNA repair processes to remove photoproducts prior to cell proliferation (Jeggo
and Löbrich, 2006). However, in the case of severe DNA damage, the cell proceeds
to undergo a process of self-destruction. This can occur via one of three closely-
related cell death pathways: apoptosis, terminal differentiation or necrosis.
12 Literature review
Exposure of skin to physical trauma or toxic chemicals can result in the
deterioration of cell membranes and organelles followed by cell death in a process
known as necrosis (Gold et al., 1994). The morphological characteristics of necrotic
cells include cellular swelling, the disruption of the cell membrane and chromatin
condensation before cellular and nuclear lysis (Wyllie, 1980). It has been shown
experimentally that high doses of UVR exposure can cause keratinocytes to undergo
necrosis, releasing proinflammatory cytokines to activate the inflammatory immune
response (Caricchio, 2003). These responses only occur at excessively high doses of
UVR exposure (in excess of 80 mJ/cm2), which are significantly above the average
MED of 25 mJ/cm2 (Tadokoro, 2003). At physiologically-relevant UVR doses,
however, the accumulation of DNA mutations generally causes keratinocytes to
undergo apoptosis or terminal differentiation (Zhuang et al., 2000).
The phenomenon of apoptosis, or the active, energy dependent, genetically-
programmed cellular self-destruction mechanism, was first discovered as a
morphologically distinct pattern of cell death, dissimilar from that observed in
necrosis (Kerr, 1971). Apoptotic keratinocytes formed as a consequence of UVR
exposure are known as ‘sunburn cells’, and are characteristic markers of UVR-
induced damage. At biologically-relevant doses of UV irradiation, it has been
proposed that intrinsic pathways of apoptosis are the principle mode of removing
damaged keratinocytes (Assefa, 2005). The process of apoptosis is vital in the
maintenance of skin homeostasis, particularly facilitating the constant turnover of
keratinocytes required to maintain a constant epidermal barrier. Terminal
differentiation in keratinocytes is a similar form of programmed cell death, in which
basal proliferative keratinocytes at the dermal-epidermal junction undergo an
irreversible series of changes, ultimately differentiating into corneocytes that
constitute the stratum corneum. Following UVR exposure, this thickening of the
stratum corneum is also observed, a consequence of increased keratinocyte
differentiation (Kligman et al., 1985; Kligman, 1996). This protective response is
believed to scatter the penetrating UVR, thus reducing the intensity of radiation
reaching the basal keratinocytes. The thickening of the epidermis is supported by an
upregulation of genes associated with differentiation, such as involucrin, calgranulin
and elafin (Li et al., 2001).
13 Literature review
1.3.5 The role of p53 in the cellular UV-response
The nuclear phosphoprotein gene, p53, is the most commonly mutated gene
among human cancers and disruptions to its normal function have been implicated in
a wide variety of sporadic and inherited malignancies (Levine et al., 1991). In terms
of the UVR response, this tumour suppressor gene is known to play a pivotal role in
both the regulation of Gap-1 phase (G1) cell cycle arrest and in the initiation of
apoptotic pathways (Livingstone et al., 1992). Accordingly, exposure to UVR has
been shown to upregulate both the transcription of p53, as well as its activation via
the phosphorylation of serine 15 and serine 20 (Unger et al., 1999). The p53-
regulated expression of the cyclin-dependent kinase (CDK) inhibitor
p21/WAF1/CIP1 has been implicated in orchestrating the G1 phase arrest (Poon et
al., 1996). Additionally, p53 is also capable of upregulating key apoptotic genes such
as Fas/Apo-1 and Bax (O'Grady et al., 1998). This is coupled with the p53-induced
downregulation of cell survival genes like Bcl-2. The maintenance of genomic
stability and the safeguarding of DNA integrity during replication are also influenced
by the downstream upregulation of GADD45 (Carrier et al., 1994).
Furthermore, p53 has been shown to counteract ROS-damage by upregulating
the expression of proteins with antioxidant activity such as N-acetylcysteine
(Sablina, 2005). To protect against further photodamage, studies also reveal the
contribution of p53-mediated activity in the regulation of melanogenesis (Cui, 2007).
Binding of p53 directly to pro-opiomelanocortin (POMC), a precursor polypeptide
which is ultimately processed to form α-MSH, is a potent stimulator of melanin
production. Hence, p53 and its associated molecular interactors are critical in the
inhibition of photocarcinogenesis.
1.3.6 UVR-induced inflammation and tanning
Inflammation during the sunburn reaction is a key feature of the acute UV
response. However, the process of sunburn initiation and the factors that influence its
severity have yet to be precisely defined. It has been shown that the cutaneous
expression of proinflammatory cytokines, such as TNF-α, interleukins, growth
factors and endothelin-1 are upregulated as a result of UV irradiation (Figure 1.4).
These act in concert to activate systemic inflammatory and local epidermal responses
to counteract cellular damage (Kuhn et al., 1999). This response peaks within the
first two days after irradiation, in which a spike in the proliferation of both epidermal
14 Literature review
and dermal cells occurs. The transient p53-regulated cell cycle arrest phase is
therefore pivotal in ensuring the repair of DNA damage before this proliferative burst
to prevent potentially carcinogenic cell divisions.
Cytokines, hormones and various other bioregulators are also responsible for
stimulating the production of photoprotective melanin, through the process of
melanogenesis (Duval, 2001; Matsumura and Ananthaswamy, 2002). The epidermal-
melanin unit is composed of one melanocyte to about 30 keratinocytes (Fitzpatrick
and Breathnach, 1963). Shortly after irradiation, melanin granules are redistributed in
keratinocytes, forming ‘nuclear caps’ above the nuclei to help protect against further
DNA damage (Lin and Fisher, 2007). A delayed tanning response involving the
further activation and proliferation of melanocytes occurs next, which is also
associated with enhanced melanocyte tyrosinase activity, increased melanin synthesis
and the elongation and branching of melanocyte dendrites. In view of this, the
actions of tyrosinase and tyrosinase-related proteins present on the melanosomal
membrane, and melanogenic regulatory proteins like the microphthalmia-associated
transcription factor (MITF) and the melanocortin 1 receptor (MC1R) are also
important stimulators of melanin production (Liu and Fisher, 2010). Inhibitors of
melanogenesis, such as serotonin and melatonin, act as negative regulators via the
control of melanocyte proliferation and melanocyte responses to α-MSH
respectively (Slominski et al., 2002). These complex and highly-interconnected
processes are still being investigated using a number of in vitro models.
1.3.7 UVR-induced immunosuppression
The inflammatory reactions to UVR have been well-documented on a cellular
and molecular level. On the other hand, sub-erythemal doses of both UVA and UVB
radiation have also been shown to produce an immunosuppressive response. Trans
urocanic acid, a histidine derivative in the stratum corneum, helps to absorb some of
the penetrating UVB wavelength. The isomerised cis form of trans urocanic acid,
together with the generation of CPDs has been demonstrated to cause a local
immunosuppressive effect (De Fabo, 1983). Moreover, UV-irradiated keratinocytes
have been shown to secrete soluble immunosuppressive mediators, such as IL-10,
which may add to the suppression of immunity on a systemic scale (Nishigori et al.,
1996). Cellular and molecular modulators of this phenomenon have yet to be fully
defined. However, clinical data suggests that the dampening of local and systemic
15 Literature review
immunity by UV exposure may have important implications in skin cancer
development (Hoxtell et al., 1977).
1.4 FIBROBLAST-KERATINOCYTE INTERACTIONS DURING THE UVR-
RESPONSE
1.4.1 Epithelial-mesenchymal interactions in skin
Interactions between the epithelium and the mesenchyme are critical during
development and organogenesis (Kong et al., 2006). In fact, this close connection
extends to homeostatic maintenance, growth and repair of the adult cutaneous
epithelium, especially in supporting its constant renewal via proliferation, migration
and differentiation (Billingham and Silvers, 1967). It is known that dermal
fibroblasts not only promote the growth and stratification of keratinocytes, but also
play a role in preserving the organisation of the epidermis (Boyce, 1988). Studies
monitoring the development of chick skin show a strong dependency on
mesenchymal signals for epidermal morphogenesis, differentiation patterns (via
keratin synthesis) and the formation of skin appendages (Cadi et al., 1983). This is
also supported by observations from experiments with adult keratinocytes, showing
that diseased fibroblasts isolated from patients with psoriasis could induce
hyperproliferation in these cells (Saiag et al., 1985). Due to the complexity of this
communication spanning numerous systems and pathways, the precise inductive
signals that orchestrate cross-talk in normal and diseased tissue are still not
completely understood. Extensive experimental evidence indicates, however, that
cell and matrix adhesion molecules, diffusible agents (such as retinoids) and growth
factors are among the key players involved (Boukamp et al., 1990; Brockes, 1990;
Yamaguchi et al., 2005).
The BM at the epidermal-dermal junction is a major facilitator of these
interactions, in part through the action of cellular adhesion, the extracellular matrix
(ECM) and its components. Cell attachment has been shown to be closely-linked to
changes in cellular behaviour by altering morphology, cytoskeleton organization and
receptor clustering (O'Toole et al., 1997; Egles et al., 2010). Matrix components like
laminin, fibronectin and syndecans interact with adhesion molecules on keratinocytes
including integrins, cadherins and selectins, which then affect various aspects of
cutaneous development and homeostasis (Cabrijan and Lipozencic, 2011). Indeed,
blocking these interactions results in the disruption of certain epidermal-
16 Literature review
mesenchymal signalling pathways (Hirai et al., 1989). The roles of matrix molecules
and growth factors in modulating epithelial function are also inextricably linked.
Components of the extracellular matrix can bind and sequester growth factors to
induce cellular responses (Akhurst et al., 1990). Additionally, the expression of these
matrix molecules themselves has been associated with the actions and functionality
of growth factors in inducing downstream effects on epithelial cells (Upton et al.,
1999; Kricker et al., 2003).
1.4.2 Growth factors mediate epithelial-mesenchymal cross-talk
As mentioned previously, growth factors directly affect several aspects of the
developing and adult epithelium by regulating proliferation, differentiation,
migration, ECM production and numerous other processes (Aaronson et al., 1990;
Wenczak et al., 1992; Beer et al., 2000; Beyer et al., 2003). The secretion of
mitogenic growth factors in the mesenchyme activates epithelial cells in a paracrine
manner. This interaction is vital in regulating and directing repair, maintenance and
regeneration in skin, and is believed to be conserved from embryogenesis
(Briggaman, 1982).
In terms of activating keratinocyte proliferation, fibroblast-synthesized KGF,
HGF, EGF, interleukins-6 (IL-6) and -8 (IL-8) are among the principal mediators of
epidermal regeneration (Mackenzie and Fusenig, 1983). Also central to epidermal-
mesenchymal communication system is the insulin-like growth factor (IGF) family.
The potent effects of this system, in particular through the actions of insulin-like
growth factor I (IGF-I), have been extensively studied by our research group in
various epithelial tissues including the skin and breast (Noble et al., 2003; Hyde et
al., 2004; Ainscough et al., 2006; Dawson et al., 2006; Van Lonkhuyzen et al., 2007;
Hollier et al., 2008). Interactions between IGF-I and the ECM protein vitronectin and
keratinocytes have been shown to significantly enhance epidermal repair in a wound
healing context and have been used in the development of a novel wound healing
therapeutic (Van Lonkhuyzen et al., 2007; Upton et al., 2008; Upton et al., 2011; Xie
et al., 2011). With several parallels existing between the healing wound and
epidermal regeneration following UVR-damage, the potential of the IGF system in
enhancing the photorepair response was therefore studied.
17 Literature review
1.5 THE INSULIN-LIKE GROWTH FACTOR SYSTEM
The action of the IGF family is well-known as being integral to many epithelial
processes, such as migration, proliferation, attachment and invasion (Ando, 1993). It
is also involved in many pathological conditions, such as chronic inflammation,
impaired wound healing and cancer (Samani, 2007). The IGF system consists of the
single-chain polypeptide ligands IGF-I and IGF-II, which bind to cell surface
receptors, namely, the insulin-like growth factor receptor type 1 (IGF-IR), insulin-
like growth factor receptor type 2 (IGF-2R) and the insulin receptor (IR) via
autocrine, endocrine and paracrine pathways (Perona, 2006). Both IGF-I and IGF-II
are present at varying concentrations in most tissues, as well as in blood plasma with
concentrations of approximately 20 to 100 nM (Daughaday and Rotwein, 1989;
Humbel, 1990). The bioavailability of IGFs is tightly regulated by six IGF binding
proteins (IGFBPs).
The IGF-IR is expressed ubiquitously in most cell types throughout the body
(Sjögren, 2002). Ligand-receptor binding activates a range of signal transduction
pathways within the cell, such as the phosphatidylinositide 3-kinase (PI3-K) and
mitogen activated-kinase (MAPK) pathways, which have known regulatory roles in a
variety of cell types in vivo (Werner, 2008). The IGF system has a potent mitogenic
and anti-apoptotic effect on cells and is involved in somatic growth processes
(Rotwein, 1999). These properties are reflected in malignant tumour cells which
ubiquitously over-express IGF-IRs, thus enhancing tumour growth and progression
(Lopez, 2002). Experimental evidence has also shown that cells which under-express
IGF-IR are more susceptible to the induction of apoptotic cell death following
exposure to stress factors (Li, 2009).
The IGF-IR is a transmembrane tyrosine kinase receptor that is structurally and
functionally related to the insulin receptor (Abbott, 1992). The three ligands that bind
to the IGF-IR are IGF-I, IGF-II and insulin. Cysteine-rich domains of the α-subunits
on the IGF-IR determine ligand-binding affinity and specificity. Of the ligands, IGF-
I has the greatest affinity, while that of insulin is 100-fold lower (Gustafson, 1990).
Ligand-receptor binding activates the IGF-IR, causing the phosphorylation of two
main cytoplasmic membrane substrates, insulin receptor substrate I (IRS-I) and Shc,
which in turn activates downstream signal transduction cascades (Kaulfuss, 2009).
The major regulatory mechanisms in place to regulate and control the IGF-I
18 Literature review
stimulation of IGF-IR are the down-regulation of IGF-IR expression following IGF-I
stimulation, and the presence of IGF-binding proteins (IGFBPs) which competitively
bind to IGF-I.
The IGF ligands, IGF-I and IGF-II, are small (approximately 7.5 kDa) poly-
peptides which are structurally similar to insulin. They have a diverse range of
mitogenic and metabolic effects in multiple cell and tissue types. The IGFs have the
ability to exert both systemic and local effects via autocrine and paracrine actions
(Butler, 2001). The IGF system is tightly regulated through the action of the six
binding proteins (IGFBP-1 to IGFBP-6), which possess similar structures, but have
individually distinct functional properties (Perks, 2008). Most of the IGF ligands
found in the circulation or the ECM are bound to IGFBPs with high affinities
(Baxter, 1994). An interesting characteristic of IGFBPs is their ability to modulate
cell survival independent of IGF-I interactions (Wheatcroft, 2009). Most IGFBPs
have been demonstrated to possess intrinsic properties which allow them to affect
aspects of cell growth, adhesion, migration and apoptosis (Perks, 2008).
Circulating IGF-I is predominantly synthesized by the liver, with other tissues
capable of its local production (Sjögren et al., 1999). In skin, IGF-I is synthesized
mainly by dermal fibroblasts and at lower levels by epidermal melanocytes, together
acting in a paracrine manner on basal proliferating keratinocytes (Flier et al., 1986;
Martin, 1990; Philpott et al., 1994). During tissue repair and remodelling, fibroblasts
synthesize IGF-I to increase keratinocyte proliferation for tissue repair and
remodelling (Gartner et al., 1992). The complexities of IGF-I-mediated crosstalk
between keratinocytes and other dermal and epidermal cell types require further
study. Parathyroid hormone-related protein (PTH-rp), secreted by keratinocytes, is
thought to influence the upregulation of IGF-I in fibroblasts (Shin, 1997). Also
produced by basal keratinocytes, IGFBP3 is thought to moderate the pro-survival
effect of fibroblast-secreted IGF-I (Hollowood, 2000). It has been reported that
IGFBP-3 transcription is upregulated following cellular stress via a p53-mediated
mechanism, with this binding protein also able to independently initiate apoptosis in
UV-damaged keratinocytes (Williams, 2000). In addition to stimulating changes to
keratinocyte behaviour, IGF-I also enhances the survival, migration and synthesis of
melanin in melanocytes following UV-irradiation (Archambault et al., 1995;
Edmondson, 1999). Taken together, this evidence suggests that IGF-I regulated
19 Literature review
mechanisms may be a fundamental part of the skin’s protective strategies against
UV-damage.
1.5.1 IGF-IR signalling pathways
Intrinsic tyrosine kinase activity of the IGF-IR is induced by the IGF-I ligand
binding to this receptor and its resultant conformational change (Figure 1.6). This
then causes the autophosphorylation of tyrosines in the juxtamembrane domain,
which represent a docking site for IRS 1/2 and Shc, thus initiating the transmission of
the IGF-IR signal (Samani, 2007). Phosphorylated IRS 1/2 interacts with the
regulatory subunit of PI3-K, p85, which leads to the subsequent phosphorylation of
protein kinase B (AKT) (Butler et al., 1998b; Doepfner et al., 2007). Next, the
activation of the mammalian target of rapamycin (mTOR) by phospho-AKT
stimulates protein synthesis and the inhibition of pro-apoptotic signals in the cell, via
the inactivation of the pro-apoptotic protein Bad (Vaira et al., 2006; Tao et al.,
2007).
Additionally, the phosphorylation of IRS-1 or Shc can also recruit Ras, thus
activating the extracellular signal-regulated kinase (ERK) signalling pathway, and
consequently promoting cellular proliferation, growth and survival (Butler et al.,
1998a). Furthermore, the c-Jun N-terminal kinases (JNK) and p38 MAPK pathways
have also been shown to be activated by IGF-IR ligand-binding (Cheng and
Feldman, 1998; Vincent and Feldman, 2002). The downstream effects of the
activation of these pathways include the recruitment of transcription factors,
including jun, fos and E twenty-six (ETS)-like transcription factor 1 (ELK-1), which
upregulate the transcription of genes associated with mitogenesis (Hermanto et al.,
2000). Therefore, it is not surprising that the dysregulation or hyperactivation of
these pathways have been linked to the development of various cancers (Surmacz,
2000; Burroughs et al., 2002). Accordingly, the molecular events surrounding the
activation of the IGF system are tightly regulated by processes such as
phosphorylation, dephosphorylation and degradation of the signalling proteins
involved (Vincent and Feldman, 2002).
20 Literature review
Figure 1.6. Signalling cascades associated with IGF-IR activation
The insulin-like growth factor receptor type-1 (IGF-IR) is activated by binding with its ligand, insulin-like growth
factor-I (IGF-I), upon its release from insulin-like growth factor binding protein (IGFBP) and extracellular matrix
(ECM) interactions. This then stimulates its intrinsic tyrosine kinase activity in the intracellular region.
Complexes such as Shc/SHP-2 and IRS 1/2 form substrates which autophosphorylate and trigger downstream
Ras/Raf/MEK/ERK and PI3K/AKT pathways. The latter serves as a central regulator of cellular synthesis and
proliferation, as well as the inhibition of apoptosis through blocking the activities of pro-apoptotic factors such as
Bad. Together with ERK, the JNK and p38 MAPK pathways are also activated by IGF-IR, which through the
recruitment of transcription factors such as ELK-1, jun and fos, upregulate the transcription of genes associated
with mitogenesis.
1.5.2 IGF-I and its role in influencing keratinocyte responses to UVR
To further research the role of the IGF system in photobiology, a number of
studies have been conducted characterising the impact of this growth factor on
irradiated keratinocytes. The findings of these studies include that protection against
UVB radiation-induced keratinocyte apoptosis has been found to be associated with
an increased synthesis of IGF-I and the IGF-IR, together with a decrease in pro-
apoptotic factors and an amplification of pro-survival factors (Figure 1.7) (Kulik,
1997; Assmann, 2009). This apoptosis-prevention mediated by IGF-I is known to be
activated by the PI3-kinase and MAPK pathways (LeRoith et al., 1995).
Furthermore, studies on irradiated primary human keratinocytes demonstrate that
21 Literature review
IGF-IR activation not only promotes post-mitotic cell survival, but also arrests cell
proliferation to facilitate DNA repair (Kuhn et al., 1999; Heron-Milhavet et al.,
2001; Decraene, 2002). On top of this, the phosphorylation of p53 and the
subsequent activation of various signalling cascades are significantly dependent on
the functional status of the IGF-IR (Lewis, 2008). Diminished p53 activity (as a
result of mutations) is thought to be closely linked to photocarcinogenesis
(Greenblatt et al., 1994). This then further supports the theory that IGF-IR activation
may be important during cancer development, as discussed in 1.5.3.
Figure 1.7. Downstream cell survival effects from IGF-IR activation.
Binding of the IGF-I ligand to IGF-IR results in the autophosphorylation of the tyrosine residues on the
intracellular region of the receptor. Once phosphorylated (denoted by ‘P’), this forms a docking site for IRS.
Phosphorylated IRS activates PI3-K which leads to the downstream activation of other effector proteins such as
phosphoinositide-dependent kinase-1 (PDK-1) and AKT. The PI3-kinase/Akt pathway increases the levels of
anti-apoptotic proteins including Bcl-2 and Bcl-x. The activation of Akt also inhibits apoptosis through the
inactivation of pro-apoptotic factors such as caspases, Bad, members of the Forkhead (FKH) family and glycogen
synthase kinase 3 beta (GSK-3β).
22 Literature review
1.5.3 A potential role of the IGF system in photocarcinogenesis
In light of this evidence, a new school of thought has emerged regarding the
influence of the IGF system on NMSC development. The IGF system has highly
proliferative and growth-promoting cellular effects which suggest that its
overexpression may be linked to tumorigenesis (DiGiovanni et al., 2000). Indeed,
this has been shown to be true in several epithelial cancers including that of the lung,
breast and colon (Donovan, 2008; Dziadziuszko, 2008; Lann, 2008). However, it is
also likely that the dysregulation of IGF-I-induced cell survival pathways may lead
to the proliferation of cells before the complete removal of photolesions, thus
representing a critical step in photocarcinogenesis (Lewis et al., 2010).
As illustrated in Figure 1.8, alterations to the activation status of IGF-IR at the
time of UV-exposure may result in inappropriate keratinocyte responses to UVR.
Dermal fibroblasts isolated from geriatric adults display characteristics of cellular
senescence and have been shown to have an impaired ability to synthesize IGF-I
(Ferber et al., 1993). Therefore, a connection between a reduced supply of IGF-I to
initiate keratinocyte photorepair and the elevated incidence of NMSCs occurring in
older individuals may exist (Green, 1990). Experimental evidence using cultured
keratinocytes also shed light on the possible mechanistic aspects of the connection
between IGF-IR activation and UV-damage (Lewis et al., 2010). In this study,
irradiated keratinocytes without IGF-IR activation continued to display mitotic
activity despite harbouring unrepaired UVB radiation-induced damage. Finally,
epidemiological evidence from patients undergoing insulin therapy provides further
clinical supporting evidence. In patients with type 2 diabetes mellitus being treated
with exogenous insulin therapy, elevated serum insulin levels (and hence, greater
levels of IGF-IR activation) have been correlated with a decreased incidence in
NMSC development (Chuang et al., 2005).
23 Literature review
Figure 1.8. A potential role of the IGF system in photocarcinogenesis
(A) In skin, dermal fibroblasts secrete a variety of factors, including IGF-I (yellow arrow) to activate various
physiological and repair processes in epithelial keratinocytes. (B) Following exposure to UVB radiation, studies
have shown that IGF-I plays an important role in ensuring that keratinocytes harbouring DNA damage undergo
appropriate cell cycle arrest, repair or apoptotic pathways to prevent the replication of damaged cells. (C) In
situations whereby the fibroblast-expression of IGF-I is compromised, such as in aging skin, diminished IGF-IR
activation may lead to inappropriate keratinocyte responses to UVB radiation-induced damage. (D) Keratinocytes
harbouring genetic damage may be allowed to divide and multiply, thus forming a critical initiating factor in non-
melanoma skin cancer (NMSC) development.
As yet, however, the precise significance of the IGF family during the acute
epidermal response to UVB radiation requires further investigation to establish more
concrete associations. Furthermore, understanding the role of IGF-I as a facilitator of
epidermal-mesenchymal interplay on endocrine, paracrine and autocrine levels in
skin is an essential next step.
1.6 EXPERIMENTAL MODELS OF PHOTOBIOLOGY
The ability to model human skin experimentally is fundamental in enhancing
our understanding of the mechanisms underlying skin cancer development.
Traditional in vivo and in vitro systems that take advantage of increasingly elegant
cell culture and animal systems have provided valuable insights into the cellular
responses to UVR exposure. Nonetheless, incorporating the intricate biology of
human skin into a physiologically-relevant experimental platform remains a
significant challenge. The advantages and limitations of currently-used in vitro
systems are discussed in the following sections and summarised in Table 1.1.1.
24 Literature review
1.6.1 Cell culture systems
One of the most extensively-used and easily-accessible models for
photobiology and photocarcinogenesis research involves the use of 2-dimensional
(2D) cell cultures. The ease of use, cost-effectiveness and the diversity of cell lines
available make such systems highly attractive to researchers. Both cancer cell lines,
including BCC (Yang, 2011), SCC (Wood, 2011) and melanomas (Kosztka, 2011)
and continuous non-malignant cells like HaCaT keratinocytes (Thumiger et al.,
2005) are utilised experimentally. Cell lines have a wide-spread range of applications
in photobiological studies, particularly in the preclinical developmental and
screening stages for developing potential photoprotective and anticancer agents
(Zattra et al., 2009; Yoshihisa, 2010).
Fundamentally, however, studies employing conventional cell monolayer
systems possess several intrinsic limitations, particularly in their application to
primary cell culture. The maintenance of primary keratinocytes in culture involves
the presence of an irradiated mouse fibroblast feeder layer to encourage attachment
and stimulate proliferation (Rheinwald, 1975; Alitalo et al., 1982). This then poses a
challenge for isolating ‘fibroblast-free’ keratinocyte populations as the two cell types
are normally grown on a single monolayer. The immortalized human keratinocyte
cell line, HaCaT, has been extensively used in the monolayer format for applications
such as Vitamin D studies (Lehmann, 1997; Park et al., 2011), as well as in
conjunction with 3D skin organotypic models (Schoop et al., 1999; Gotz et al.,
2012). However, in the context of photobiology studies, HaCaT cells demonstrate
significant differences compared to normal human keratinocytes in terms of survival
after irradiation (Thumiger et al., 2005). Therefore, this limits their use in the study
of keratinocyte regeneration post-irradiation, under physiological conditions.
Importantly, monolayer cultures do not adequately reflect the complex
architecture (Figure 1.1) and the diverse array of intercellular interactions occurring
in native skin. Such systems are therefore limited in their ability to model epidermal-
mesenchymal paracrine interactions. Another consideration of 2D culture systems is
that artificially-treated tissue culture vessels may lack vital components of the native
cellular microenvironment, which can alter cells’ responses to irradiation.
25 Literature review
Table 1.1.1. Characteristics of commonly used in vitro and in vivo models for studying skin cancer biology.
Relative differences between models are represented by (present), − (absent) and from least (+/$) to most (+++/$$$), as summarised from text. Table modified from (Fernandez et al., 2012b).
In vitro models In vivo models
Primary cell
culture
Cell line
culture
Ex vivo skin
explants
HSE-DED
model
HSE (synthetic
scaffold)
Pigmented HSE Mouse models Recruited
volunteers
Experimental considerations
Costs involved $ $ $ $ $$ $ $$$ $$$
Lifespan of culture + NA + + + + +++ NA
Specialised facilities −
Specialised skills ++ + ++ ++ ++ ++ +++ +++
Ethical considerations ++ NA ++ ++ ++ ++ +++ +++
Inter-sample variation +++ + +++ +++ +++ +++ ++ +++
Sample processing time ++ + ++ ++ ++ ++ +++ +++
Limitations to treatments + + ++ ++ ++ ++ +++ +++
Physiological relevance to human biology and pathology
3D tissue environment − −
Cell population heterogeneity − −
Primary skin tissue − − (xenografts)
Immune component − − − − Available
Phylogenetic similarity +++ +++ +++ +++ +++ +++ ++ +++
Carcinogenesis mechanism +++ ++ +++ ++ ++ +++ + +++
Use as pre-clinical model −
26 Literature review
As described in 1.4.1, elements of the ECM coupled with the tissue
organisation are known to be integral in the maintenance of cellular homeostasis
(Dawson, 1996; Cheng, 2011). In keratinocytes, the disruption of any of these factors
(such as in the absence of a fibroblast feeder layer) may result in the inappropriate
initiation of a wound-healing response such as premature differentiation (Dawson,
1996). In the long-term, due to potential modifications to cell morphology and
signalling processes, repeated subculture is also known to alter the UV-response
(Briske-Anderson et al., 1997; Chang-Liu and Woloschak, 1997). While this can be
overcome to some extent by pre-coating cell culture surfaces with ECM components
(Hollier et al., 2005), the use of most traditional 2D systems may not always be fully
representative of the in vivo situation.
2D co-culture systems
Co-culture systems, such as the use of Transwell® tissue culture plates,
overcomes some of the limitations of monolayer culture by allowing the
simultaneous cultivation of two cell types that are physically separated but that share
a common growth medium. These co-culture systems have been described in the
cultivation of both primary keratinocytes and malignant cells (De Luca et al., 1988;
Wei et al., 1999; Papini et al., 2003). Such models have advantages over
keratinocytes grown on mouse feeder layers in that ‘pure’ cultures of keratinocytes
can be isolated since they are physically separated from the mesenchymal cells. This
is of particular benefit in preventing contamination from other cell types when
extracting keratinocyte RNA or proteins, for instance. Furthermore, because
keratinocytes are grown on membranes in detachable inserts, this allows for the
removal of inserts to expose individual monolayers of cells to UVR in the case of
photobiology studies.
1.6.2 Animal models
Whole organism models have empowered researchers with the ability to
investigate complex local and systemic processes in an in vivo setting (Ha et al.,
2005). Despite this, to date no one particular animal model faithfully represents the
molecular pathogenesis and aetiology of human skin cancers. The potential for
genetic and immunologic alteration in the animal model of choice is also highly
beneficial for dissecting the mechanisms of photocarcinogenesis. Accordingly,
certain mouse strains have been developed with specific parallels to human
27 Literature review
photocarcinogenesis pathways. For example, the SKH-1 albino mouse strain has
been extensively applied in elucidating the mechanisms of SCC initiation and
progression due to similarities in the genetic profile of skin tumours and
tumorigenesis compared to humans (Verkler et al., 2008). Likewise, the hepatocyte
growth factor/scatter factor (HGF/SF) transgenic mouse strain is not only
histologically similar to human skin, but also displays similar patterns of melanoma
development, progression and the associated gene alterations (Takayama, 1996;
Noonan et al., 2003). Another approach in maintaining biological relevance to
humans involves xenografting human tissue onto immunodeficient mice to analyse,
for instance, the factors influencing tumour invasion (Walsh, 2011). A two-stage
carcinogenesis protocol is usually used in inducing mouse tumours. This involves
firstly initiating cancer cells with UVR treatment together with a chemical
carcinogen such as 7,12-Dimethylbenz(a)anthracene (DMBA) (Sauter, 1998) as
UVR exposure alone does not directly act as a cancer initiator and promoter in
mouse skin (Halliday, 2005). Next, the repeated administration of a tumour-
promoting agent, commonly 12-O-tetradecanoylphorbol-13-acetate (TPA), induces
the amplification of the initiated cancer stem cell population (Kemp, 2005). This
technique may therefore diverge from the pathways of human photocarcinogenesis.
Additionally, murine tumours manifest mainly as non-epithelial sarcomas and
lymphomas as opposed to the majority of human tumours which are epithelial in
origin (Artandi et al., 2000). Mice also differ to humans in their skin biology, for
example, possessing different growth fractions of actively proliferating basal
keratinocytes, of around 60% (Potten, 1974), as compared to approximately 20% in
human keratinocytes (Heenen et al., 1998).
Besides mice, fish models such as the Xiphophorus, zebrafish and medaka have
been frequently used, in particular to study factors influencing the development of
CMM (Stanton, 1965; Naruse et al., 2004; Geiger et al., 2008). This model holds
considerable potential for the screening for future genetic and chemical inhibitors
and modifiers of melanoma progression. Critically, however, due to the evolutionary
separation from humans, results obtained from non-mammalian systems such as
these may not necessarily be directly translated to humans. In the case of resistance
to UVR-damage, for instance, Xiphophorus possess certain unique innate defences
28 Literature review
including DNA photolyase which is photoactivated to repair nuclear DNA lesions
(Ahmed and Setlow, 1993).
On the other hand, several considerations need to be made before adopting the
use of animal models. Besides the ethical considerations, several cardinal anatomical
differences exist between the skin biology of most animal models and humans. For
example, the abundance of fur, relatively thinner dermal and epidermal layers,
distinct localisations of melanocytes and metabolic rates separate mice models from
humans (Khavari, 2006). To summarise, skin cancers are not naturally occurring in
many commonly-used laboratory animals and have to be artificially-induced
chemically, often with non-physiological doses of UVR (Halliday, 2005). Among
species in which skin malignancies are naturally occurring, the zebrafish and medaka
fish models are frequently employed, and have been instrumental in studies into the
biology of melanoma development (Naruse et al., 2004; Fernandez et al., 2012a).
However, the evolutionary separation between humans and distinctive DNA repair
mechanisms in these fish species, limit their use as a comparative model (Mitchell,
2010). Moreover, there has been a global shift towards more stringent laws
regulating and limiting the use of laboratory animals as testing models (Ponec,
2002). Indeed, recent amendments to the European Union Cosmetic Directive
prohibit the testing of therapeutic or cosmetic products on animals (Adler et al.,
2011). Therefore, there is an urgent need for reproducible and effective alternative in
vitro skin models.
1.6.3 In vivo clinical studies
Examining cutaneous and systemic changes in consenting volunteers is the
gold standard model of photobiology research. Indeed, studies into the local immune
response (Brink et al., 2000a), DNA damage (Snellman et al., 2003) and gene
expression (Beattie et al., 2005) have been carried out in vivo on participants.
However, major ethical concerns regarding the use of human volunteers limit such
studies due to the risks involved and the requirement for painful skin biopsies.
Further limitations include the number of UVR treatments per participant and the
dosage that can be safely administered. In order to overcome these issues, a number
of skin substitute research models have been established.
29 Literature review
1.6.4 In vitro skin models
Skin explant models
Explant cultures consisting of ex vivo human skin tissue are a simple yet
versatile model that have been widely adopted to monitor keratinocyte damage and
the efficacy of photoprotective treatment with UVR (Arad, 2007). Such ex vivo
cultures are also advantageous for analysing complex processes such as
melanogenesis (Dong et al., 2010) and UVR-induced modulation of the cutaneous
endocrine system (Ito et al., 2005), as the native skin biology is retained. Explant
keratinocytes in culture conditions have been demonstrated to be capable of many
processes associated with metabolism, proliferation and DNA repair after irradiation
(Reus, 2011). Nonetheless, a major drawback of this system is the limited lifespan of
the explant cultures in vitro. The viability of cells within the excised tissue decreases
rapidly with an associated increase in apoptosis (Reus, 2011). Under culture
conditions, explants have a lifespan of around five days before displaying a marked
drop in cell proliferation and the thickening of the stratum corneum from increased
terminal differentiation is observed (Onuma, 2001). Besides this, there is a limited
capacity for post-irradiation regeneration, with only about 5% of the total
keratinocyte population shown to be actively proliferating in ex vivo explant models
(Onuma, 2001).
Tissue-engineered models
The method of isolating and culturing primary human keratinocytes represents
a major cornerstone in the evolution of tissue-engineered skin equivalent models.
Cultivated keratinocytes are reconstituted onto either naturally-derived or synthetic
dermal scaffolds, thus creating a 3-dimensional (3D) human skin equivalent (HSE)
organotypic model, encompassing both the epidermal and dermal elements. These
skin composites were initially developed in clinical practice during the 1980s for
grafting onto victims of burns who sustained extensive full-thickness skin damage
(Burke, 1981; Chakrabarty, 1999). Since its inception, the HSE has also evolved into
a powerful research tool with numerous applications in skin biology research (Ponec,
2002; Supp and Boyce, 2005; MacNeil, 2007). These models have been used to
study aspects of skin pigmentation (Hedley et al., 2002; Bernerd et al., 2012), wound
healing (Falanga et al., 2002), cancer progression (Meier et al., 2000) and various
cellular interactions (Harrison et al., 2006a).
30 Literature review
Fundamentally, most HSEs retain the native structural and cellular
organisation. The key function of the scaffold component is to mimic the dermis and
ECM, which is lacking in many simple 2D models (Morita, 2002). Synthetic
biomaterials have been applied for this purpose, which include the use of chitosan-
gelatin-hyaluronic acid (Liu et al., 2004), collagen (Valenta, 2004) and fibrin
(Mazlyzam, 2007). Natural polymers are usually preferred over synthetic compounds
that may affect cell proliferation due to a lack of biocompatibility. Dermal substitutes
like Alloderm™ (Lifecell Corporation, Branchberg, NJ), Biobrane™ (Dow
Hickham/Bertek Pharmac., Sugar Land, Tx) and Integra™ (Integra LifeScience,
Plainsborough, NJ) are commercially-available, but have mixed results in terms of
cellular interactions with the dermal matrix (Schafer et al., 1989). Cell-based skin
substitutes, such as EpiDerm (MatTek), which are comprised of both the epidermal
and dermal components, are also obtainable. It has been documented, however, that
HSEs consisting of biopolymer or animal-derived dermal components, such as rat
tail collagen, have altered tissue microenvironments in terms of the presence of
glycosaminoglycans, proteoglycans, lipids and fibrin (El Ghalbzouri et al., 2009).
Attempts to create pigmented bioengineered skin equivalents to investigate
melanocyte biology and interactions with keratinocytes have progressed significantly
in recent years. Early work conducted on the isolation and culture of primary human
melanocytes (Eisinger and Marko, 1982; Marko et al., 1982) have led to the
development of specialised culture mediums and conditions for the propagation of
these cells (Halaban et al., 1988; Medrano and Nordlund, 1990; Na et al., 2006; Li et
al., 2012). The pigmentary system, however, is still a challenge to recreate
structurally and functionally in vitro. Proliferation and functional characteristics of
melanocytes (migration, melanogenesis and dendrite formation) in vivo are highly
regulated by signals from ECM molecules, UVR-exposure and various paracrine
factors, especially those secreted by surrounding keratinocytes (Yaar and Gilchrest,
1991; Scott et al., 1997). In cultures solely consisting of melanocytes, it is not
surprising therefore, that these cells are observed to have altered morphologies and
functionality (Shin et al., 2012). Cultured melanocytes often lose dendrites and
become hyperproliferative, thus losing intrinsic properties of native cells (Herlyn et
al., 1988).
31 Literature review
Pigmented HSEs consisting of a keratinocyte and melanocyte epidermal layer
constructed on collagen gels (Archambault et al., 1995; Bessou et al., 1997), and de-
epidermised dermal (DED) scaffolds (Todd et al., 1993; Bessou et al., 1996; Bernerd
et al., 2012) have been applied to model reactions to UVR and pigmentation. Indeed,
the incorporation of melanocytes into both 2D and 3D culture systems has aided in
the discovery of many regulators and pathways involved in skin pigmentation
(Hedley et al., 2002; Smith-Thomas et al., 2004; Balafa et al., 2005). However,
limitations in the pigmented skin models and technical difficulties have been
encountered in these studies. Varying results regarding the proliferation-potential,
survival and differentiation status of melanocytes in the HSE, as well as the
consistency of the melanocyte to keratinocyte ratio have been reported (Bertaux et
al., 1988; Nakazawa et al., 1997; Zhao et al., 2012). This has been attributed to the
seeding ratio, culture conditions and variation between melanocyte donor skin types
(Nakazawa, 1998). It has proven to be even more challenging to maintain native
melanocyte biology with high seeding densities of melanocytes and other culture
conditions being linked to melanocyte hyperproliferation and a shift towards a
neoplastic phenotype (Larue et al., 1992; Oba-Shinjo et al., 2006). Overall, while
pigmented HSEs have potential as a powerful model for studying melanocyte
biology and function, considerable work needs to be conducted into preserving the
delicate equilibrium of the melanocyte microenvironment to maintain the consistency
and reliability of results obtained.
Commercially-available HSE composites are advantageous as they negate the
need for ethical approval, access to human tissue donors and laborious primary cell
isolation and culture methods. In Australia, however, strict quarantine restrictions
limit the entry of human cell-based materials, thus making such commercial products
unattainable to researchers (Poumay, 2004). Ultimately, due to logistical issues and
high costs involved, the import of such skin cultures may not be viable as a globally-
accepted research tool.
1.6.5 HSEs as in vitro photobiology models
The structure, cellular organisation, biochemistry and cell-cell interactions in
native skin each play a vital role in the cutaneous responses to irradiation. Therefore,
an experimental model which reflects these properties as faithfully as possible to the
in vivo situation would generate the most physiologically-relevant data on cutaneous
32 Literature review
photodamage. The classic markers of UVB radiation-induced skin damage, such as
alterations to keratinocyte cell viability (Gujuluva et al., 1994), proliferation (El-
Abaseri, 2006) and apoptosis (Zhuang et al., 2000) and the formation of CPDs, have
been detected in HSE models. Likewise, UVB radiation-induced expression changes
in the epidermal proteins like keratins (Smith and Rees, 1994), filaggrin, loricrin,
transglutaminase type 1 and involucrin (Lee et al., 2002) have also been observed in
various irradiated HSE models (Bernerd, 1997). Keratin expression patterns can be
used as a useful marker of a range of characteristics of the epidermis, including
keratinocyte differentiation and thickening in response to UVR (Baba et al., 2005).
Keratin 10 is expressed by keratinocytes in the early stages of differentiation and its
expression has been shown to diminish in the days following UVB irradiation
(Bernerd, 1997). During the regenerative period, the expression of keratin 10
progressively resumes, and thus can also be used as a marker of epidermal repair.
Other keratinocyte differentiation markers that are modulated by irradiation are
filaggrin and loricrin. Filaggrin is a precursor for the generation of urocanic acid
(UCA), a UVB radiation-absorbing substance which reduces the amount of radiation
that can penetrate the epidermis (Zenisek, 1955). Both filaggrin and loricrin (found
in the cornified cell envelope of terminally-differentiated keratinocytes) in HSE
show a similar pattern of post-irradiation decrease, followed by a gradual increase
over about 10 days (Bernerd, 1997). Importantly, the previously used HSE models
possess the capacity for post-irradiation repair and regeneration, with epidermal
normalisation taking place over a period of around 1 to 2 weeks in vitro (Bernerd,
1997).
1.6.6 HSE-DED as an organotypic model
To create a physiologically-relevant in vitro representation of human skin, it is
fundamental to recreate a tissue environment that recapitulates as closely as possible
the morphology, function and cell-cell interactions that occur in vivo. In view of this,
a DED scaffold, derived from ex vivo tissue has been chosen to create this
organotypic model for the studies in this thesis. The HSE-DED consists of primary
human skin cells grown on a DED base and cultured at the air-liquid interface to
promote epidermal formation (Figure 1.9). Importantly, the HSE-DED has been
found to closely approximate native skin both in terms of the morphology and
biochemistry of the dermis and epidermis (Chakrabarty, 1999; Ponec, 2002; Huang,
33 Literature review
2004; Poumay, 2004; Welss et al., 2004; Harrison et al., 2006b). This system has
been established as a model of cutaneous wound healing in our research group
(Kairuz et al., 2007; Xie et al., 2010; Xie et al., 2011). In particular, the HSE-DED
has been used in the evaluation of potential therapeutics to promote
reepithelialisation via enhancing keratinocyte proliferation and migration (Xie et al.,
2011). Additionally, it has been used to investigate regeneration following burn
injury, the impact of hyperbaric oxygen therapy on wound healing and in the
development of chemically-defined culture media (Topping, 2006; Kairuz et al.,
2007; Borg et al., 2009; Mujaj et al., 2010).
Figure 1.9. HSE-DED composites in culture
Human skin equivalent on de-epidermised dermal scaffold (HSE-DED) composites are cultured at the air-liquid
interface of media to promote the maturation of the epidermal component. The scale bar represents 1.5 cm.
Briefly, construction of this model involves the decellularization of ex vivo skin
tissue while preserving BM components and the organization of collagen in the
dermis (Ghosh et al., 1997; Huang, 2004). Primary keratinocytes and fibroblasts are
isolated and cultured and are then seeded back onto the DED (Black, 2005). The
processing of the DED removes cells from the tissue while retaining essential ECM
factors. This is important as cellular interactions with the BM are known to be
paramount in regulating the function and viability of basal keratinocytes and
underlying dermal fibroblasts (Jones, 1995). Experimental evidence shows that
keratinocytes grown on HSE composites lacking a complete BM, such as in artificial
matrices, exhibited lower levels of attachment, proliferation, atypical epidermal
architecture and significantly greater numbers of apoptotic cells (Chakrabarty, 1999;
34 Literature review
Segal, 2008). Once primary cells are seeded onto the DED scaffold, the initial
presence of BM proteins enhances subsequent retention and remodelling of the BM
layer by cells to mimic the in vivo situation (Ralston et al., 1999). It has been
observed that elements of the BM including collagens IV, VII and laminin, are
maintained with the use of the DED (Medalie et al., 1996).
Keratinocytes in HSE-DEDs when in contact with air have been shown to
differentiate and form a mature epidermis (Prunieras et al., 1983). Moreover, multi-
photon microscopy has revealed that the epidermal microtopology and architecture in
the HSE-DED cultured at the interface of air and culture medium is akin to that of
native skin, with the preservation of structural features such as rete ridges (Xie et al.,
2010). Other epidermal markers, such as the presence of the natural epidermal layers
and the expression of keratins 1/10/11, 6, 14, 16, loricrin, involucrin and filaggrin
have also been identified in the HSE-DED (Monteiro-Riviere et al., 1997; Parnigotto
et al., 1998; Ponec et al., 2000). The stratum corneum is absent in submerged and
conventional 2D keratinocyte cultures and hence deviate from the native epidermal
organisation (Parnigotto et al., 1998). Lamellar bodies, which play a critical role in
maintaining the skin’s barrier against irritants, were also found to be present in the
HSE-DED (Fartasch and Ponec, 1994). A histological comparison between native
skin and HSE-DED tissue sections is shown in Figure 1.10.
Figure 1.10. Comparison of histology between native skin and HSE-DED sections.
The histology of HSE-DED (B) shows similarities to that of native skin (A), stained with haematoxylin and eosin.
The epidermis in the HSE-DED shows the preservation of keratinocyte organisation patterns as in native skin,
including the presence of the stratum corneum. The arrow indicates the presence of rete ridges at the dermal-
epidermal junction.
26 Literature review
While HSE models with synthetic dermal scaffolds have been used in
photobiology as described previously, the model proposed for use in the studies in
this thesis, consisting of a DED and a reconstructed epidermis has yet to be fully
characterised as a potential research tool for studying the effects of UVR. The HSE-
DED possesses an ex vivo dermal component which, as an in vitro model, more
closely mimics the in vivo situation as compared to synthetic scaffolds (Topping,
2006). Specifically, the DED together with the preservation of elements of the BM
are considered to be advantageous in terms of promoting and maintaining a
multilayered epidermis (Chakrabarty, 1999; Pianigiani et al., 1999). Moreover,
structural and organisational aspects of the DED and HSE epidermis have been
shown to be very similar to native skin, which is critical in particular for
photobiology studies to model the path of UVR in skin (Dawson, 1996). As a major
aim of this study was to investigate how dermal fibroblasts influence epithelial
responses to UVB radiation, this cell-based platform was adopted to model this
crosstalk in a physiologically-relevant setting.
27
1.7 SUMMARY AND SIGNIFICANCE
The development of more sophisticated preventative and treatment strategies
against skin cancer is closely linked with advancing our understanding on the
biology of photocarcinogenesis. While the long-term clinical implications of UVR
exposure and the resulting damage to the skin on a cellular level have been well-
documented, the mechanisms behind this photodamage have yet to be fully
understood. In particular, the paracrine interactions between mesenchymal and
epidermal cells and their role in the development of NMSCs have not been defined.
In part this is a result of limitations in current experimental models, including cell
culture and animal models (Table 1.1.1.). However, advancements in tissue-
engineering techniques have led to the application of increasingly elegant HSE
constructs to model native skin in the study of cellular responses to UVR.
The significance of the HSE-DED model lies in the ability to detect specific
markers of photodamage in irradiated primary skin cells grown in a physiologically-
relevant model. This allows flexibility to study cellular alterations at a range of
different UV doses and post-irradiation time points with varying combinations of
incorporated skin cell types. Besides providing an alternative to animal models for
studying the mechanisms surrounding cellular responses to UVR, the HSE-DED
model can provide an accessible substitute for commercially-available skin
substitutes and 2D culture systems. Importantly, this represents a biologically-
relevant platform for investigating paracrine interactions between epidermal and
dermal cells to more accurately understand the role that this interplay has on the
development of NMSCs. With the incidence of skin cancers on the rise both globally
and nationally, the HSE-DED model of photobiology may aid in the urgent need to
bridge current knowledge gaps in the field. Moreover, this system can be applied to
creating improved treatment and preventative strategies against skin damage, thus
reducing the risk of skin cancer development.
28
1.8 PROJECT HYPOTHESES
The underlying hypotheses of this project are as outlined below. Firstly, it was
hypothesized that a human cell-based in vitro skin equivalent model can be utilised
as a physiologically-relevant platform for photobiological studies. Next, it was
hypothesized that dermal fibroblasts influence the responses of epithelial
keratinocytes to UVB radiation by enhancing cell survival and repair processes. I
was also hypothesized in the studies reported herein that the IGF system may play an
important role in this crosstalk during the acute response to UVB radiation.
1.9 PROJECT AIMS
The following outline the major aims of the research conducted in this project:
1. Validation of 2D and 3D in vitro models of cutaneous photobiology to
study keratinocyte responses to UVB radiation.
2. Investigation of the influence of paracrine interactions between dermal
fibroblasts and keratinocytes during the acute UVB irradiation response.
3. Studying the role of IGF-I in the fibroblast-mediated protection from UVB
radiation-damage in keratinocytes.
29
Chapter 2: Materials and methods
2.1 INTRODUCTION
The protocols and materials used throughout this project have been detailed in
this Chapter. Any specific deviations to the procedures outlined in the following
sections are described where they appear. Where not specified, laboratory grade
general regents were purchased from commercial suppliers. Technical details on
specialised experimental procedures are also described in this Chapter.
2.2 EXPERIMENTAL PROCEDURES
2.2.1 Ex vivo skin tissue collection
Human skin tissue (from surgical discard) was obtained from informed and
consenting donors undergoing elective abdominoplasty and breast reduction surgical
procedures. Ethical approval for the collection of human tissue samples was granted
by the Queensland University of Technology, as well as the Institute of Health and
Biomedical Innovation and the hospitals from which patient samples were obtained.
These included ethical approval for sample collection from St Andrew’s War
Memorial Hospital (Approval number: 2004/46) and the Princess Alexandra and
Brisbane Private Hospitals (Approval number: QUT 3865H). Donor skin tissue from
surgical discards was superficially cleaned with isopropyl alcohol-impregnated wipes
and subcutaneous fat was subsequently removed using a scalpel blade. Split-
thickness skin grafts (of approximately 1 mm thickness) were sectioned using a
surgical dermatome. Skin tissue was collected in sterile transport medium containing
antibiotic/antimycotic solution (ABAM) (10,000 U/mL penicillin G, 10,000 µg/mL
streptomycin sulphate and 25 µg/mL amphotericin B in 0.85% sterile saline
(Invitrogen)). All subsequent processing of ex vivo skin tissue was conducted under
sterile conditions.
30
Processing of skin tissue samples
Pieces of donor skin tissue were treated with serial antimycotic and
antimicrobial washes to prevent fungal and bacterial culture contamination. Skin was
immersed in a wash medium (4% ABAM in 500 mL phosphate buffered saline
(PBS) with 160 mg gentamicin sulphate) for five minutes, followed by a second
wash (2% ABAM in 500 mL PBS with 80 mg gentamicin sulphate), and a final wash
in 1% ABAM in 500 mL PBS for five minutes each. Thinner regions of skin tissue
were cut into approximately 3 mm by 3 mm pieces for skin cell isolation and culture
as described in the next section. Other pieces of skin with thicker, more uniform
dermal thicknesses were cut into approximately 15 mm by 15 mm squares for use as
a dermal scaffold, as outlined in Section 2.2.3.
Fixation of control native skin samples
Ex vivo native skin samples were obtained to use as comparative controls. Skin
tissue obtained in 2.2.1 was immersed in 10% neutral buffered formalin (United
Biosciences, Queensland, Australia) overnight at 4°C. Fixed skin tissues were then
transferred to tissue processing cassettes before being immersed in 70% ethanol.
Subsequent processing for histological and immunohistochemical studies was
performed as described in 2.2.6.
2.2.2 Primary cell culture
Isolation of keratinocytes from ex vivo tissue
A modification of the protocol originally described by Rheinwald and Green
(1975) was used for the isolation of primary keratinocytes. Briefly, after processing,
3 mm by 3 mm pieces of skin tissue were incubated in 0.125% trypsin solution
(Invitrogen) in PBS at 4°C overnight to digest skin at the dermal-epidermal junction.
After enzymatic digestion, the epidermis was physically detached from the dermis
using forceps. Cells at the epidermal-dermal junction were gently scraped off using a
scalpel blade. Collected cells were then resuspended in Full Green’s medium (FGM)
(Rheinwald, 1975), consisting of Dulbecco's Modified Eagle Medium (DMEM) with
Ham’s F12 Medium (in a 3:1 ratio). The following additives were also added: 10%
foetal calf serum (FCS) (Hyclone); 10 ng/mL human recombinant epidermal growth
Chapter 2: Materials and methods 31
factor (EGF) (Invitrogen); 1% v/v penicillin/streptomycin solution (Invitrogen); 1
µg/mL insulin (Sigma-Aldrich, Australia); 0.2 µM triiodothyronine (T3) (Sigma-
Aldrich); 0.01% non-essential amino acids solution (Invitrogen); 5 µg/mL transferrin
(Sigma-Aldrich); 180 mM adenine (Sigma-Aldrich); 0.4 µg/mL hydrocortisone
(Sigma-Aldrich) and 0.1 µg/mL cholera toxin (Sigma-Aldrich) (Chakrabarty, 1999).
The freshly-isolated cell suspension was centrifuged at 1000 rpm for 5 minutes and
resuspended in fresh FGM, before being passed through a 70 µm cell strainer. Cell
numbers were then counted using a haemocytometer (Neubauer) and the live:dead
cell ratio assessed using Trypan blue (Sigma) staining.
Culture of human keratinocytes
Keratinocytes were cultured using a protocol based on the original technique
described by Rheinwald and Green. Primary keratinocytes were co-cultured on a pre-
seeded feeder layer of the murine 3T3 fibroblast cell line (J2 clone, American Type
Culture Collection, Manassas, VA, USA). These 3T3 fibroblasts were cultured
routinely in DMEM supplemented with 5% FCS, 1% v/v penicillin/streptomycin
solution (Invitrogen) and 2 mM L-glutamine (Invitrogen) at 37°C with 5% CO2.
Before being seeded as a feeder layer, the 3T3 fibroblasts were trypsinised,
resuspended in FGM and irradiated with 50 Gy gamma radiation (Australian Red
Cross Blood Service, Brisbane, Australia). The irradiated 3T3 fibroblasts were
seeded at a density of 2 x 106 cells per 75 cm
2 (T75) tissue culture flask in 10 mL
FGM and were incubated at 37°C with 5% CO2 for 24 hours prior to the addition of
2 x 106 freshly-isolated epidermal cells. Primary cell cultures were maintained at
37°C with 5% CO2. The cell culture medium (FGM) was replaced every two to three
days, until the keratinocytes reached a confluency of about 80%. This usually took
around seven to nine days.
Culture of primary human dermal fibroblasts
After the removal of the epidermis for keratinocyte isolation (as outlined in the
previous section), the remaining dermal tissue was finely minced using a scalpel
blade into approximately 0.5 mm x 0.5 mm pieces and was then enzymatically-
digested in 0.05% collagenase A (Type I) (Invitrogen) in DMEM at 37°C, 5% CO2
overnight. This digested dermal tissue in solution was then centrifuged at 2000 rpm
for 10 minutes. The tissue was resuspended in DMEM supplemented with 10% FCS,
1% v/v penicillin/streptomycin solution and 2 mM L-glutamine and cultured in a T75
32 Materials and methods
cell culture flask. Dermal fibroblast cultures were passaged upon reaching a
confluence of approximately 80%, and passage 2-5 (P2-5) cells were used for all
further experiments.
2.2.3 Construction of human skin equivalent
Preparation of de-epidermised dermal scaffold
The de-epidermised dermis (DED) from ex vivo skin tissue was prepared using
previously-reported methods (Dawson et al., 2006). In brief, larger pieces of ex vivo
skin tissue (1.5 cm x 1.5 cm) were decellularised by immersion in sterile hypertonic
1 M sodium chloride solution (Sigma-Aldrich) and incubated overnight at 37°C to
decellularise the tissue. The non-viable epidermis was then separated from the
dermis using forceps and discarded. The remaining DED was immersed in three
successive antibiotic washes for 24 hours each, as described in Section 2.2.1, and
stored at 4°C until further use. Before the construction of the HSE, the DED was
submerged into the experimental medium of choice for 4 hours at 37°C to allow for
diffusion and equilibration of the medium in the DED.
Construction of HSE-KC
The HSE composites were constructed using a previously described protocol
(Topping, 2006; Xie et al., 2010). The DED tissue (outlined in the previous section)
was immersed in FGM for 4 hours at 37°C, before being placed into the wells of a
24-well tissue culture plate (Nagle Nunc International, Roskilde, Denmark) with the
papillary surface facing upwards. Sterile stainless steel rings with 6.7 mm internal
diameter silicone washer bases (Aix Scientific, Aachen, Germany) were rinsed in
PBS and positioned centrally onto the papillary surface of the DED (Topping, 2006).
Primary keratinocytes at Passage 1 (P1) were trypsinised upon reaching 80%
confluence and resuspended at a concentration of 1 x 105 cells/mL in FGM. Next,
200 µL of this P1 cell suspension was added to the internal well of each stainless steel
ring. The external space between the ring and the well was topped up with FGM. The
HSE-KC constructs were cultured with the rings containing keratinocyte suspensions
for 48 hours at 37°C with 5% CO2. To culture HSE composites at the air-liquid
interface, sterile stainless steel grid inserts with 1 mm diameter circular pores were
placed into 6-well tissue culture plates (Nunc). The stainless steel rings were then
removed from the surface of the DED and the newly formed HSE-KC constructs
were placed on top of these grid inserts. Approximately 6 mL of FGM was then
Chapter 2: Materials and methods 33
added to the wells such that the DED was partially submerged and the upper
epidermal surface was at the air-liquid interface. The HSE-KC composites were then
cultured for 9 days at 37°C with 5% CO2, and the medium was replenished every two
to three days, maintaining the surface of the HSE-KC epidermis at the air-liquid
interface.
Construction of HSE-KCF
The HSE-KCF model was constructed using a similar protocol to that
described in the previous section. Briefly, dermal fibroblasts at P2-5 (as described in
Section 2.2.2) were trypsinised and resuspended in FGM. Next, 1 x 104 cells in 200
µL of medium was added to the internal well of the stainless steel rings on the
papillary surface of the DED. These were cultured for 48 hours at 37°C with 5%
CO2. Half of the medium was then aspirated from the stainless steel wells and 2 x 104
keratinocytes at P1 in 100 µL were added to the wells. The composites were then
cultured for a further 48 hours at 37°C with 5% CO2, before the rings were removed
and HSE-KCF composites transferred to stainless steel grids within 6-well tissue
culture plates, with the papillary surface at the air-liquid interface. The HSE-KCF
composites were cultured in these conditions for 9 days at 37°C with 5% CO2, with
the medium replenished or replaced every two to three days. A diagram outlining the
timelines of the HSE-KC and HSE-KCF construction is depicted in Figure 2.1.
34
Figure 2.1. Timelines of 2D and 3D keratinocyte in vitro culture methods
This diagram illustrates the timelines associated with the construction of the 3D HSE-KC skin culture (Section
2.2.3) and the 2D co-culture method described in Section 2.2.4. The first step of method of HSE construction is
the isolation and culture of primary keratinocytes, a process taking about seven days. For the HSE models, the
keratinocytes are seeded onto the DED scaffold for 2 days before being raised to the air-liquid interface for 9
days to promote epidermogenesis. Upon the maturation of the epidermis, the composites are then exposed to
UVB radiation. Samples are then harvested during the five-day post-irradiation period to study the acute effects
of UVB irradiation on these composites. However, HSE samples may be maintained in culture for up to three
weeks (Ng and Ikeda, 2011). For the Transwell® co-cultures, keratinocyte monolayers are irradiated after the
two-day seeding period with samples harvested during the post-irradiation period for further analysis.
2.2.4 Co-culture of keratinocytes and dermal fibroblasts using a Transwell® cell
culture system
The co-culture of primary keratinocytes and fibroblasts using Transwell® cell
culture plates (Corning, Costar, Cambridge, MA, USA) followed a modified protocol
previously described by Kandyba and colleagues (2008). Primary dermal fibroblasts
(P2-5) were seeded at a density of 5 x 104 cells in 900 µL FGM per well into the
bottom wells of the 12-well co-culture plates. Fibroblasts were maintained in culture
for 24 hours at 37°C with 5% CO2. Transwell® inserts with 0.4 µm pore size
polyester membranes were then inserted into these wells, and 3 x 105 P1 primary
keratinocytes in 400 µL FGM were added to each insert and then returned to the
culture conditions (Figure 2.2). After 24 hours from the time of keratinocyte seeding,
the FGM was aspirated, and both the insert membranes and the bottom wells were
washed once in warmed PBS. The medium was then replaced with serum-free
medium (SFM), consisting of FGM without FCS, hydrocortisone, insulin, EGF,
hydrocortisone and T3. Growth factors used in subsequent studies were diluted to the
appropriate concentrations in this SFM. In experiments on keratinocyte monolayers
cultured without the presence of fibroblasts, Transwell inserts were removed at day 2
Chapter 2: Materials and methods 35
post-keratinocyte seeding and transferred to fresh 12-well plates without cells in the
lower chambers. Keratinocyte monolayers were exposed to UVB radiation as
described in Section 2.2.5.
Figure 2.2. Transwell® co-culture of primary human keratinocytes and fibroblasts
Schematic diagram showing the co-culture of fibroblasts (Fib) in lower well and keratinocytes (KC) cultured on a
membrane in the upper insert well (A). Both cell types share common medium (FGM or SFM) through the
membrane with 0.4 µm diameter pores. In experiments requiring the culture of keratinocytes only, inserts were
transferred to new wells in the absence of fibroblasts upon reaching confluency (B). For the UVB irradiation of
keratinocytes (C), inserts were removed from the wells and exposed to UVB radiation as described in Section
2.2.5.
2.2.5 UVB irradiation protocols
Irradiation of HSE composites with UVB radiation
The HSE composites were irradiated with single doses of UVB radiation using
a TL40 medical UVB bulb (Philips) with a stabilised power supply inside a Class II
Biological Safety Cabinet (Figure 2.3). The UVB bulb was encased in a layer of
cellulose acetate (Kodacel TA-407 clear 0.015 inch, Eastman-Kodak), thus
efficiently blocking any contaminating wavelengths below 290 nm (Therrien et al.,
1999). The precise output of UVB radiation transmitted was assessed using a 0.5 m
focal length action double spectroradiometer (Princeton Scientific Instruments, New
Jersey, USA) calibrated to deliver 9.27 mJ/cm2/min UVB radiation, at a distance of 6
cm from the external surface of the bulb. The HSE constructs were transferred to
Petri dishes containing a thin layer of pre-warmed PBS to 37˚C with the epidermal
surface facing upwards. All UVB irradiation treatments were conducted in PBS as
the exposure of cell culture medium to UVR has been demonstrated to result in the
generation of cytotoxic photoproducts (Wang, 1976; Zigler et al., 1985). To exclude
the effects of heat and visible light, the HSE composites were irradiated in the dark
and the temperature at the surface of the HSE was monitored and found to be
36 Materials and methods
constant at room temperature of about 23°C. The irradiation protocol for a 50 mJ/cm2
dose, as used in this project, required HSEs to be placed in the irradiation set-up for a
period of 5 minutes and 24 seconds. Non-irradiated controls were treated to the same
experimental conditions for this duration, but in the absence of a UVB radiation
source.
37
Figure 2.3. UVB irradiation of cells and skin tissue samples
Diagrammatic representation of the UVB irradiation method. A medical UVB lamp was set up inside a Class II
Biological Safety Cabinet to maintain sterile conditions during irradiation. The distance from the surface of the
lamp to the surface of the samples (as indicated by the arrow) was fixed at 6 cm, based on the calibrated output of
the lamp. Non-irradiated controls were placed under the same irradiation conditions, but in the absence of a
UVB-emitting source.
Irradiation of keratinocyte monolayers in Transwell® co-cultures
To irradiate keratinocyte monolayers in the co-culture studies, the Transwell®
inserts (from 2.2.4) were removed from their respective cell culture wells, washed
once in 100 µl of pre-warmed PBS and exposed to UVB radiation (as described in
2.2.5), under 50 µL PBS. After UVB irradiation, this layer of PBS was removed and
fresh culture medium was replaced into the inserts and wells. Co-culture plates were
then returned to culture conditions before samples were harvested at specific time
points post-irradiation.
2.2.6 Histological analysis
Sections of HSE and native skin tissue were analysed histologically using a
routine haematoxylin and eosin staining method. Briefly, tissues were fixed in 10%
neutral buffered formalin (United Biosciences, Queensland, Australia) overnight at
4°C. Fixed HSE composites were cut in half using a scalpel blade and transferred to
tissue processing cassettes before being immersed in 70% ethanol. The tissues were
then paraffinised using an automated vacuum tissue processor (Excelsior ES, Thermo
Scientific), embedded in paraffin using a tissue embedder (Shandon Histocentre 3,
Thermo Electron Corporation) and 3 µm sections were cut using a rotary microtome
(Leica RM2245, Leica Microsystems, Australia). Tissue sections were deparaffinised
in xylene (Merck), washed in a graded ethanol series and rehydrated in distilled
38 Materials and methods
water. The sections were then stained with Harris haematoxylin (HD Scientific,
Australia) and alcoholic eosin phloxine (HD Scientific) following a standard
haematoxylin and eosin staining protocol (Coleman et al., 2012). Stained sections
were mounted with Pertex® mounting medium (Medite, Burgdorf, Germany) and
glass coverslips (#1.5, HD Scientific). Stained sections were imaged using an
Olympus BX41 microscope with attached digital camera (Micropublisher, 3.3 RTV,
QImaging).
2.2.7 Immunohistochemical analysis
Immunohistochemical analysis was performed as previously documented
(Topping, 2006). Briefly, paraffin sections of irradiated HSEs, of 3 µm thickness
were mounted onto Superfrost® Plus microscope slides (Menzel-Glässer). Sections
were then deparaffinised and rehydrated as described in the previous section. Heat-
induced epitope retrieval (HIER) in either tris-ethylenediaminetetraacetic acid
(EDTA) buffer at pH 9.0, or sodium citrate buffer at pH 6.0 for 4 minutes at 97°C,
followed by 10 minutes at 70°C in a decloaking chamber was used to pre-treat
sections. Endogenous peroxidases were blocked with hydrogen peroxide solution
(Biocare Medical, Pike Lake, CA, USA), and non-specific antibody binding was
blocked with blocking reagent (Biocare Medical). Primary antibodies were diluted in
sample diluent solution (Biocare Medical) and incubated for either 60 minutes at
37°C, or overnight at 4°C in a humidified chamber. Primary antibody binding was
detected using a mouse probe secondary antibody (Biocare Medical) and
3,3’diaminobenzidine (DAB) chromogen (Biocare Medical). The sections were
counterstained with haematoxylin Gill 1 (G1) nuclear stain (HD Scientific). The
sections were then dehydrated in graded alcohols and xylene washes with the stained
tissue sections mounted and coverslipped as described in the previous section.
2.2.8 Immunofluorescent analysis
The fluorescent detection of antibody-labelled tissue sections was used to
visualise the presence of specific targets. Formalin-fixed paraffin-embedded tissue
sections were rehydrated and treated with HIER as described in the previous section.
Following the incubation with the primary antibody, the sections were washed
thoroughly. Primary antibody binding was visualised using fluorescently-labelled
secondary antibodies (Alexa Fluor®, Invitrogen) at 1:5000 dilutions in an antibody
diluent solution (MAXBind™, Active Motif) for 60 minutes at 37°C in a humidified
Chapter 2: Materials and methods 39
chamber, protected from light. Following the wash steps, the sections were incubated
with PBS containing 0.5 µg/mL 4',6-diamidino-2-phenylindole (DAPI) solution for
10 minutes at room temperature to label all cell nuclei within the tissue. The tissues
were then washed once in PBS and mounted onto microscope slides in anti-fade
mounting medium (Prolong® Gold, Invitrogen). The sections were mounted under
glass coverslips (#1.5, HD Scientific). The immunofluorescently-labelled tissues
were then imaged using either fluorescence microscopy (Eclipse, Nikon, Japan) or
confocal microscopy (TCS SP5, Leica).
2.2.9 Image analysis
Digital image analyses of stained and antibody-labelled tissue sections and cell
monolayers were conducted using ImageJ software (public domain software
downloaded from http://rsb.info.nih.gov/ij/ (Abramoff, 2004).
2.2.10 Immunofluorescent analysis of Transwell® insert membranes
Immunofluorescence was used to detect specific target antigens in keratinocyte
monolayers grown in Transwell® co-cultures. At various time points following
irradiation, the Transwell® inserts were removed from the cell culture plates and
were washed in cold PBS. The cells on the membrane were fixed by immersing the
inserts in 4% paraformaldehyde (PFA) solution for 30 minutes at room temperature
(Lanier and Warner, 1981). The membranes were cut out from the insert casing using
a scalpel blade and stored submerged in cold PBS at 4°C until the staining procedure.
Keratinocytes on the membrane were permeabilised with 0.2% Tween-20 (Merck,
Australia) in PBS for 60 minutes at room temperature. Cells were then washed in
PBS containing 1% bovine serum albumin (Sigma-Aldrich) and blocked with a
blocking medium (Active Motif, Carlsbad, CA, USA) for 60 minutes at 37°C.
Primary antibodies were diluted in staining medium (MAXbind™, Active Motif) and
incubated with the antibody solution for 60 minutes at 37°C in a humidified
chamber. The membranes were then washed in PBS containing 1% bovine serum
albumin before the addition of fluorescent secondary antibodies (diluted 1:1000 in
staining medium). The membranes were incubated with secondary antibodies for 60
minutes at 37°C in a humidified chamber, protected from light. The insert
membranes were washed to remove residual secondary antibody and immersed in
PBS containing 0.5 µg/mL DAPI solution for 10 minutes in the dark at room
temperature. The membranes were then washed once in PBS and mounted onto
40 Materials and methods
microscope slides in anti-fade mounting medium (Prolong® Gold, Invitrogen). Glass
coverslips (#1.5, HD Scientific) were placed on top of the membranes and the
mounting medium was left to set at room temperature in the dark for at least six
hours. Immunofluorescently-labelled membranes were imaged using fluorescence
microscopy (Eclipse, Nikon, Japan).
2.2.11 TUNEL Assay for the detection of apoptotic cells
Detection of apoptotic keratinocytes in irradiated HSE
Tissue sections of irradiated HSE composites were probed for the presence of
apoptotic cells using a terminal deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) assay (Roche Applied Science, Penzberg, Germany) according to the
manufacturer’s protocol. Briefly, the sections were deparaffinised and rehydrated as
described in 2.2.6. The tissues were permeabilised with 20 µg/mL Proteinase K
solution (Ambion, Life Technologies) for 20 minutes at 37°C. As a positive control,
tissue sections were pre-treated with 2 U DNase I (Invitrogen). The negative controls
were treated using the same protocol, but without the addition of the terminal
deoxynucleotidyl transferase enzyme. The sections were then incubated with the
TUNEL enzyme label solution for 60 minutes at 37°C, protected from light. The cell
nuclei were counterstained with DAPI solution for ten minutes at room temperature.
The sections were imaged using fluorescence microscopy (Eclipse, Nikon) at an
excitation wavelength of 450 to 500 nm and a detection range from 515 to 565 nm.
Detection of apoptotic keratinocytes in irradiated 2D co-cultures
Apoptotic keratinocytes were detected in irradiated Transwell® insert
membranes using the protocol detailed in 2.2.11. Prior to the TUNEL assay being
performed, cell monolayers in the Transwell® inserts were fixed in 4% PFA for 30
minutes at room temperature. The insert membranes were cut out of the insert casing
(as described in 2.2.10) and stored in PBS at 4°C until the assay was performed.
2.2.12 Assessment of cellular viability
Cellular viability assays in HSE
Metabolically active cells in UVB-irradiated HSE composites were detected
using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay (Denizot and Lang, 1986). Briefly, the HSE composites were immersed in 1
mL of 0.5 mg/mL MTT solution in PBS (Sigma-Aldrich) and incubated for 90
Chapter 2: Materials and methods 41
minutes at 37°C. The MTT reaction results in a colorimetric change from yellow to
purple. This colour change in the epidermis of the HSE composites was imaged
using a digital camera.
Cellular viability assays in 2D co-cultures
Keratinocyte monolayers cultured on Transwell® insert membranes were
assayed for cell viability using a CellTitre 96® AQueous Assay (Promega). After
exposure to UVB radiation, 200 µL of cell culture medium from the Transwell®
insert, and 900 µL of medium from the lower chamber were removed from all the
wells. Next, 40 µL of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium) (MTS) reagent was added to the insert and mixed
with cell culture medium. Cell monolayers were incubated with the reagent for two
hours at 37°C with 5% CO2 and protected from light. Controls consisted of cell
culture medium only treated with MTS under the same incubation conditions.
Following incubation, 100 µL samples of the cell culture medium was transferred
into a 96-well plate. The absorbances of the solutions were read at 490 nm using a
Beckman Coulter microplate reader (Beckman Coulter, Fullerton, CA, USA). Blank
readings were obtained from the negative control samples.
2.2.13 Detection and quantification of secreted cytokines in 2D and 3D cultures
Analysis of IL-6 concentrations in cell culture medium and cell lysates
An enzyme-linked-immunoassay (ELISA) kit for the detection of human IL-6
was used as per the manufacturer’s instructions (Quantikine®, R&D Systems, MN,
USA). Briefly, 1 mL SFM cell culture supernatant samples were obtained from HSE
cultures and 2D co-cultures prior to and after UVB irradiation as described in 2.2.5.
For the Transwell® co-cultures, the medium in the insert and the well were mixed
thoroughly before the sample was obtained. The medium was centrifuged and the
supernatant collected to remove cell debris. Samples were stored at -80°C until the
assay was performed. Serial dilutions of recombinant IL-6 standards with known
concentrations were prepared. Assay diluent was then added to all wells of a 96-well
plate which was pre-bound with a mouse monoclonal antibody against human IL-6.
Following this, 100 µL serial dilutions of standards and samples were added to
duplicate wells in the plate and were incubated for two hours at room temperature.
The samples were then aspirated and the wells were washed with the provided wash
buffer. A secondary polyclonal antibody conjugated to horse radish peroxidase was
42 Materials and methods
added and the plate was incubated for a further two hours at room temperature.
Following a similar wash step, all wells were incubated with a substrate containing
hydrogen peroxide and tetramethylbenzidine chromogen for 20 minutes in the dark.
The reaction was stopped by the addition of 50 µl of 2 M sulphuric acid. The optical
densities of the reactions were read at 450 nm using a microplate reader (Beckman
Coulter, Fullerton, CA, USA).
Analysis of IL-8 concentrations in cell culture medium and cell lysates
An IL-8 ELISA kit for the detection of human CXCL8/IL-8 was used as per
the manufacturer’s instructions (Quantikine®, R&D Systems, MN, USA). Cell
culture and cell lysates samples were collected as described in the previous section
and the assay protocol was performed as previously described. The ELISA reactions
comprised of 50 µL standards and diluted samples. A one-hour incubation step with
the conjugated secondary antibody was performed. The absorbances at the reaction
end-point were read at 450 nm following the immunoassay protocol.
2.2.14 Analysis of cell cycle phase by fluorescent-labelling of DNA content
The ratios of cells at different cell cycle phases following irradiation were
assessed using flow cytometry. The method of fluorescently-labelling cellular DNA
content allows for the estimation of the cells’ cycle phases (Nunez, 2001). Briefly,
cell culture medium was removed from the inserts and the membranes were washed
with PBS. Keratinocyte monolayers were trypsinised and these cell suspensions were
centrifuged at 1500 rpm for 5 minutes. The pellets were resuspended in ice cold PBS
and micro-centrifuged at 6000 rpm for 5 minutes, with this wash process repeated
twice. The cell pellets were then fixed by resuspending in ice cold 70% ethanol and
stored at -20°C until the DNA staining protocol was performed. Prior to staining, cell
pellets were washed twice in cold PBS as mentioned previously. Cells were then
resuspended in a solution containing 5 mL 0.01% Triton-X (in PBS), 100 µL of 10
mg/mL Rnase A (Sigma) and 100 L of 1 mg/mL propidium iodide (PI) (Sigma).
Cell suspensions were incubated in this solution for 15 minutes at 37°C, protected
from light. Keratinocytes were then filtered through a 37 µm nylon mesh (BD
Biosciences) to prevent cellular aggregation. Cell fluorescence was then measured
using a flow cytometer (Cytomics FC500, Beckman Coulter). Histograms from the
fluorescent output were analysed using the MXP Analysis Software (Beckman
Chapter 2: Materials and methods 43
Coulter) to determine the proportion of cells in G1, S, G2/M phases and apoptotic
cells present in the total cell population.
2.2.15 Extraction of total RNA from HSE composites
The RNA profile of HSE keratinocytes was analysed prior to and following
UVB irradiation. The total RNA was extracted from the HSE epidermis using a
previously-reported technique for the isolation of high-quality RNA from skin
(Clemmensen et al., 2009). The composites were immersed papillary-side down in
3.8% ammonium thiocyanate solution in PBS (Sigma), at pH 7.4 and at room
temperature for approximately ten minutes. Upon epidermal separation, the
epidermis was peeled off with forceps and immersed in 250 µL of TRI-reagent®
(Zymo). Cells in the epidermis were lysed by gently pipetting the tissue in TRI-
reagent® on ice. The papillary surfaces of the HSE composites were also scraped
into the Trizol reagent using a scalpel blade to ensure the lysis of the epidermal
keratinocytes.
2.2.16 Purification of total RNA from cell lysates
The total RNA extracted from the HSE composites (as outlined in 2.2.15) was
purified using a Direct-Zol II RNA Mini-prep kit (Zymo Research Corporation,
Irvine, CA, USA), following the manufacturer’s supplied protocol. Briefly, 100%
ethanol was added to cell homogenates in TRI-reagent® as mentioned previously at a
ratio of 1:1. This mixture was loaded onto the supplied columns and centrifuged,
with the first flow through discarded. An in-column DNase I digestion was then
performed using 1 unit of DNase I per column, incubated at 37°C for 15 minutes.
The column was washed with wash buffers and the total RNA was eluted in 25 µL of
DNase/RNase-free water. The concentration of the eluted RNA was determined
using a Nanodrop spectrophotometer (Thermo Scientific). The purity of the extracted
RNA was determined via the ratio of the absorbance at 260/280 nm and 260/230 nm,
with ratios of approximately 2 indicating high-quality RNA samples. Next, RNA
stability was assessed by separating 1 µL of sample on a 1.5% agarose/tris-
acetate/EDTA (TAE) gel electrophoresed at 115 V for 30 minutes. The presence of
two distinct 18S and 28S ribosomal RNA bands (with the 28S band about twice as
intense as the 18S band) indicated stable RNA samples. The RNA samples were
stored at -80°C until further use.
44 Materials and methods
2.2.17 Synthesis of double-stranded cDNA
First strand DNA synthesis was performed using SuperScript™ III First-Strand
Synthesis SuperMix for qRT-PCR (Invitrogen); following the manufacturer’s
supplied protocol. Briefly, standard reactions of 10 µL of the supplied reaction mix,
1 µg of RNA, 2 µL of reverse transcriptase enzyme mix and water to a final volume
of 20 µL were prepared. Reactions were performed on a thermal cycler machine (MJ
Research PTC-200 Thermo Cycler) at 25°C for 10 minutes, 55°C for 30 minutes,
85°C for 5 minutes and 4°C for 15 minutes. Two units of RNase H was then added to
each tube and the reaction incubated for 20 minutes at 37°C, and the reaction
terminated at 4°C. The resulting cDNA samples were diluted 1:10 in nuclease free
water and stored at -20°C until use in quantitative real-time PCR (qRT-PCR)
analysis.
2.2.18 Primer design
Primer-basic local alignment software (Primer-BLAST), available through the
National Centre for Biotechnology Information (accessible at
www.ncbi.nlm.nih.gov/tools.primer-blast/) was used to design primers for qRT-PCR.
The parameters used to define primer sequences included a minimum primer melting
temperature (Tm) of between 57°C and 63°C, with a maximum of 3°C difference
between primer Tm and an amplicon length between 70 and 150 base pairs. The
resulting PCR primers were purchased from Sigma with 25 nmol concentrations and
desalted purity.
2.2.19 Analysis of differential gene expression using qRT-PCR
Different expressions of specific genes of interest were determined using qRT-
PCR. Reactions of 20 µL volumes were set up in a 96-well plate format and run on
an ABI Prism 7500 Real-Time PCR System (Applied Biosystems). Each reaction
included 10 µL SYBR Green Master Mix (Life Technologies), 0.2 µM of both
forward and reverse primers, and 20 ng cDNA templates and made to 20 µL with
water. A two-step cycling protocol was used with a denaturation step at 95°C for 10
minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for one minute. To
confirm amplification of the correct sequence, post-amplification melt curve analysis
was conducted using the High Resolution Melting (HRM) Software (Applied
Biosystems) to determine the melting temperature (Tm) of the amplified PCR
Chapter 2: Materials and methods 45
product. The results were analysed using the 7500 Real-Time PCR Analysis
software, v2.0 (Applied Biosystems), using the ‘automatic’ function for baseline and
threshold values. Target gene expression was normalised to the S26 ribosomal
subunit (Forward: 5’CGCAGCAGTCAGGGACAT3’ Reverse:
5’AGCACCCGCAGGTCTAAATC3’) as it has been found to be most consistent as
a housekeeping gene in primary human keratinocytes (Bonnet-Duquennoy et al.,
2006). The relative comparative threshold cycle (CT) method (2-∆∆C
T) was used to
determine relative gene expression differences between the genes of interest.
2.2.20 Extraction of protein from HSE epidermal cell lysates
The epidermis was separated from the dermal component of the HSE
composites using the epidermal separation protocol detailed in 2.2.15. The epidermal
tissue was immersed in Radio Immuno-precipitation Assay (RIPA) buffer containing
25 mM Tris, HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1% sodium
deoxycholate, 0.1% SDS (Thermo Scientific) with 2 mM Na3VO4, 10 mM NaF and a
complete protease inhibitor cocktail (Roche Diagnostics). The papillary surface of
the HSE was gently scraped with a scalpel blade into the cell lysis buffer to collect
residual epidermal cells. The epidermis was first passed through a 1 mL pipette tip at
least ten times on ice, mixed with a vortex for 20 seconds and kept on ice for ten
minutes. This was repeated three times to lyse cells. Residual epidermal tissue,
consisting mainly of the stratum corneum, was then removed. Cell lysates were
subsequently passed through a syringe with a 26-gauge needle at least five times
before being centrifuged at 14 000 g for 20 minutes at 4°C. The supernatants were
collected and the protein concentration in the samples determined using a
bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL, USA). Cell lysates were
stored at -80°C until further use.
2.2.21 Extraction of protein from Transwell® cell monolayers
Spent growth medium from keratinocytes cultured on Transwell system
membranes was removed and the monolayers washed twice with PBS to remove
traces of medium that may interfere with downstream applications. The cells were
then lysed in RIPA buffer (Thermo Scientific) supplemented with 2 mM Na3VO4, 10
mM NaF and a complete protease inhibitor cocktail (Roche Diagnostics) as described
in 2.2.20. The cell lysates were homogenised by passing through a 26-gauge needle
at least five times prior to centrifugation at 14 000 g for 20 minutes at 4°C. The
46 Materials and methods
supernatants were collected and the protein concentration in the samples determined
using a bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL, USA). Cell lysates
were stored at -80°C until further use.
2.2.22 Protein separation of cell lysates using SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was
used to size-separate proteins contained in the cell lysate samples prepared in 2.2.20.
Pre-cast SDS-PAGE gels (NuPAGE® SDS-PAGE gel systems, Life Technologies)
containing 10% resolving gel and 4% stacking gel regions were used to enhance
band resolution. Samples containing 20 µg of total protein were pre-treated with 4x
sample loading buffer (Invitrogen) and a reducing agent (Invitrogen) for 10 minutes
at 70°C. The gels were placed in running buffer (NuPAGE®, Life Technologies) and
the samples (including a pre-stained broad range molecular weight marker (BioRad))
were loaded into SDS-PAGE gel wells. The samples were then subjected to
electrophoresis at 200 V for 35 minutes at 4°C.
2.2.23 Western immunoblotting
Following SDS-PAGE protein resolution, size-separated proteins on the gel
were transferred onto nitrocellulose membranes (BioTrace®NT, Pall Corporation,
FL, USA) using a semi-dry transfer technique. Briefly, the membrane was
sandwiched between the SDS-PAGE gel and filter paper. This was then immersed in
transfer buffer (3.03 g/L Tris Base, 14.4 g/L glycine and 20% ethanol) and placed
within a semi-dry electrophoresis machine (BioRad Trans-blot SD semi-dry transfer
cell). Transfers were run at 45 mA per gel, for a maximum of 90 minutes. After the
transfer, the nitrocellulose membranes were blocked with Odyssey blocking buffer
(LI-COR®). Primary antibody dilutions (1:10000) were performed in Odyssey
blocking buffer and incubated on the membranes overnight at 4°C. The membranes
were then washed in Tris-buffered saline Tween-20 (TBS-T) for five three-minute
washes. The fluorescently-labelled secondary antibodies were diluted 1:20000 in
Odyssey blocking buffer and incubated on the membranes for one hour at room
temperature. Fluorescently-labelled bands were then imaged using an Odyssey®
Infrared Imaging System (LI-COR®). The acquired images were analysed using
Image Studio Software (LI-COR®) for the quantification of band fluorescence
intensity.
Chapter 2: Materials and methods 47
2.2.24 Statistical analysis
Data analysis was performed using one-way analysis of variance (ANOVA)
with Tukey’s post-hoc analysis, unless otherwise stated. Statistically significant
differences were considered to exist at p<0.05.
48
Chapter 3: Validation of the HSE-KC photobiology model 49
Chapter 3: Validation of the HSE-KC
photobiology model
3.1 INTRODUCTION
A wide variety of in vitro skin models have been employed to investigate the
effects of UVR on skin, including an array of animal models, cell lines and
bioengineered skin constructs (Fernandez et al., 2012b). Nevertheless, there is still an
urgent need for more cost-effective and easily accessible experimental models
capable of more accurately representing the structural and functional aspects of
native skin for photobiology studies. The human epidermis possesses a highly
specialised spatial organisation and structure, which is a critical property in
maintaining its barrier function. Consequently, the intricacies of the epidermis, such
as the presence of the basal, spinous, granular and cornified keratinocyte layers, have
posed a challenge to recreate in vitro. With each of these layers defined by the
differentiation status, polarity, morphology and functionality of the keratinocytes
within it, the importance of incorporating these features into in vitro skin models for
representing and predicting epidermal physiology is evident. Hence, the development
of improved cutaneous biology models represents a fundamental requirement for
advancing our knowledge of UVR-induced damage, repair and regeneration
pathways and their relevance to photocarcinogenesis. Therefore, the first aim of this
study was to characterise a 3D HSE-KC tissue-engineered skin model (consisting of
primary human keratinocytes cultured on an ex vivo DED scaffold) as an in vitro
platform for studying acute epidermal responses to UVB radiation.
Increasingly elegant organotypic skin models have been developed for a range
of different experimental applications, including photobiology, toxicology studies
and cutaneous biology research (Archambault et al., 1995; Hudon et al., 2003;
Bechetoille et al., 2007). The incorporation of different cell types from dermal or
epidermal origins into a 3D scaffold has resulted in the creation of pigmented and
vascularised skin equivalent cultures, along with skin constructs with an integrated
immune component (Topol et al., 1986; Hudon et al., 2003; Laubach et al., 2011;
Liu et al., 2011). Besides the varied combinations of cell types incorporated into the
50 Validation of the HSE-KC photobiology model
epidermis, this array of skin substitutes also makes use of a range of natural and
synthetic dermal substrates, each with its own advantages and disadvantages, as
discussed in 1.6.4.
Our laboratory has previously reported the use of a HSE-KC skin culture
model for studying epithelial biology in applications such as tissue regeneration
during wound healing (Topping, 2006; Rayment et al., 2008; Xie et al., 2010).
Critically, the HSE-KC retains vital structural, functional and organisational
properties of the native dermis and epidermis, which may be lacking in other in vitro
models (Xie et al., 2010). Accordingly, a 3D HSE-KC model and its application in
mapping epidermal responses to UVB radiation are detailed in this Chapter.
Significantly, this study represents a previously unexplored method for
characterising UVB radiation-induced changes to human-derived keratinocytes in
vitro.
Many aspects of the epidermal microenvironment, for instance, elements of the
BM and ECM as well as the structural and organisational properties of the epidermis
are known to directly modulate keratinocyte responses to UVB radiation (Amano,
2009). In particular, the distinctive multilayered structure of the epidermis is thought
to be critical for blocking the penetration of harmful UVR, thus minimising DNA
damage to the actively proliferating basal cells (Everett et al., 1966). The HSE-KC,
proposed as a photobiology skin model in this project, was firstly characterised to
analyse the expression of key features of the native epidermis in these constructs, in
comparison to that observed in samples of ex vivo skin tissue. Indeed, investigating
the preservation of morphological and biological cutaneous markers in the HSE-KC
known to be essential in the skin’s UV response was an essential first step in
validating this model as a physiologically-relevant tool for photobiological studies.
The next stage of validation involved the identification of key photodamage
markers in HSE-KC composites exposed to UVB radiation. The generation of CPD
DNA photolesions in the nuclei of irradiated keratinocytes is known to be a
characteristic feature of UVB radiation-induced DNA damage (You et al., 2000).
These premutagenic molecular lesions arising from the formation of C=C double
bonds in thymine or cytosine bases, result in structural DNA modifications which
can interfere with its replication (Ruven et al., 1993; Vreeswijk et al., 1994). Hence,
the subsequent repair of these DNA photolesions has been shown to be among the
Chapter 3: Validation of the HSE-KC photobiology model 51
most critical processes in protecting against photocarcinogenesis (Svobodova et al.,
2012). The formation of apoptotic keratinocytes, also known as sunburn cells, is
another distinctive marker of UVB radiation-induced epidermal damage, often used
as a qualitative and quantitative measure of acute photodamage (Young, 1987;
Weatherhead et al., 2011). Also, central to the early responses of skin to UV-
exposure is the induction of the cutaneous inflammatory response (Young, 2006; Bijl
et al., 2007). While clinical features of sunburn such as erythema and enhanced
melanogenesis are not able to be detected in the avascular and unpigmented HSE-
KC, DNA damage and immunomodulatory factors secreted by irradiated
keratinocytes that initiate these inflammatory effects can be analysed (Kondo et al.,
1993; Enk et al., 1995; Nishigori et al., 1996). Accordingly, these detectable markers
of UVB radiation-induced damage, which have been well-documented in vivo and in
vitro (reviewed in 1.3), were investigated using the irradiated HSE-KC model in this
Chapter (Young, 1987; Miyauchi-Hashimoto et al., 1996; Svobodova et al., 2006).
During the acute phase of UVB photodamage, the activation of cellular repair
mechanisms to remove UVB radiation-induced DNA photolesions is an innate
feature of the native epidermis (reviewed in 1.3.3). Repair and regeneration
processes are an essential component in maintaining the integrity of the epidermal
barrier, while preventing the oncogenic transformation of proliferative cells (Liu et
al., 1983; Kraemer et al., 1990; Bernerd, 2005; Mouret et al., 2008). In irradiated
keratinocytes, many of these repair pathways are known to be orchestrated by the
activity of the p53 tumour suppressor protein (Cui, 2007).
Hence, in addition to the generation of UVB photodamage in the HSE-KC, this
study also investigates whether keratinocytes in the HSE-KC epidermis retained their
ability to repair this damage. This included the removal of CPD photolesions, as well
as shifts in keratinocyte proliferation and differentiation following UVB radiation
exposure. Other changes, such as the modification of epidermal protein expression,
are known to occur in the post-irradiation period (Bernerd and Asselineau, 1997).
For example, an increase in the number of terminally differentiating keratinocytes
following irradiation results in the associated rise in the expression levels of specific
keratins such as keratins 1, 10 and 11 (Fuchs et al., 1987). Hence, UVB radiation-
induced alterations were analysed on both an epidermal tissue level, as well as at the
cellular level in the irradiated HSE-KC constructs.
52 Validation of the HSE-KC photobiology model
Broadly, the aims of this Chapter were therefore to:
1) Compare the expression of various epidermal markers between native skin
and HSE-KC samples;
2) Characterise the responses of the HSE-KC to UVB radiation; and
3) Identify epidermal changes over time during the acute UVB radiation
response using the HSE-KC model.
Chapter 3: Validation of the HSE-KC photobiology model 53
3.2 MATERIALS AND METHODS
The description of protocols and methods used in this section are outlined in
Chapter 2. Details surrounding specific materials utilised are described in the
following sub-sections.
3.2.1 Fixation of native skin samples
Ex vivo native skin samples used in comparative studies were obtained, fixed
and processed as detailed in 2.2.1. For immunohistochemical studies, native skin
samples and reconstructed HSE-KC tissue from the same patient donor were used.
3.2.2 Construction and culture of the HSE-KC
The isolation and culture of primary human keratinocytes and the construction
of the HSE-KC model has been detailed previously in 2.2.1 to 2.2.3.
3.2.3 Irradiation of the HSE-KC
All HSE-KC were irradiated with specific doses of UVB radiation as per the
irradiation protocol described in 2.2.5.
3.2.4 Histology
All histology was performed as per routine haematoxylin and eosin staining
protocols, previously outlined in 2.2.6.
3.2.5 MTT assay of cell viability
Viable keratinocytes on the irradiated and non-irradiated HSE-KC were
visualised using the protocol described in 2.2.12 and photographed using a digital
camera.
3.2.6 Immunohistochemistry
Immunohistochemistry was performed as per previously described protocols
(2.2.7). The following primary antibodies were used in the detection of tissue
markers: mouse monoclonal anti-cytokeratin 1/10/11 (USBioLogical), mouse
monoclonal anti-cytokeratin 2e (Abcam), mouse monoclonal anti-cytokeratin 14
(Novus Biologicals), mouse monoclonal anti-filaggrin antibody (Santa Cruz
Biotechnology), mouse monoclonal anti-thymine dimer antibody (clone KTM53,
Kamiya Biomedical Company), mouse monoclonal anti-human p53 (clone DO-7,
Dako), monoclonal antibody against human collagen IV (Clone CIV22, Dako) and
54 Validation of the HSE-KC photobiology model
monoclonal mouse anti-human Ki-67 (Dako). All tissue sections were pre-treated
with HIER as previously described (2.2.7). Antibody-binding was visualised using
DAB, and tissue counterstained with haematoxylin G1.
3.2.7 Detection and quantification of secreted cytokines in 2D and 3D cultures
The concentrations of IL-6 and IL-8 in cell culture medium samples obtained
from the irradiated Transwell® culture inserts, HSE-KC and HSE-KCF cultures were
quantified using an ELISA technique, as described in 2.2.13.
3.2.8 TUNEL assay
The TUNEL assay for the in situ detection of cells undergoing apoptosis was
performed using the method described in 2.2.11.
Chapter 3: Validation of the HSE-KC photobiology model 55
3.3 RESULTS
3.3.1 Immunohistochemical characterisation of the HSE-KC epidermis
The human epidermis is comprised of distinct differentiated layers and a
characteristic polarized pattern of keratinocyte growth, with actively proliferating
cells situated at the dermal-epidermal junction. The expression of various
keratinocyte differentiation markers in ex vivo skin and HSE-KC tissues (from the
same donor) were analysed using immunohistochemistry (Figure 3.1). The
expression of keratins 1/10/11 was examined as these are known markers of
terminally-differentiated keratinocytes in the epidermis (Freedberg et al., 2001). In
both native and reconstructed skin sections, keratinocytes expressing keratin 1/10/11
were present throughout the middle layers of the epidermis, with the exception of the
stratum corneum and the stratum basale (Figure 3.1A).
Cytokeratin 2e has been shown to be expressed in the keratinocytes of the
upper spinous and granular cells of the native epidermis (Collin et al., 1992; Smith et
al., 1999). Similar expression patterns of cytokeratin 2e were detected in both the
native skin and HSE-KC samples (Figure 3.1B). However, a more intense and
uniform distribution of this keratin was observed in the ex vivo skin samples relative
to the HSE-KC. Similarly, the presence of keratin 14 (Figure 3.1C) appeared to be
more strongly detected around the basal keratinocytes in native skin as compared to
the immunostaining observed in HSE-KC. In ex vivo tissue, there was a gradual
decrease in the expression of keratin 14 from the suprabasal cell layers towards the
stratum corneum. In the HSE-KC, on the other hand, there was a more uniform
distribution and slightly diminished expression of keratin 14 among the basal and
suprabasal cells.
Filaggrin is a late differentiation epidermal protein with an expression highly
localised to the stratum corneum in vivo (Sandilands et al., 2009). Positive
immunostaining of filaggrin was detected in the cornified envelope of keratinocytes
within the stratum corneum in both HSE-KC and skin tissue samples alike (Figure
3.1D). In the HSE-KC, however, the stratum corneum appeared to be more densely
packed in comparison to that of native skin. In addition, filaggrin in the skin
equivalent model was observed to be more strongly expressed at the junction of the
stratum corneum and stratum lucidum.
56 Validation of the HSE-KC photobiology model
Figure 3.1. Comparison of cutaneous markers in HSE-KC and native skin
Immunohistochemical analysis was conducted to detect the expression of various epidermal and BM markers on
both HSE-KC composites (left column) and native skin sections (right column). (A) Expression of keratin
1/10/11 shows the presence of differentiated keratinocytes in the stratum spinosum, stratum lucidum and stratum
granulosum within the epidermis. (B) Cytokeratin 2e expression in HSE-KC and native skin show localisation
within the upper suprabasal keratinocytes. (C) Keratin 14 was detected in basal and suprabasal keratinocytes in
both sections of HSE-KC and native skin, with the latter showing a more intense immunoreactivity in the stratum
basal. (D) Expression of filaggrin was limited to the stratum corneum in both HSE-KC and ex vivo skin. (E)
Collagen IV was detected at the epidermal-dermal junction and lining dermal structures in both the skin
equivalents and native skin. Images are representative of immunohistochemical analyses of native skin and HSE-
KC constructs from three independent skin donor samples. The scale bar shown (bottom right hand corner)
represents 50 µm in length.
Chapter 3: Validation of the HSE-KC photobiology model 57
The presence of type IV collagen, a major constituent of the BM zone in skin,
was detected at the dermal-epidermal junction in both the HSE-KC and skin tissue
samples (Figure 3.1E). Besides separating the epidermis and dermis, collagen IV is
also known to line sweat glands and blood vessels, which accounts for its positive
immunostaining in these structures within the dermis and DED (Konomi et al.,
1984).
Overall, the immunohistochemical study of epidermal and BM markers show
strong parallels in the expression and localisation of these markers between the HSE-
KC and native skin.
3.3.2 Optimisation of UVB-irradiation protocol
Several previously documented studies investigating UVB radiation responses
and photocarcinogenesis involve the use of excessive doses of radiation (Tadokoro,
2003; Garcia et al., 2010; Jeong et al., 2012). In view of our aim to develop a
biologically relevant photobiology model, therefore, we treated the HSE-KC
composites with a range of UV doses known to be experienced from solar UVR in
Australia (Kimlin et al., 1998; Kimlin and Parisi, 2001). This dose optimisation
study aimed to generate histological markers of UVB photodamage in irradiated
HSE-KC, while at the same time, maintaining enough viable epidermal cells to
support post-irradiation regeneration.
The histological analysis of irradiated HSE-KC tissues indicates a marked
increase in the morphological characteristics of epidermal damage with increasing
doses of UVB radiation (Figure 3.2). Composites exposed to 1 MED of UVB
radiation developed ‘sunburn cells’ located in the suprabasal layer of the epidermis
(Figure 3.2C). These apoptotic keratinocytes are characterised by the presence of
pyknotic nuclei and cytoplasmic shrinkage (Häcker, 2000). The presence of
apoptotic cells were not observed in haematoxylin and eosin-stained sections of non-
irradiated controls and HSE exposed to suberythemal (below 1 MED) doses of UVB
radiation. In composites exposed to an excess of 3 MED UVB radiation and above, a
proportion of the suprabasal keratinocytes also displayed cytoplasmic swelling,
strongly suggesting radiation-induced necrosis (Figure 3.2 D to F) (Caricchio, 2003).
In tissue exposed to 5 and 10 MED UVB radiation, the basal keratinocytes displayed
58 Validation of the HSE-KC photobiology model
high levels of cell death at 24 hours post-irradiation, resulting in separation at the
epidermal-dermal junction during histological processing (Figure 3.2 D and E).
The histology of irradiated HSE-KC therefore suggests that UVB radiation
doses below 3 MED induce histological markers of photodamage without the
complete destruction of the proliferative stratum basale as a result of UVB-induced
cell death.
Figure 3.2. Dose-response of HSE-KC to UVB radiation.
Haematoxylin and eosin staining of UVB-irradiated HSE-KC sections subjected to increasing doses of UVB are
shown. Stained sections of (A) non-irradiated HSE-KC controls, and skin equivalents exposed to a single dose of
(B) 0.5 MED, (C) 1 MED, (D) 3 MED, (E) 5 MED and (F) 10 MED at 24 hours post-exposure were imaged.
Arrows indicate cells showing morphological characteristics of apoptotic keratinocytes. The circled region shows
the presence of keratinocytes showing signs of cytoplasmic swelling, which is indicative of radiation-induced
necrosis. Irradiation with UVB doses in excess of 3 MED (75 mJ/cm2) results in the destruction of basal
keratinocytes and epidermal-dermal separation during histological processing. Scale bar represents 50 µm.
Histology is representative of experimental triplicates conducted on three independent skin donors.
3.3.3 Assessment of cell viability in irradiated HSE-KC
Metabolically active epidermal cells in the irradiated HSE-KC composites
were visualised using an MTT assay which results in a colorimetric change from
yellow to purple in viable tissue. These MTT-stained regions were then digitally-
photographed to determine the relative differences in cell viability between different
Chapter 3: Validation of the HSE-KC photobiology model 59
doses of UVB and time-points after exposure. Figure 3.3 shows images captured of
MTT-stained HSE composites exposed to increasing doses of UVB (A to F). At
UVB exposures of 3, 5 and 10 MED, a slightly decreased intensity of MTT stain was
observed in the epidermis at 24 hours post-irradiation. There were no significant
differences in the diameter of the circular areas where keratinocytes were originally
seeded onto the DED with increasing UVB dose. Figures G to L show HSEs exposed
to a single 2 MED UVB dose at Day 0 (G) and returned to culture conditions over a
period of five days (H to L) to assess whether keratinocytes remained viable in
culture post-irradiation. Visualisation of the MTT-stained epidermal layers in these
composites shows no observable differences in intensity or area over the five days.
Figure 3.3. Epidermal viability following UVB-irradiation in HSE
The viability of cells within the HSE-KC epidermis following UVB exposure was analysed using an MTT assay.
At 24 hours post-irradiation, metabolically active keratinocytes (purple staining) were visualised in HSEs
exposed to a single dose of (A) 0 MED, (B) 0.5 MED, (C) 1 MED, (D) 3 MED, (E) 5 MED and (F) 10 MED
UVB. Skin composites exposed to a single 2 MED UVB dose and kept in culture conditions post-irradiation were
assayed at (G) Days 0, (H) 1, (I) 2, (J) 3, (K) 4 and (L) 5. Images are representative of triplicate experiments
conducted on three independent skin donors. Red scale bar represents 1.6 cm.
60
3.3.4 Formation of CPDs in irradiated HSE-KC composites
One of the primary markers of UVB-induced damage in keratinocytes is the
formation of CPD molecular lesions in the DNA as a result of a photochemical
reaction forming covalent bonds in pyrimidines (as described in 1.3.1). The
formation of these dimers was probed in HSE-KCs exposed to a single 2 MED dose
of UVB radiation using immunohistochemical analysis (Figure 3.4). At 24 hours
post-irradiation, strong positive nuclear staining was observed in the basal and
suprabasal keratinocytes, indicating the presence of CPDs within these regions.
Figure 3.4. UVB exposure induces the formation of CPDs in HSE-KC
Immunohistochemical analysis was used to detect the presence of UVB-induced CPDs in the nuclei of cells in the
epidermis of HSE-KC at 24 hours after UVB treatment. The figure shows sections of (A) non-irradiated HSEs,
antibody-probed sections of HSE-KC exposed to 2 MED of UVB at (B) 100x and (C) 200x magnification
respectively. The presence of CPDs, indicated by brown staining, was observed predominantly within the nuclei
of basal and suprabasal cells of the epidermis. Black and red scale bars represent 50 µm. Images from
immunohistochemical analyses are representative of experimental triplicates conducted on three independent skin
donors.
3.3.5 Formation of apoptotic keratinocytes in the irradiated HSE-KC
Keratinocytes that have sustained irreparable DNA damage as a result of UVB
damage undergo apoptosis to prevent the propagation of cells harbouring
photolesions (Young, 1987). The TUNEL assay (2.2.11) was employed to detect
DNA fragmentation, a characteristic marker of the UVB-induced apoptotic pathway.
At 24 hours after a single 2 MED UVB dose, the in situ TUNEL assay showed the
presence of epidermal keratinocytes undergoing apoptosis (Figure 3.5). Magnified
and merged images demonstrate the specific nuclear localisation of DNA
fragmentation, which is fluorescently-labelled during the TUNEL reaction. Cells
undergoing apoptosis were present in the stratum basale, with the majority localised
Chapter 3: Validation of the HSE-KC photobiology model 61
among the suprabasal keratinocyte populations. An average of 19.9 ± 4.1% of the
total number of epidermal nuclei was found to be TUNEL-positive at 24 hours post-
irradiation.
Figure 3.5. Apoptotic keratinocytes in irradiated HSE-KC
Apoptotic keratinocytes were detected in HSE-KC, 24 hours following a single exposure of 2 MED UVB (A left
to right). The first column indicates sections stained with DAPI nuclear marker. Blue fluorescence indicates
keratinocytes in the epidermis of HSE-KC. The second column shows TUNEL positive cells, indicated by the
presence of green fluorescence. The final column shows merged images. The white dotted line indicates the
epidermal-dermal junction in the skin equivalents. A region of these sections has been magnified (B left to
right), showing clearly the nuclear localisation of DNA fragmentation as detected by the TUNEL reaction.
Yellow lines indicate the scale of 50 µm. Images from the TUNEL assay analysis are representative of
experimental triplicates conducted on three independent skin donors.
3.3.6 UVB-induced expression of p53 in the HSE-KC
The expression of p53 by keratinocytes subsequent to UVB exposure is known
to be critical for regulating cellular regeneration and suppressing oncogenesis
(Maltzman and Czyzyk, 1984; Levine et al., 1991; Will, 2000). Keratinocyte
expression of p53 was detected in irradiated HSE-KC using immunohistochemical
analysis and shown to progressively increase during the acute phase of UVB-induced
damage (Figure 3.6). Sections from HSE-KCs sacrificed at 0, 3, 6 and 24 hours after
DAPI TUNEL Merged
4x
40x
62 Validation of the HSE-KC photobiology model
a single dose of 2 MED UVB display p53-positive nuclei, particularly in the
proliferative basal layer and the lower suprabasal layers of the HSE epidermis. A
weak, diffuse cytoplasmic staining was also observed; this gradually intensified in
the hours following UV exposure. Magnified views of the p53-expressing
keratinocytes reveal a shift in the localisation patterns of p53 within the cell nuclei.
At 6 hours post-irradiation, p53 was detected in a speckled manner throughout the
nuclei, or in the perinuclear regions. However, at 24 hours, there was a shift towards
a more intense centrally-located expression of p53 within the nuclei. Non-irradiated
HSE controls (G and H) showed weak cytoplasmic p53 presence, as well as
translocation to the nucleus or intensification of the staining at 24 hours.
Figure 3.6. UVB induces p53 expression in irradiated HSEs
Sections of HSE exposed to 2 MED UVB were sampled at 0 hours (A), 3 hours (B), 6 hours (C) and 24 hours (D)
post-irradiation and analysed for the presence of p53 via immunohistochemical analysis. The presence of a brown
staining indicates the presence of p53, which is elevated in the nuclei of UVB-exposed skin equivalents as
compared to the non-irradiated controls at 0 hour (G) and 24 hours (H). A weak, diffuse cytoplasmic staining was
also observed, intensifying from the 3 hour time-point. Figures E and F are magnified views of the epidermis
showing alterations in the patterns of p53 distribution within the nucleus at 6 and 24 hours respectively. The
black scale bars represent 50 µm and the red scale bar represents 25 µm. Images from immunohistochemical
analyses are representative of experimental triplicates conducted on three independent skin donors.
63
3.3.7 Secretion of inflammatory cytokines by irradiated HSE-KC constructs
The sunburn reaction in skin as a consequence of contact with UVR rays is in
part mediated by an array of keratinocyte-produced cytokines, including IL-6, IL-8,
IL-10 and TNF-α (Kock et al., 1990; de Vos et al., 1994; Grewe et al., 1995).
Upregulation of IL-6 synthesis in response to UVB irradiation has been documented
and is believed to be a central catalyst for the activation of the local and systemic
immune response (Urbanski et al., 1990). Another proinflammatory factor, the
chemokine IL-8, is known to be constitutively expressed in the epidermis, with this
expression being appreciably increased upon UVB exposure (Kondo et al., 1993).
The levels of secreted IL-6 and IL-8 were quantified in the cell culture
supernatants of HSE-KC cultures in response to UVB irradiation. These cytokines
were measured using a sandwich ELISA technique and normalised to the total
protein detected in the samples of SFM collected. As shown in Figure 3.7, significant
upregulation in the production of both IL-6 and IL-8 was observed in cell culture
medium obtained from these irradiated HSE-KC constructs. At the T0 time-point, no
significant differences in IL-6/8 levels were detected between culture medium
samples from irradiated and non-irradiated HSE-KC cultures. Levels of IL-6 were
about 100-fold higher in irradiated HSE-KC at the 24 hour time point (90.3 ± 16.8
pg/mL) as compared to those collected at 0 hour (1.00 ± 0.3 pg/mL). Synthesis of IL-
8 induced by UVB showed an approximately 3-fold increase compared to the control
(66.1 ± 5.7 pg/mL versus 24.0 ± 1.0 pg/mL). The basal unstimulated levels of IL-8 in
the culture medium was elevated as compared to that of IL-6, with about seven times
higher IL-8 detected in the non-irradiated controls.
64
Figure 3.7. Quantification of IL-6 and IL-8 concentrations in HSE-KC culture supernatants
Cell culture medium samples were obtained from HSE-KC cultures prior to and 24 hours after irradiation with 2 MED of UVB. Supernatants were analysed for the presence of interleukin-6 (IL-
6) and IL-8 using an ELISA. Data represents the average quantities of IL-6 and IL-8 detected at the two time points (mean ± SEM), in experimental triplicates from two independent skin donors,
expressed in pg/mL. All measurements were normalised to total protein levels in culture supernatants. Asterisks represent statistical signifance (p<0.05) in the difference from matched samples
at t=0 hr. The error bars are representative of the standard error of the mean (SEM).
IL-6 production in HSE-KC
Time post-irradiation
0 hr
24 h
r
0
50
100
150
IL-6
co
ncen
trati
on
(p
g/m
L)
IL-8 production in HSE-KC
Time post-irradiation
IL-8
co
ncen
trati
on
(p
g/m
L)
0 hr
24 h
r
0
50
100
1500 MED
2 MED*
65 Validation of the HSE-KC photobiology model
3.3.8 Removal of CPDs in HSE-KC post-irradiation
Exposure of HSE-KC composites to UVB radiation results in the formation of
CPD photolesions in keratinocyte nuclei, as described in 3.3.4. Due to the frequency
of their exposure to environmental UVR, keratinocytes posses highly sophisticated
repair mechanisms to cope with the formation of UVB-induced damage (D’Errico et
al., 2007). The efficient removal of these CPDs has been strongly associated with the
prevention of oncogenic transformation in irradiated keratinocytes (Marionnet et al.,
1998; Katiyar et al., 2000; Zhao et al., 2002). In view of this, immunohistochemistry
was performed to determine whether UVB-induced CPDs in HSE-KC keratinocyte
nuclei post-irradiation were removed over time. As shown previously, control non-
irradiated HSE-KC were negative for the presence of CPDs (Figure 3.8 A to C). At
Day 0, CPD-positive keratinocyte nuclei were shown to be present in cells of the
stratum basale and suprabasal layers (Figure 3.8D). From 24 hours post-irradiation
there was a gradual decrease in the number of keratinocytes bearing photolesions,
and the intensity of immunostaining, with very few CPD-positive cells observed after
Day 4 (Figure 3.8 E to I). Overall, immunohistochemical analysis showed that
keratinocytes in the irradiated HSE-KC were able to repair CPD photolesions in
approximately three to four days after a single dose of 2 MED UVB.
66 Validation of the HSE-KC photobiology model
Figure 3.8. Detection of CPDs in irradiated HSE-KC over time
The presence of UVB-induced CPD in the nuclei of HSE-KC irradiated with 2 MED UVB (D to I) was detected using immunohistochemical analysis. Images A to C show non-irradiated
controls at the 0, 1 and 2 day time-points, showing no detectable CPDs. Images D to I indicate a clear reduction in the numbers of CPD-positive keratinocytes from Days 0 to Day 5 post-
irradiation. Images from immunohistochemical analyses are representative of experimental triplicates conducted on three independent skin donors.
67 Validation of the HSE-KC photobiology model
The numbers of CPD-positive cells were quantified using image analysis of
immunohistochemically-stained sections, as described previously (see section 2.2.6).
An average number of stained cells in five fields of view from three independent
experiments were counted and are represented in Figure 3.9. This graph shows the
gradual decrease in the numbers of HSE-KC keratinocytes harbouring photolesions
over a period of five days post-irradiation. The largest decrease in the number of
observed CPD-positive cells occurred between 24 and 48 hours after UVB exposure,
with an average decrease of about 52.7 ± 7.3% during this time period. There was a
statistically-significant drop in the numbers of CPD-positive cells as compared to
Day 0 at Days 2, 3, 4 and 5.
Figure 3.9. Repair of CPDs in irradiated HSE-KC
The average numbers of CPDs present in the nuclei of irradiated HSE-KC were quantified post-irradiation. The
graph indicates a decrease in the numbers of CPD-positive cells over time. The greatest number of CPDs repaired
occurred between 24 and 48 hours post-exposure. The asterisks represent statistically-significant (p<0.05)
differences compared to the number of CPD-positive cells at Day 0. Results are representative of the average of
five fields of view in three independent experiments with three independent keratinocyte donors. The error bars
are representative of the SEM.
68 Validation of the HSE-KC photobiology model
3.3.9 Expression of Ki-67 in irradiated HSE-KC
At the same time that UVB induces cell death and cycle arrest, the opposing
effects of survival and the stimulation of proliferation also takes place in UV-
exposed skin. Epidermal hyperplasia triggered by UVB is thought to be a
consequence of the activation of various growth factor receptors and the actions of
cytokines (Rosette and Karin, 1996; Peus et al., 2000). These include fibroblast-
produced factors, such as EGF, IGF-I and IL-1, which are thought to contribute to
orchestrating the death-survival balance in irradiated keratinocytes (Rosette and
Karin, 1996).
The proliferative capacity of irradiated keratinocytes was investigated in the
epidermis of the HSE-KC. In this study, the Ki-67 nuclear proliferation marker was
used to quantify the proportion of keratinocytes within this epidermal layer actively
undergoing cell division in sections of HSE-KC tissue. The Ki-67 antigen is strongly
associated with actively proliferating cells, and has been shown to be present within
the cell’s nucleus during all active phases of the cell cycle and mitosis (Heenen et al.,
1998; Scholzen and Gerdes, 2000; Burcombe et al., 2006).
Keratinocytes expressing Ki-67 were mainly localised to the stratum basale in
immunostained sections of the HSE-KC (Figure 3.10). Non-irradiated HSE-KCs
exhibited a high proportion of Ki-67-positive keratinocytes over Days 0 to 2 during
the five days at which samples were obtained (Figure 3.10 A to C). Under normal
culture conditions the number of actively proliferating keratinocytes was observed to
diminish after this time (Figure 3.10 D to F). The HSE-KC composites exposed to a
single dose of 2 MED UVB showed a relatively decreased number of Ki-67-
expressing basal keratinocytes in the initial two days post-irradiation (Figure 3.10 G
to I). After this time point, a greater proportion of basal keratinocytes was observed
to express Ki-67 as compared to the non-irradiated controls (Figure 3.10 J to L).
The number of Ki-67-expressing keratinocytes was then quantified using
image analysis software (Figure 3.11). From the immunohistochemically-stained
sections shown previously, the average number of proliferating cells per field was
counted over a five day period following UVB exposure.
69 Validation of the HSE-KC photobiology model
Figure 3.10. Detection of Ki-67 in irradiated HSE-KC
The proliferation marker, Ki-67, was detected in non-irradiated controls (A to F) and 2 MED UVB irradiated HSE-KC (G to L). Images are representative of sections obtained sequentially every
24 hours from Day 0 to Day 5 after irradiation. Brown stained keratinocyte nuclei indicate the presence of the Ki-67 protein, expressed during active phases of the cell cycle. Images from
immunohistochemical analyses are representative of experimental triplicates conducted on three independent skin donors. The scale bar represents 50 µm.
70 Validation of the HSE-KC photobiology model
Non-irradiated HSE-KC show a gradual increase in the numbers of
proliferating keratinocytes from Day 0 to Day 2, followed by a decrease to below 20
Ki-67-positive cells per field. In comparison, irradiated HSE-KC exposed to 2 MED
UVB demonstrated a decreased number of proliferating cells in the days immediately
following irradiation. At Day 2, the average number of proliferating cells in these
HSE-KC composites was significantly lower than that observed in the non-irradiated
HSE-KC controls. Subsequently, there was a gradual increase in the proportion of
cells in the active phases of the cell cycle. This returned to levels similar to those
seen in non-irradiated control HSE-KCs by Day 5. Hence, this data indicates the
irradiation of HSE-KC with UVB induces changes in the numbers of proliferating
keratinocytes. This may be linked to the onset of CPD repair during this period as
described in the previous section.
Figure 3.11. Quantification of Ki-67 expression in irradiated and non-irradiated HSE-KC
The numbers of keratinocytes with positive immunostaining for the presence of Ki-67 were counted in irradiated
and non-irradiated HSE-KC. At Day 2, there was a significant decrease in the number of Ki-67-positive
keratinocytes in HSE-KC composites exposed to a single 2 MED dose at Day 0. Data represents an average of
five fields counted in experimental triplicates from three independent experiments. Error bars represent SEM
values. Asterisks represent statistically significant differences (p<0.05) as compared to the non-irradiated controls
at the same time point.
Days post-irradiation
Av
era
ge
Ki-
67
+ c
ells
/fie
ld
0 1 2 3 4 5
0
20
40
600 MED
2 MED
*
* *
*
71 Validation of the HSE-KC photobiology model
3.3.10 Keratinocyte differentiation
The expression of keratin 1/10/11 (a marker of differentiated keratinocytes)
was analysed using immunohistochemistry (Figure 3.12). The HSE-KC composites
were exposed to either a single 2 MED or 0 MED control UVB dose, and were
sacrificed every day for five days. Using a protocol described in 3.2.6, tissue sections
were probed for the presence of keratin 1/10/11 to monitor potential changes in the
differentiated keratinocyte layers within the epidermis. The areas of these regions
were quantified using image analysis software, with the mean immunoreactive areas
expressed in Figure 3.13.
Analysis of the differentiating keratinocyte layer in the HSE-KC epidermis
revealed that UVB significantly altered the thickness of this region following
irradiation. In the first three days during this post-irradiation period, there was a
steady decline in the average area of the spinous and granular layers of the epidermis.
From the immunohistochemical analysis (Figure 3.12 G to L), this was associated
with a thickening of the stratum corneum layer. The thickness of the stratum basale,
however, remained relatively unchanged during this period. Hence, these results
indicate that UVB radiation induced the differentiation of this keratinocyte layer into
corneocytes which constitute the stratum corneum. Physiologically, it has been
demonstrated that a single moderate dose of UVB can induce up to a three-fold
thickening of the stratum corneum (Diffey, 1991). Together with the tanning
response, the increased thickness of this outermost layer of the epidermis aids in
shielding the basal keratinocytes from the harmful effects of UVR (Elias, 2005).
72 Validation of the HSE-KC photobiology model
Figure 3.12. Time-course analysis of keratin 1/10/11 expression in irradiated and control HSE-KC
Immunohistochemical staining of keratin 1/10/11 in non-irradiated sections of HSE-KC at Day 0 (A), and 2 MED irradiated tissue at Day 0 (B). Figures C to D and H to L show keratin 1/10/11
positive keratinocytes detected from Day 1 to Day 5 in non-irradiated and irradiated HSEs respectively. Scale bar represents 50 µm. Images from immunohistochemical analyses are
representative of experimental triplicates conducted on three independent skin donors.
73 Validation of the HSE-KC photobiology model
Figure 3.13. Quantification of keratin 1/10/11 expression in HSE-KC over time
The area of keratin 1/10/11 expression within the epidermis from immunohistochemical analysis was quantified
using image analysis software (ImageJ). The mean area of keratin 1/10/11 positive staining was expressed in µm2
from five microscopy fields in three independent experiments. Error bars represent SEM values. Asterisks
represent statistically significant differences (p<0.05) as compared to the non-irradiated controls at the same time
point. Experiments were conducted on samples from three independent skin tissue donors.
Days post-irradiation
Are
a o
f K
1/1
0/1
1 im
munore
activ
ity (m
2)
0 1 2 3 4 5
0
10000
20000
30000
40000
500000 MED
2 MED
*
*
*
*
Chapter 3: Validation of the HSE-KC photobiology model 75
3.4 DISCUSSION
The creation of improved in vitro photobiology models involves both aligning
physiological and biological parallels with human skin in vivo, as well as combining
the ability to induce classical photodamage markers in the model. Therefore, the first
aim of this study was to compare the HSE-KC with ex vivo skin to determine the
expression of key epidermal markers known to influence the UVB response. The
HSE-KC organotypic model has previously been characterised by our research group
in relation to the study of a diverse range of epithelial processes, primarily those
associated with wound-healing (Topping, 2006; Xie et al., 2010). Hence, the aim of
this study was to further validate the HSE-KC as a photobiology model to address
some of the current limitations of conventional cell-based UV biology models.
The generation of various types of tissue-engineered skin constructs capable of
modelling the morphology and organisation of the human epidermis, has previously
been documented (Prunieras et al., 1983; Ponec et al., 1988b; Bernerd et al., 2012).
In accordance with such previously reported work, morphological features including
the rete ridges and the epidermal organisation were found to be similar in both the
HSE-KC and native skin sections (Figure 3.1). To confirm the presence of
keratinocytes at various stages of differentiation in the HSE-KC epidermis, the
expression of keratins 1/10/11 and cytokeratin 2e was analysed using
immunohistochemistry (Figure 3.1A and B). These two keratin sub-types are known
to be upregulated in differentiating keratinocyte populations (in the high and
intermediate suprabasal layers) and in the granular layer respectively (Paladini et al.,
1996; Lane and McLean, 2004; Luo et al., 2011). In the immunohistochemical
analysis performed in this study, it was found that both these keratins showed similar
expression patterns in the HSE-KC epidermis as compared to that observed in the
sections of native skin tissue.
Next, the expression of keratin 14 in these tissues was visualised using
antibody-based labelling. This keratin is known to predominantly constitute the
cytoskeleton of basal keratinocytes (Torma, 2011). In the native skin samples, there
was a more pronounced gradient of keratin 14 expression, decreasing in intensity
from the basal to suprabasal keratinocytes (Figure 3.1C). However, in the HSE this
keratin appeared to be more uniformly expressed in the basal and transient
76 Chapter 3: Validation of the HSE-KC photobiology model
amplifying cells. Indeed, this differential pattern of keratin 14 expression has also
been observed between other organotypic skin cultures and ex vivo skin (Smola et
al., 1993; Hinterhuber et al., 2002). Promisingly, however, the expressions of keratin
14, along with the cytokeratin 2e and keratin 1/10/11 differentiation markers showed
strong parallels between both the reconstituted and ex vivo tissues, thus confirming
the formation of a multilayered epithelium with a polarised differentiation gradient in
the HSE-KC.
The stratum corneum functions to scatter and absorb penetrating UVR to limit
the amount of damaging radiation reaching the proliferating basal keratinocytes
(Anderson and Parrish, 1981). Expressed in terminally differentiated layers of the
epidermis, filaggrin is thought to directly aid in maintaining this barrier function of
skin (Kanitakis et al., 1988; Sandilands et al., 2009; Mildner et al., 2010).
Immunohistochemical analysis of filaggrin expression shows similar patterns of
distribution in the HSE-KC as compared to that of native skin (Figure 3.1D). In the
HSE-KC, however, there appeared to be a decreased intensity of filaggrin staining in
the stratum corneum. This is in line with previous experimental data showing slightly
diminished filaggrin expression in various organotypic skin models (Hinterhuber et
al., 2002; Ponec et al., 2002). Nonetheless, the expression of filaggrin as part of a
functional stratum corneum barrier is significant in the validation of the HSE-KC as
an improved model of photobiology as compared to 2D cultures that lack this
feature. Indeed, it has been shown that irradiated tissue-engineered models lacking
filaggrin have elevated levels of DNA damage and apoptosis observed in the stratum
basale (Mildner et al., 2010). Hence, in the study of UVB-induced changes to the
proliferative cells of the epidermis, a multi-layered epidermis is a critical aspect to
include in the experimental model of choice.
The culture of the HSE-DED constructs at the air-liquid interface has been
shown to promote the differentiation of keratinocytes during the formation of the
stratified epidermal layer (Ponec, 1991). All the same, aspects of the in vitro culture
conditions required for HSE-DED culture deviate from those of the epidermis in
vivo. For example, the constant sloughing of the outermost layer of the skin due to
contact friction is absent in these skin equivalents, which has been shown to result in
hyperplasia of the epidermal component in HSE-DED constructs (Ponec, 2002).
Furthermore, the temperature at the surface of the skin is generally lower than the
Chapter 3: Validation of the HSE-KC photobiology model 77
internal body temperature. Indeed, skin cultures maintained at 37˚C have been shown
to have altered expressions of certain epidermal markers (Gibbs et al., 1997).
Collectively, these factors may contribute to the minor differences in the expression
of certain epidermal markers between the HSE-KC and native skin samples.
Collagen IV, along with other ECM factors (including laminin and collagen
VII) make up the BM region that separates the epidermis and dermis. The expression
of collagen IV has been detected in a variety of in vitro organotypic skin cultures and
is thought to be synthesized by both epithelial and mesenchymal skin cells (Bell et
al., 1979; Asselineau et al., 1985; Thomas and Dziadek, 1993). The localisation of
collagen IV in the HSE-KC in this study was observed at the epidermal-dermal
junction, akin to expression patterns in native skin sections (Figure 3.1E). This
outcome was significant for two major reasons. Firstly, BM components have been
found to profoundly influence keratinocyte responses to external stimuli such as
UVB (Stenback and Wasenius, 1985; Amano, 2009). Secondly, this finding validates
the use of the HSE-KC model in subsequent experiments investigating paracrine
interactions across the epidermal-dermal junction, as this model preserves the native
architecture and biology of skin in this respect.
An issue associated with several previous studies on keratinocyte behaviour
post-UV exposure has been the use of non-physiological doses of UVR to induce
photodamage in skin cells (Bender et al., 1997; Leverkus et al., 1997). To address
this, we aimed to develop an irradiation protocol using biologically-relevant doses of
UVB. In this study, only the UVB wavelength was used as a radiation source as this
particular wavelength has been shown to be the main source of UVR-induced
damage in epidermal cells (Muramatsu et al., 1992; Bernerd and Asselineau, 2008).
The quantitative measurement of 1 MED, or the amount of radiation required to
produce mild erythema 24 hours post-irradiation has been found to be about 25
mJ/cm2 on average in white-skinned individuals (Diffey, 1992; Tadokoro, 2003). In
Queensland, studies have shown facial solar UVR exposure can range from 2 to 11
MED (Kimlin et al., 1998; Parisi et al., 2000).
The histology of UVB-irradiated HSE-KC composites shows increasing levels
of epidermal damage as larger doses of UVB were applied (Figure 3.2). The skin
equivalents exposed to doses in excess of 3 MED of UVB showed widespread
damage to keratinocytes, and displayed characteristics of tissue necrosis (Wyllie,
78 Chapter 3: Validation of the HSE-KC photobiology model
1980). This observation can be attributed to a number of factors. Firstly, the HSE-KC
does not contain melanocytes incorporated into the constructs, which are a
fundamental cell type in protecting keratinocytes from the damaging effects of UVB
(Bertaux et al., 1988; Liu, 2007; Santiago-Walker, 2009). Indeed, pigmented skin
equivalents with incorporated melanocytes have been shown to withstand UV doses
in excess of 5 MED without extensive necrotic damage to the epidermis (Todd et al.,
1993; Nakazawa et al., 1997). This can be partly attributed to the shielding effect of
melanin, which is thought to absorb 50 to 75% of penetrating UVR via the formation
of supranuclear caps in melanosomes (Brenner, 2008). In addition to this, the
regeneration of the functional epidermal barrier following photodamage is also
partially maintained by paracrine signals from mesenchymal fibroblasts
(Archambault et al., 1995; Jost et al., 2001; Mildner et al., 2007). Therefore, the
cellular toxicity displayed with higher doses of UVB in the HSE-KC may in part be
due to the absence of both melanin and the activation of dermal-derived protective
pathways.
Accordingly, the dose of UVB that was chosen for subsequent experiments was
2 MED (50 mJ/cm2), as it was within the physiological range, and was shown to
induce UVB damage while maintaining enough viable keratinocytes for subsequent
epidermal regeneration. The detection of cellular viability (via MTT staining) after
this UVB dose showed no significant changes in intensity in HSE-KC up to 5 days
post-irradiation (Figure 3.3). However, while the MTT assay has been frequently
used as a measure of skin viability, it should be used in conjunction with other
histological and immunohistochemical techniques to determine the status of
keratinocyte viability more accurately (Castagnoli et al., 2003). This has also been
demonstrated in other experimental systems showing that cell viability assays such as
the lactate dehydrogenase release assay more accurately reflect the presence of
apoptotic cells, as compared to the MTT assay (Lobner, 2000).
The classical markers of acute cutaneous photodamage have been well-
documented (Matsumura and Ananthaswamy, 2002; Matsumura and Ananthaswamy,
2004). The principal signs of UVB-induced damage include the formation of DNA
lesions, cell cycle arrest, cell death via apoptosis, inflammation and the tanning
response (Black et al., 1997). In view of this, irradiated HSE-KC constructs were
analysed for the presence of keratinocyte-specific damage markers (Section 3.3.4).
Chapter 3: Validation of the HSE-KC photobiology model 79
The formation of CPD lesions in the stratum basale and suprabasal keratinocytes
were detected 24 hours after exposure in HSE-KC exposed to UVB (Figure 3.4). The
CPDs were localised within the nuclei of these cells and did not appear to be present
in non-irradiated controls. Both the distribution and the localisation of CPD
formation post-irradiation were consistent with results from similar studies
conducted in vivo (Chadwick et al., 1995; Katiyar et al., 2000). Skin sections from
volunteers exposed to 2 MED of UVR were reported to show similar formations of
these thymine dimers in keratinocyte DNA, with approximately 60% of CPD-
positive cells present in the epidermis at 48 hours after exposure. The results
documented herein were also comparable to those obtained from in vitro studies
using UVB-irradiated HSE constructs, in which UVB radiation-induced CPDs were
observed in the epidermal regions of irradiated skin composites (Marionnet et al.,
2010; Bernerd et al., 2012).
The process of apoptosis is a protective mechanism in skin to prevent the
proliferation of severely DNA-damaged cells following UV-exposure (D'Errico et
al., 2003). The majority of apoptotic basal and suprabasal keratinocytes appeared 24
hours after a single dose of 2 MED UVB in the HSE-KC, as detected using the
TUNEL assay (Figure 3.5). This technique to detect UVB radiation-induced
apoptosis in keratinocytes from fixed skin tissue has been widely-used in the study of
photodamage (Wrone-Smith et al., 1997; Yamaguchi et al., 2006). Interestingly, it
was observed that more pigmented skin showed fewer TUNEL-positive cells present
in the stratum basale following irradiation, as compared to skin from less pigmented
individuals (Yamaguchi et al., 2006). This suggests that the relatively elevated levels
of apoptotic keratinocytes present in the basal layer of irradiated HSE-KCs may be in
part due to an absence of melanocytes in these skin equivalents.
The activation of both DNA damage-induced apoptosis and repair mechanisms
(via cell cycle arrest) are highly-dependent on the function of p53 (Ananthaswamy
and Kanjilal, 1996). These p53-associated repair mechanisms are central in
counteracting UVB-induced damage (Cotton and Spandau, 1997; Will, 2000; van
Laethem et al., 2009). Accordingly, the activation and upregulation of p53
expression has been observed in a variety of experimental systems within the initial
24 hours post-irradiation, with peak levels of detection apparent after 6 hours (Tron
et al., 1998a; Tron et al., 1998b; D'Errico et al., 2005). In the study reported herein,
80 Chapter 3: Validation of the HSE-KC photobiology model
sections of irradiated HSE-KCs were probed for the presence of p53, and also
showed the accumulation of p53 within keratinocyte nuclei within 24 hours of UVB
exposure (Figure 3.6). The majority of p53-positive keratinocytes were basal and
suprabasal cells, in line with observations from previous studies (Lu et al., 1999).
Moreover, localisation patterns of nuclear p53 in the HSE-KC appeared to shift from
being predominantly perinuclear to being more centrally-located within keratinocyte
nuclei. This phenomenon has been previously reported in experiments conducted on
irradiated keratinocyte monolayers, showing clear evidence of p53 translocation into
the nucleus following high doses of UVB (Cotton and Spandau, 1997). It is
important to note that the p53 response in irradiated keratinocytes is highly sensitive
to a variety of factors including cellular adhesion and differentiation status (Kumar et
al., 1999). Additionally, major differences in the expression patterns of p53 and its
target genes have been identified between irradiated cell monolayers and in vivo skin
cells (Enk et al., 2006). Therefore, physiologically-similar p53 expression patterns
detected in the irradiated HSE-KC suggest that this model may be a more
representative model of the acute UV response.
The secretion of an array of cytokines by keratinocytes during the acute UV-
response has been well-documented in both in vivo and various in vitro techniques
(Enk et al., 1995; Strickland et al., 1997; Brink et al., 2000b; Saade et al., 2000;
Bechetoille et al., 2007; Liebel et al., 2012). These inflammatory and anti-
inflammatory cytokines include IL-1, IL-6, IL-8, IL-10, IL-12 and tumour necrosis
factor-α (Kock et al., 1990; Grewe et al., 1995; Enk et al., 1996; Petit-Frere et al.,
1998). This response is closely-linked to the erythemal effects observed post-
exposure and is thought to be triggered by DNA damage (Petit-Frere et al., 1998).
Cell culture supernatants from irradiated HSE-KC cultures were found to have
significantly elevated levels of IL-6 at 24 hours post-exposure (Figure 3.7). It was
also found that IL-8 secreted by HSE-KC composites had increased in response to
UVB radiation. In the case of IL-8, relatively higher baseline levels may be attributed
to the fact that keratinocytes have been found to constitutively produce IL-8 (Kondo
et al., 1993). Notably also, the upregulation of IL-8 production in irradiated
keratinocytes has been demonstrated to occur as early as one hour after exposure
(Kondo et al., 1993). This may then account for the lack of a significant increase in
IL-8 levels compared to baseline observed after 24 hours. The secretion of these
Chapter 3: Validation of the HSE-KC photobiology model 81
keratinocyte-secreted soluble mediators of inflammation have been previously
detected in both HSE and monolayer culture supernatants as a result of UVB
exposure (Grandjean-Laquerriere et al., 2003; Bechetoille et al., 2007). This
represents another central marker of the acute UVB response that was successfully
detected in the HSE-KC in vitro model.
Due to their frequent exposure to damaging radiation, keratinocytes are
equipped with sophisticated and efficient DNA repair strategies. During the post-
irradiation repair phase in skin, UVB radiation-induced photolesions, such as CPDs,
are repaired via the nuclear excision repair system (Setlow, 1966; Nijhof et al.,
2007). These processes have been well-documented in several keratinocyte culture
systems and together with cell cycle checkpoint mechanisms, function to guard the
integrity of the cell’s genetic information (Liu et al., 1983; Zhou and Elledge, 2000;
D'Errico et al., 2003). The rate of DNA damage repair is highly dependent on the
type of photolesion, the cell type, as well as the efficiency of the repair mechanism
(Svobodova and Vostalova, 2010). Lesions such as 6-4PP have been observed to be
fully repaired within 6 hours, whereas the more abundant CPDs can take up to 24
hours for the activation of repair systems (Costa, 2003).
The next aim in the validation of the HSE-KC photobiology model was to
characterise repair and regeneration responses following UVB exposure. It was
previously shown that the UVB irradiation protocol was able to illicit the formation
of CPDs in keratinocyte nuclei in the HSE-KC. Therefore, the removal of these DNA
damage markers was monitored at time-points subsequent to irradiation as an
indication of photolesion repair. Irradiated HSE-KCs sacrificed every day for five
days after UVB treatment displayed a gradual decrease in the presence of nuclear
CPDs, as shown via immunohistochemical analysis (Figure 3.8). The majority of the
CPDs occurring in basal and suprabasal keratinocyte nuclei were observed to be
eliminated between 24 and 48 hours post-irradiation (Figure 3.9). At Day 2 after
irradiation, approximately 50% of the CPD photoproducts were observed to have
been repaired in the HSE-KC. Similar experiments using irradiated keratinocyte
monolayers revealed that an average of only 20% of the initial numbers of CPD-
positive cells remained 24 hours after UVB exposure (D'Errico et al., 2003).
Prior studies have shown significant differences in the repair rates of CPDs
between native skin and cultured keratinocyte models (Mouret et al., 2008). These
82 Chapter 3: Validation of the HSE-KC photobiology model
comparative studies showed higher rates of CPD removal in monolayer cultures of
keratinocytes, as compared to that observed in irradiated skin in vivo. Results from
the analysis of CPD removal in the HSE-KC, however, showed more parallels with
the observed rate of CPD repair in native skin, with experimental data demonstrating
54.3% of CPD-positive cells remain present at 48 hours post-irradiation (Mouret et
al., 2008). This observation may be attributed to differences in the capacity for CPD
repair between different keratinocyte subpopulations in the epidermis. Proliferative
cells in the stratum basale are known to have relatively higher efficiencies of DNA
repair as opposed to differentiating keratinocytes in the suprabasal layers (Liu et al.,
1983). Therefore, it is to be expected that monolayer cultures (with a larger
population of proliferative keratinocytes) would be selective for cells with enhanced
DNA repair capacities.
While immunohistochemical staining and subsequent image analysis is
commonly used for detecting the presence of photolesions over time, more sensitive
techniques allow for an increased accuracy in the quantification of CPDs. For
example, techniques such as high performance liquid chromatography methods
associated with tandem mass spectrometry (HPLC-MS/MS) have been used to more
accurately quantify photoproducts in irradiated human skin (Bykov et al., 1999; Zhao
et al., 2002). However, these more sensitive methods reveal similar rates of
photolesion repair as found in whole skin models (Bykov et al., 1999; Mouret et al.,
2006).
An important aspect of the acute UVB response is the regulation of the cell
cycle to ensure that damaged DNA is repaired before being replicated. Specifically,
G1 cell cycle arrest ensures that photolesions are successfully removed from DNA
before the cell enters the S and M phase (Morgan and Kastan, 1997). The Ki-67
proliferation marker is a nuclear antigen shown to be expressed in the active phases
of the cell cycle (Heenen et al., 1998; Scholzen and Gerdes, 2000). Previous
investigations into epidermal proliferation patterns in native skin reveal a substantial
increase in the number of Ki-67-expressing basal cells at 48 hours post-UVB
irradiation (Lee et al., 2002). The study reported herein has shown that the number of
Ki-67-positive cells in the HSE-KC show a similar proliferative burst occurring
around three to four days after UVB-irradiation which subsequently returned to
baseline values (Figure 3.11). At Day 2 after irradiation, there were significantly
Chapter 3: Validation of the HSE-KC photobiology model 83
fewer proliferating cells in the UVB-exposed HSE-KC compared to matched non-
irradiated controls. Taken together with observations of CPD repair in the irradiated
HSE-KC, it is likely that this decrease in the number of actively cycling
keratinocytes is associated with repair processes in these cells. Also, the presence of
paracrine signals from other skin cell populations in vivo may be responsible for the
relatively accelerated post-irradiation proliferative phase as compared to the HSE-
KC (Werner and Smola, 2001).
Apart from the regulation of cell cycle progression, the processes of apoptosis,
cellular senescence and terminal differentiation are also critical in suppressing the
proliferation of UVB-damaged cells (Gandarillas, 2000). After prolonged and
repeated UVB exposures, an increase in keratinocyte terminal differentiation not
only prevents the duplication of DNA-damaged cells, but also results in the
thickening of the stratum corneum to prevent further photodamage (Gambichler et
al., 2005). However, the mediators of such responses and the mechanisms behind the
regulation of keratinocyte terminal differentiation during the UV response have yet
to be fully defined.
Keratins 1 and 10 are early markers of terminal differentiation and are
constitutively expressed in the suprabasal keratinocyte layers (Woodcock-Mitchell et
al., 1982). Hence, an antibody against keratin 1/10/11 was used to probe tissue
sections of irradiated HSE-KC and revealed changes in the proportions of
differentiated keratinocyte populations in the epidermis in the days following UVB
exposure (Figure 3.12). A diminished expression of these keratins was observed
during the 48 hours following irradiation, which then appeared to increase in
intensity from Day 3. Also, a thickening of the stratum corneum was observed during
this period, in which this layer readily detached from the HSE-KC composites, in a
process resembling blistering.
Previous in vivo and in vitro studies show a similar progressive decrease in
keratin 10 expression around 48 to 72 hours post-irradiation, which was subsequently
strongly expressed in suprabasal keratinocytes (Rosario et al., 1979; Bernerd and
Asselineau, 2008). In experiments using irradiated HSE models, it was observed that
keratin 10 expression appeared to normalise to baseline levels at about 14 days after
UVB-exposure (Bernerd, 1997). Interestingly, in vivo testing using small animal
models also showed decreased keratin 1 expression in the epidermis subsequent to
84 Chapter 3: Validation of the HSE-KC photobiology model
UVB exposure (Horio et al., 1993; Kambayashi et al., 2001). Therefore, UVB is
likely to cause alterations to early markers of keratinocyte differentiation, such as
keratins 1, 10 and 11 during the acute sunburn phase. The thickening and blistering
of the stratum corneum may be associated with the upregulation of other epidermal
proteins, such as cytokeratin 6, filaggrin, loricrin and involucrin (Lee et al., 2002).
Indeed, an increase in late stage differentiation markers such as filaggrin and loricrin
has been previously observed in irradiated skin, indicating keratinocyte
differentiation may be accelerated and promoted as a photoadaptive response (Bosca
et al., 1988; Kanitakis et al., 1988; Mehrel et al., 1990). Thus, changes to other
markers of epidermal differentiation can be further investigated using the HSE-KC
model in order to more fully characterise these processes as a result of UVB
exposure.
The HSE-KC model holds potential as a versatile and powerful photobiology
tool due to the retention of several physiological cutaneous properties. Moreover, the
HSE-KC represents a basic epidermal model into which additional cell types can be
incorporated resulting in the creation of an improved biologically-relevant skin
model. In the next Chapter, the addition of fibroblasts into the DED component of
the HSE-KC is described, which allows for the investigation of paracrine interactions
between the dermal and epidermal cells during the UVB response. It is important to
bear in mind that despite the HSE-DED model being flexible in terms of the origin
and type of primary cells that may be incorporated, this may also contribute to inter-
donor variability, a phenomenon that has been reported in a variety of skin studies
(Basketter et al., 1996; Judge et al., 1996; Hiroshima et al., 2011). Indeed, studies
have shown variability to exist, particularly in keratinocyte UVB-responses between
individuals (Sesto et al., 2002). We attempted to limit these effects in our study by
acquiring donor skin from female donors and from non-sun exposed anatomical
regions. The use of an ex vivo dermal component, however, can also contribute to
variability between HSE-DED constructs due to, for example, regional differences in
the health of the tissue and the composition of the BM. In the future, the
development of increasingly elegant synthetic dermal substitutes may provide
improved dermal scaffolds which can reduce variability while preserving the
physiological properties of the dermis more faithfully (Liu et al., 2004; Valenta,
2004; Supp and Boyce, 2005; Mazlyzam, 2007).
Chapter 3: Validation of the HSE-KC photobiology model 85
3.5 CONCLUSION
In summary, the HSE-KC in vitro skin model has been successfully validated
as a tool for photobiology in terms of the ability to experimentally induce
characteristic UVB damage markers, as well as its capacity for post-irradiation repair
and regeneration (Figure 3.14). Additionally, comparative studies on the HSE-KC
and native skin show similar expression in terms of various epidermal markers. In
optimising the irradiation protocol, a physiologically-relevant dose of UVB radiation
was determined. Therefore, the baseline measures obtained in the analysis of
irradiated HSE-KC composites formed the basis for the next aim which was to
investigate the effect of paracrine interactions with fibroblasts on these responses.
Chapter 3: Validation of the HSE-KC photobiology model 86
Figure 3.14. Validation of the HSE-KC as a photobiology model
Diagrammatic representation of the results obtained from the HSE-KC validation study reported in Chapter 3.
Histological and immunohistochemical analysis of the HSE-KC revealed similarities in terms of the structure and
composition of the epidermal component in comparison to the native epidermis. These include the presence of a
stratified and organised epidermal layer, positively expressing native epidermal markers, such as keratin 1/10/11,
cytokeratin 2e, keratin 14 and filaggrin. Also, the dermal-epidermal junction showed the retention of
morphological properties like the rete ridges as well as components associated with the BM zone, such as
collagen IV. Exposure of these skin composites to UVB resulted in the formation of classical markers of UV
damage in the HSE-KC epidermis, including CPD photolesions, apoptotic keratinocytes, the expression of p53
and the secretion of IL-6 and IL-8. Additionally, markers of epidermal repair and regeneration such as changes to
keratinocyte proliferation, the removal of CPD and blistering through increased keratinocyte differentiation were
also observed.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 87
Chapter 4: Fibroblast-keratinocyte
interactions and the acute UVB
response
4.1 INTRODUCTION
While the aetiology and risk factors associated with skin cancer development
have been extensively explored, knowledge gaps still exist on the understanding of
pathogenesis at the cellular and molecular levels. An important factor influencing the
progress of photocarcinogenesis research is the urgent need for more robust in vitro
experimental approaches (as reviewed in Section 1.6). To address this, we have
successfully demonstrated that a primary cell-based HSE-KC model is applicable in
photobiology research to study keratinocyte changes following UVB irradiation. This
model represents a physiologically-relevant platform to study these responses to
UVB, by preserving several anatomical and functional aspects of native skin, as is
commonly lacking in many previously-employed photobiology models. The
complexity of the human epidermis, in terms of its barrier function against harmful
environmental agents, is reflected in both its intricate structure and the inherent
network of intercellular interactions which, in combination, aid in its protection and
regeneration from UVR-damage. While the shielding effect on keratinocytes from
UVR is known to be primarily conferred by melanin-producing melanocytes, it is
less well-understood how signals from mesenchymal cell types may contribute to
preventing and repairing photodamage. Hence, the aim of the study described herein
was to characterise the novel application of 2D and 3D cell-based in vitro
technologies to study fibroblast-keratinocyte interactions during the acute UVB
response in primary human skin cells.
Fibroblasts are known to be central in the regulation of many epithelial
processes, for example, proliferation, differentiation and keratinocyte migration, as
discussed in Section 1.4 (Aaronson et al., 1990; Wenczak et al., 1992; Beer et al.,
2000; El-Ghalbzouri et al., 2002; Beyer et al., 2003). The dermis consists of a
heterogeneous population of fibroblasts, with the papillary fibroblasts (situated in the
88 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
more superficial layers) being primarily responsible for secreting the ECM
molecules, growth factors and cytokines associated with epidermal maintenance and
regeneration (Sorrell et al., 2007). Besides being critical during phases of wound-
healing, these fibroblast-keratinocyte interactions are also thought to play a vital role
in epidermal regeneration after UVB-induced damage (Kuhn et al., 1999; Hedley et
al., 2002; Bernerd, 2005). While the exact mediators and mechanisms involved in
these interactions have yet to be comprehensively defined, various growth factor
systems such as that of EGF, KGF and IGF have been shown to be among the
principal players (Mackenzie and Fusenig, 1983; Lewis and Spandau, 2008). These
and other fibroblast-secreted factors have been observed to promote cellular
proliferation and orchestrate anti-apoptotic and repair processes post-irradiation in
vitro (Grundmann et al., 2010; Lewis et al., 2010). However, further analysis of
these processes using more robust, biologically-relevant models are still required to
characterise the complex process of fibroblast-protection during the acute
keratinocyte response to UVB more accurately.
Previous studies aimed at uncovering the role of epidermal-mesenchymal
interactions in the cutaneous UVB response predominantly rely on the use of
conventional cell culture methods (Kuhn et al., 1999; Lewis and Spandau, 2008).
These traditional 2D cell-line and primary cell monolayers possess various
limitations associated with their use for such investigations. In particular, these
experimental models lack the capacity to adequately represent some of the more
complex interactions between the dermal and epidermal cell populations. As a result
of such drawbacks, the application of monolayer culture methods in uncovering the
intercellular processes involved in the acute keratinocyte UV-response has been
limited. Nonetheless, 2D cell cultures also possess significant advantages in terms of
their ease of use, shorter incubation periods required to attain confluent monolayer
cultures, reduced variability between sample replicates and enhanced versatility
associated with the size and number of experimental replicates.
Henceforth, in order to address both the utilities and constraints of
conventional cell cultures, we proposed the use of a Transwell® co-culture model,
constituting both primary human fibroblasts and keratinocytes grown in a two-tiered
cell culture system. Comprised of two physically separated cell populations in a
common growth medium and separated by a porous membrane, this co-culture
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 89
system has previously been successfully employed by our research group to study
cellular processes including growth factor-mediated cell migration (Hyde et al.,
2004; Simpson et al., 2010; Kashyap et al., 2011). While its application in
photobiological studies has been limited thus far, the Transwell® system is
increasingly being employed to map the relationships between distinct cell
populations in modulating the UV-response. For instance, this system has been taken
advantage of in modelling keratinocyte and adipocyte interactions subsequent to
UVR exposure (Kim et al., 2011). Importantly, this model encompasses the technical
ease-of-use, reproducibility and versatility of conventional 2D cell cultures, while
more closely mimicking in vivo interactions between epidermal and mesenchymal
cell populations. Therefore, the Transwell® model was adopted in this project as an
in vitro model to characterise the influence of primary fibroblasts on the responses of
keratinocytes to UVB radiation.
In the previous Chapter, the application of a 3D HSE-KC model of primary
keratinocytes cultured on an ex vivo DED scaffold was shown to demonstrate similar
UVB irradiation damage and regeneration effects as those commonly observed in
vivo. Accordingly, the use of this 3D tissue-engineered model was also employed in
the study reported herein as an additional platform for dissecting the mechanisms
behind epidermal-mesenchymal paracrine interactions during the photoresponse to
UVB. Fundamentally, the application of these 3D organotypic photobiology models
represents a novel experimental approach, more closely retaining various
physiological properties of native skin which are believed to be critical to the UVB
irradiation response (as discussed in Section 1.3). Indeed, several physical and
functional characteristics of skin in vivo, including the BM zone at the dermal-
epidermal junction, are also known to be instrumental in facilitating fibroblast-
keratinocyte interactions, and are also retained to some degree in the HSE-DED
(Ponec et al., 2000; Amano, 2009). Additionally, from a technical viewpoint, the
process of HSE-DED culture allows for the incorporation of various combinations of
cell types into the epidermal and dermal components. This flexibility allows for the
examination of alterations in keratinocyte responses with and without the presence of
fibroblasts in the DED component in this study.
Importantly, recent theories into a possible correlation between skin aging and
NMSC development suggest potential implications of the disruption of these dermal-
90 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
epidermal interactions during the early stages of photocarcinogenesis (Lewis et al.,
2008). Notably, the lack of keratinocyte activation by fibroblast-secreted factors in
aged and chronically sun-exposed skin has been linked to an inappropriate response
to UV-damage (Albert and Weinstock, 2003; Lewis et al., 2010). Moreover, the
activation of repair mechanisms following UVB exposure has been shown to be
heavily dependent on the activation status of the IGF-IR in keratinocytes (Kuhn et
al., 1999). Furthermore, analysis of dermal fibroblasts in elderly individuals show
signs of cellular senescence and an associated diminished functionality in these cells,
thus compromising the synthesis of growth factors important during the epidermal
UVB radiation photoresponse (Lewis et al., 2010; Bigot et al., 2012; Spandau et al.,
2012). Hence, altered fibroblast-keratinocyte interactions may be related to the
higher risk of photocarcinogenesis observed in these elderly individuals (Lewis et al.,
2008). Understanding the relationship between these epithelial-mesenchymal
networks following photodamage is therefore an essential first step in the
development of targeted strategies to prevent photocarcinogenesis in such susceptible
individuals.
A critical cellular safeguard against oncogenic transformation is the activation
of appropriate repair or cell death pathways upon DNA damage (Bernerd et al.,
2001a; Bernstein et al., 2002). Direct genetic photodamage by UVB exposure occurs
in part via the generation of pyrimidine dimers, which if left unrepaired, can result in
the accumulation of mutations in various proto-oncogenes and tumour suppressor
genes (Ananthaswamy and Kanjilal, 1996). Likewise, changes to the initiation of
apoptotic pathways that function to prevent the proliferation of DNA-damaged cells
may also be responsible for skin cancer initiation (Matsumura and Ananthaswamy,
2002). While keratinocyte survival is critical for maintaining a functional epidermal
barrier post-irradiation, the removal of severely damaged cells through programmed
cell death is also crucial to prevent the propagation of transformed cells in this highly
proliferative tissue. The factors which orchestrate this delicate balance between cell
survival and death following UVR exposure, however, have yet to be extensively
mapped. Therefore, the next aim of the study reported herein was to investigate
keratinocyte damage, repair and death pathways using the novel photobiology
models as described earlier. In particular, this study aims at defining some of the
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 91
mechanisms behind fibroblast-mediated regulation of these processes using novel in
vitro strategies.
Broadly, the aims of this Chapter are therefore to:
1. Investigate the impact of dermal fibroblast co-culture on UVB-induced
keratinocyte damage, viability and apoptosis using a Transwell® system;
2. Develop and characterise a 3D HSE-DED model incorporating human
dermal fibroblasts and keratinocytes (HSE-KCF), to be used as a novel
platform for identifying how fibroblasts can modulate keratinocyte
survival, repair and apoptosis following irradiation, and
3. Identify some key pathways and molecular mechanisms involved in
fibroblast-mediated modulation of the acute UVB response in the
epidermis.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 92
4.2 MATERIALS AND METHODS
All the methods and materials used in this Chapter are as previously described
in Chapter 2. Any variation to these procedures is outlined in the following sections.
4.2.1 Primary human cell isolation and culture
The procedures for the isolation and culture of primary human keratinocytes
and fibroblasts from ex vivo skin tissues are detailed in 2.2.2.
4.2.2 Transwell® fibroblast and keratinocyte co-cultures
The co-culture of primary keratinocytes and fibroblasts using the Transwell®
cell culture system is described in 2.2.4.
4.2.3 Analysis of cellular viability
The MTS assay was used to quantify cellular viability in irradiated
keratinocytes, as a function of metabolic activity, as described in 2.2.12. The MTS
assay was performed 24 hours after the administration of a UVB dose as detailed in
4.2.7.
4.2.4 TUNEL assay for the detection of apoptosis
The TUNEL assay was used to detect apoptotic cells in the irradiated
keratinocyte monolayers, as previously described in 2.2.11. This technique was also
used to visualise the presence of sunburn cells (apoptotic keratinocytes) in formalin-
fixed, paraffin-embedded tissue sections, as outlined in 2.2.11.
4.2.5 Detection and quantification of secreted cytokines in 2D and 3D cultures
The concentrations of IL-6 and IL-8 in irradiated Transwell®, HSE-KC and
HSE-KCF cultures were quantified using an ELISA, as described in 2.2.13.
4.2.6 HSE-KC construction and culture
The HSE-KC composites were constructed and maintained in culture using the
protocol outlined in 2.2.3.
4.2.7 Irradiation of keratinocyte monolayers on Transwell® inserts
Keratinocyte monolayers were irradiated with a single UVB dose (as outlined
in 2.2.5). In the dose optimisation experiments described in 4.3.1, keratinocyte
monolayers were exposed to the UVB-emitting bulb for 0, 162, 324, 486, 648 and
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 93
810 seconds to administer a single dose of 0, 1, 2, 3, 4 and 5 MED respectively. To
investigate the protective and reparative effects of fibroblast co-cultures on
keratinocytes, three different irradiation and culture protocols were utilised, as
depicted in Figure 4.1. Keratinocytes were either cultured in Transwell® inserts
without the presence of fibroblasts (Figure 4.1A), with fibroblast co-culture prior to
irradiation (B) or transferred to co-culture conditions after UVB exposure (C). All
experiments were conducted in SFM.
Figure 4.1. Irradiation of keratinocyte monolayers cultured in different co-culture conditions
Keratinocyte cultures in SFM without fibroblasts in the lower chamber were irradiated with UVB (A and C).
Under the first condition (A), irradiated keratinocyte monolayers were returned to cultures containing SFM only.
In the second condition (B), keratinocytes grown in co-culture conditions with fibroblasts were irradiated and
transferred to wells containing SFM only. In the final culture condition (C), fibroblast-free keratinocyte cultures
were irradiated and subsequently transferred to fibroblast co-culture conditions following UVB-exposure.
4.2.8 Optimisation of HSE-KCF culture protocol
The protocol for the construction of the HSE-KCF (consisting of keratinocytes
and fibroblasts on a DED scaffold) was firstly developed to identify the optimum cell
seeding conditions required for fibroblast incorporation into the HSE-KCF. The
primary human fibroblasts were isolated and cultured as detailed in 2.2.2. These
dermal fibroblasts at P2-5 were seeded on either the reticular or papillary surface of
the DED in stainless steel rings at various time points prior to the addition of cultured
keratinocytes. The optimal culture conditions (as detailed in 2.2.3) for the
construction of the HSE-KCF were determined via histological and
immunohistochemical assessment.
4.2.9 Irradiation of the HSE-DED composites
The HSE-KC and HSE-KCF composites were irradiated with specific doses of
UVB as per the irradiation protocol described in 2.2.5.
94 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
4.2.10 Histological analysis of tissues
All histology was performed using routine haematoxylin and eosin staining
protocols, previously outlined in 2.2.6.
4.2.11 Immunofluorescent analysis
Fixed native skin and HSE-DED tissues were processed as described in 2.2.6 to
obtain 5 µm thick tissue sections. For the immunofluorescent detection of vimentin,
these tissue sections were pre-treated with HIER in sodium citrate buffer (as
previously outlined in 2.2.7). The sections were then incubated with a polyclonal
rabbit anti-vimentin antibody to Arg28 in human vimentin (Cell Signaling
Technology) overnight at 4°C. The tissue was then treated with a fluorescently-
labelled goat anti-rabbit secondary antibody (Alexa Fluor™ 594, Invitrogen) for 60
minutes at room temperature. Cell nuclei were counterstained with DAPI as
previously described in 2.2.10.
The immunofluorescent labelling of cell monolayers cultured in Transwell
inserts was performed using the protocol detailed in 2.2.10. The presence of cleaved
caspase-3 was visualised in these keratinocyte monolayers using a polyclonal
antibody specific against the large fragment (17/19 kDa) of activated caspase-3
resulting from cleavage at Asp175 (Cell Signaling Technology). Cell monolayers
were incubated with 1:50 dilutions of the primary antibody overnight at 4°C. The cell
monolayers were also fluorescently-labelled with the DAPI nuclear marker as
described in 2.2.10. Immunofluorescently-labelled cell monolayers were visualised
using confocal microscopy (TCS SP5 confocal microscope, Leica). The formation of
CPD DNA dimers was also detected in these irradiated keratinocyte monolayers
using a mouse monoclonal antibody against CPDs (clone KTM53, Kamiya
Biomedical Company, Japan) at a 1:1000 dilution and incubated overnight at 4°C.
These keratinocyte monolayers were simultaneously labelled with DAPI and
visualised using fluorescence microscopy as outlined in 2.2.10.
4.2.12 Immunohistochemical analysis
All immunohistochemical analysis conducted on the tissue sections of the
HSE-KC and HSE-KCF were performed as described in 2.2.7.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 95
4.2.13 Analysis of apoptotic pathways using ELISA
A sandwich ELISA apoptosis array was used to detect the presence of elements
of the apoptotic cascade in cell lysates from irradiated HSE-KC and HSE-KCF (as
described in 2.2.20). The PathScan® Apoptosis Multi-target Sandwich ELISA kit
(Cell Signaling Technology, Beverly, MA) was used as per the manufacturer’s
protocol. Briefly, cell lysates (diluted to 250 pg/µl in the manufacturer’s supplied
sample diluent) were then added to a 96-well plate pre-bound with primary
antibodies to various protein antigen targets, and incubated overnight at 4°C. The
wells were then incubated for one hour with the corresponding detection antibodies
and HRP-linked secondary antibodies. The supplied 3,3',5,5'-tetramethylbenzidine
(TMB) substrate was then added to each well and incubated for 10 minutes at 37°C,
after which the reaction was stopped using the stop solution provided. The
absorbances of the ELISA reactions were read at 450 nm using a spectrophotometer.
The magnitude of the reaction absorbance was directly proportional to the quantity of
the bound target protein.
4.2.14 qRT-PCR analysis of gene expression
The protocol for qRT-PCR performed in this study is described in 2.2.19. The
primer sequences for p53 were TAACAGTTCCTGCATGGGCGGC (5’ to 3’) and
AGGACAGGCACAAACACGCACC (3’ to 5’).
4.2.15 Western immunoblotting analysis
Western blots were performed as previously outlined in 2.2.23.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 97
4.3 RESULTS
Aim 1: To study the impact of fibroblast co-culture on the survival, repair and
death in irradiated keratinocytes using a Transwell® cell culture system
4.3.1 Optimisation of a Transwell® co-culture system photobiology model
In order to address the first aim of this Chapter, a 2D co-culture system was
firstly characterised and optimised for its use in assessing UVB-induced keratinocyte
responses. The co-culture of primary mouse keratinocytes and fibroblasts using this
Transwell® system has been previously described in an investigation into epidermal
morphogenesis during wound healing (Kandyba et al., 2008). The use of this
Transwell® cell culture system addresses some of the limitations of conventional 2D
monolayer cultures; in particular, the potential to culture two physically-separated
cell populations which share a common cell culture medium (Ami et al., 1995). In
the study reported here, this technique was characterised for its use as a novel
photobiology co-culture model using primary human skin cells.
Notably, it has been previously demonstrated that cells grown in a 2D
monolayer format respond differently to various treatments and stimuli, including
UVR exposure, in comparison to those within an in vivo setting (Sun et al., 2006).
Therefore, while the optimal experimental UVB dose for 3D HSE-KC composites
was previously assessed (Section 3.3.2), the UVB dose required to reduce cellular
viability by 50% in the Transwell® keratinocyte monolayers was firstly determined.
This would thus provide baseline measures from which fibroblast-induced changes to
keratinocyte UV-responses can be assessed in the subsequent Transwell®
experiments described in this Chapter.
The dose-response of keratinocyte viability to increasing levels of UVB are
shown in Figure 4.1A. The MTS assay used here to assess the relative cell survival in
irradiated keratinocyte populations is widely used to attain experimental dose-
response curves to UVR exposure (Ho et al., 2010; Staples et al., 2010; Han et al.,
2011). The relative cell viabilities in these keratinocyte populations were shown to
significantly decrease compared to non-irradiated cultures, 24 hours after a single
dose of 4 and 5 MED of UVB. At these doses, the percentage of viable cells
decreased to 65.2 ± 19.8% and 29.6 ± 14.0% respectively (Figure 4.2A).
98 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
Accordingly, a 4 MED UVB dose was applied in the subsequent experiments to
analyse changes to keratinocyte photoresponses as a result of fibroblast co-culture.
Figure 4.2. Cell viability of keratinocytes in UVB-irradiated Transwells®
(A) The relative cell viabilities of irradiated keratinocytes exposed to increasing doses of UVB were measured
using the MTS assay. Cells were assayed at 24 hours post-irradiation, and the absorbances expressed as a
percentage of values obtained from the non-irradiated control. Asterisks represent statistical significance (p<0.05)
relative to the non-irradiated control. (B) The effects of culture conditions and UVB exposure on keratinocyte
viability are expressed as a percentage of results obtained from non-irradiated SFM cultures. The asterisks
represent statistical significance of p<0.05. Error bars indicate SEM values. Results are representative of
experimental triplicates using cells obtained from experiments on three separate skin donors.
4.3.2 Effects of fibroblast co-culture on cell viability in irradiated keratinocytes
In the previous experiments (Section 4.3.1) the UVB dose observed to decrease
the overall viability in keratinocyte monolayers by 50% was successfully established
as being 4 MED (Figure 4.2A). Next, the changes to keratinocyte UVB responses as
a result of fibroblast co-culture were further investigated. Specifically, whether
fibroblasts conferred a protective effect to keratinocytes prior to their exposure to
UVB, or played a role in increasing cellular survival after irradiation was studied
(Figure 4.2B).
Exposure of the keratinocyte monolayers to UVB irradiation was shown to
decrease cell viability 24 hours post-irradiation under all culture conditions.
Keratinocyte monolayers cultured without the presence of fibroblasts were observed
A B
UVB dose (MED)
Pe
rce
nta
ge
ce
ll vi
ab
ility
0 1 2 3 4 50
50
100
150
*
*
UVB dose (MED)
Per
cent
age
cell
viab
ility
0 4 0 4 0 4
0
50
100
150 *
SFM + Fib (before UV) + Fib (after UV)
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 99
to have the greatest decrease in metabolic activity following irradiation, with a mean
overall decrease of 53.7 ± 11.9%. Cells co-cultured with fibroblasts before being
exposed to UVB radiation showed the most significant resistance to cell death with a
89.3 ± 19.6% of viable cells remaining post-irradiation. When transferred to
fibroblast-containing cultures after the irradiation process, the keratinocytes showed
no significant change in cell viability as compared to those cultured in SFM only
(67.0 ± 23.1%). These results suggest that keratinocyte activation by fibroblast-
secreted factors prior to UVB-induced damage may be more vital for resisting cell
death and maintaining cell viability following insult, thus conferring a protective
effect on irradiated keratinocytes.
4.3.3 Effects of fibroblast co-culture on cell death in irradiated keratinocytes
Keratinocyte apoptosis resulting from the effects of UVB exposure is a
characteristic tissue damage marker both in vivo and in vitro (Wrone-Smith et al.,
1997; Matsumura et al., 2005; Svobodova et al., 2012). The activation of apoptotic
pathways after UVB photodamage represents a fail-safe cellular mechanism
triggered predominantly by DNA damage, to reduce the risk of malignant
transformation (van Laethem et al., 2005; Claerhout et al., 2006; van Laethem et al.,
2009). Using the Transwell® co-culture system, it was reported in the previous
section that dermal fibroblast co-culture positively enhanced keratinocyte viability
subsequent to UVB exposure (4.3.2). This augmentation of cell viability in irradiated
keratinocytes was therefore characterised to determine a possible link to the
inhibition of UVB-induced apoptosis. Using an in situ TUNEL assay to detect the
presence of DNA fragmentation as a marker of apoptosis, keratinocytes undergoing
cell death post-irradiation were thus visualised and quantified. Figure 4.3 shows
TUNEL-positive keratinocytes detected in irradiated monolayers cultured using
different fibroblast co-culture conditions, as described in Figure 4.1. Using image
analysis software, the percentage of apoptotic cells per field under different culture
conditions were then determined and are represented in Figure 4.4.
A relatively low percentage of keratinocytes undergoing apoptosis was
observed in mock-irradiated controls in all the samples (1.04 ± 0.08%). In all the cell
monolayers exposed to 4 MED of UVB, however, keratinocyte nuclei containing
DNA fragmentation were detected at 24 hours post-irradiation, which represents a
characteristic cellular feature of late stage apoptosis (Cotton and Spandau, 1997).
100 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
The greatest ratio of TUNEL-positive cells was observed in keratinocytes cultured in
the absence of fibroblasts in SFM only (27.9 ± 12%) (Figure 4.3, Figure 4.4).
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 101
Figure 4.3. UVB-induced keratinocyte apoptosis in a 2D co-culture model
Apoptotic keratinocytes were detected using a TUNEL assay in Transwell® insert monolayers 24 hours after
exposure to a single dose of 4 MED UVB (B,C and D, left to right) and an non-irradiated control (A, left to
right). Images A, B and C show representative images of keratinocyte monolayers in different culture conditions
(as shown in Figure 4.1) labelled with the nuclear fluorescent label, DAPI (first column), cells treated with the
TUNEL reaction (second column) and the corresponding merged images (third column). Row B shows TUNEL-
positive keratinocytes from irradiated cultures grown in SFM only. Row C represents identical keratinocyte
cultures co-cultured in the presence of fibroblasts before the irradiation protocol. Row D shows apoptotic
keratinocytes in monolayers co-cultured with fibroblasts in the Transwell® system after UVB irradiation.
Fluorescence detected following the TUNEL reaction shows a localisation of DNA fragmentation within the cell
nuclei, as observed in the merged images. Images are representative of experiments conducted in triplicate from
three independent skin cell donors. The scale bar represents 50 µm.
102 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
Figure 4.4. Fibroblast co-culture inhibits apoptosis in irradiated keratinocyte monolayers
Apoptotic keratinocytes from irradiated Transwell® insert membranes in different co-culture conditions (Figure
4.3) were quantified. Data represents the mean number of TUNEL-positive keratinocytes counted per microscope
field. Keratinocytes were grown in SFM only (-Fib), with fibroblasts in the lower chamber of the Transwell®
(+Fib (Before)) or transferred to fibroblast co-culture conditions after irradiation (+Fib (After)). No significant
numbers of apoptotic cells were observed in non-irradiated controls in both co-culture (data not shown) and
single monolayer cultures of keratinocytes. Results shown are representative of experimental triplicates, from
experiments performed on cells from three independent donors. Asterisks represents statistical significance at
p<0.05. Error bars indicate SEM values.
In monolayers transferred to fibroblast co-culture conditions immediately
following the irradiation protocol, similar levels of keratinocyte apoptosis were
observed as those cultured only in SFM (24.8 ± 9.6%). However, in keratinocytes
grown in co-culture conditions with fibroblasts prior to their exposure to UVB
radiation, significantly fewer apoptotic cells were detected at this time-point (12.1 ±
4.3%).
These results paralleled those obtained in the cell viability assays performed in
the previous section, showing that fibroblast co-culture before cellular insult with
UVB radiation appears to have the greatest protective effect in terms of enhancing
keratinocyte survival. This is further supported by the observation that co-culture
with fibroblasts post-irradiation did not appear to have a significant effect on
inhibiting apoptosis following UVB-exposure. The activation of keratinocytes by
fibroblast-produced factors at the time of UVB-insult may therefore be critical in
Pe
rce
nta
ge
T
UN
EL
+ K
C/fi
eld
Non-ir
radia
ted
cont
rol
SFM +
UV
+ Fib
(bef
ore
UV)
+ Fib
(afte
r UV)
0
10
20
30
40
50
**
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 103
promoting cell survival and preventing widespread epidermal damage as a result of
increased keratinocyte apoptosis.
The induction of the apoptotic program involves several biochemical pathways
which eventually lead to hallmark morphological changes including cell shrinkage
and DNA fragmentation (Young, 1987; Hengartner, 1997a). The TUNEL assay is
useful in its ability to quantify DNA fragmentation as a measure of a late stage in the
apoptotic cascade, however, this morphological characteristic of apoptosis overlaps
with other cell death programs, including necrosis (Kerr, 1971; Gold et al., 1994).
The activation of these processes is heavily dependent on the sequential activation of
a class of cysteine-aspartic acid proteases known as caspases (Peter et al., 1997;
Zhang et al., 2000). Caspase-3, activated by both intrinsic and extrinsic apoptotic
pathways, is known as an executioner caspase and is a key mediator of UVB-induced
apoptosis upon its cleavage and subsequent activation (Taylor et al., 2008). To
further investigate the cell death pathway triggered by UVB exposure in these
irradiated keratinocytes, the presence of cleaved caspase-3 was detected using
immunofluorescence. Figure 4.5 shows images obtained from confocal microscopy
analysis, showing the expression of cleaved caspase-3 in Transwell®-cultured
keratinocytes as a result of UVB radiation exposure.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 104
Figure 4.5. Detection of cleaved caspase-3 in irradiated keratinocyte monolayers
Cleaved caspase-3, an early cellular apoptotic marker, was detected in Transwell® insert monolayers 6 hours
after the exposure to a single dose of 4 MED UVB (B,C and D, left to right) and a non-irradiated control (A, left
to right). Images A, B and C show representative images of keratinocyte monolayers in different culture
conditions (as shown in Figure 4.1) labelled with the nuclear fluorescent label, DAPI (first column), labelled with
a fluorescently-labelled antibody to cleaved caspase-3 (second column) and the corresponding merged images
(third column). Row B shows keratinocytes from irradiated cultures grown in SFM only. Row C represents
identical keratinocyte cultures co-cultured in the presence of fibroblasts prior to the irradiation protocol. Row D
shows apoptotic keratinocytes in monolayers co-cultured with fibroblasts in the Transwell® system after UVB
irradiation. Images were obtained using confocal microscopy and are representative of three experiments
conducted using samples obtained from three independent skin cell donors. The scale bar represents 50 µm.
A
B
C
DAPI Cleaved caspase-3 Merged
Non-irradiated
control
6 hr
SFM
D
6 hr
+ Fib (after)
6 hr
+ Fib (before)
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 105
Keratinocyte monolayers were fixed 6 hours after exposure to a single dose of
4 MED UVB, as the cleaved caspase-3 is known to be detected in apoptotic cells
relatively earlier in the apoptotic cascade (Jänicke et al., 1998b). The activated form
of the caspase was not readily detected in non-irradiated keratinocyte monolayers,
including serum-starved cultures. Following UVB irradiation, however, cleaved
caspase-3 was observed to be present in all UVB-exposed cells. At 6 hours after
exposure, cleaved-caspase 3 was found to be most abundant in keratinocyte
monolayers cultured in SFM only, in the absence of fibroblast co-culture. This
activated caspase was observed to be mainly localised within the cytoplasmic regions
of the keratinocytes. In Transwell® monolayers transferred to fibroblast co-culture
conditions subsequent to UVB-exposure, similar levels of cleaved caspase-3 as those
present in SFM cultures were observed. Interestingly, caspase-3 appeared to be either
diffusely distributed in a granular manner throughout the cell cytoplasm, or
concentrated around the periphery of the cell in these samples. In the keratinocyte
cultures maintained in the presence of fibroblast co-culture prior to UVB treatment,
there was an appreciable decrease in the numbers of cleaved caspase-3-expressing
cells. Due to the cytoplasmic expression of cleaved caspase-3 and the apoptotic
morphology of irradiated keratinocytes, the quantification of cells expressing
activated caspase-3 could not be accurately performed, and was hence omitted.
Overall, the immunofluorescent analysis revealed that fibroblast co-culture
prior to cellular insult by UVB radiation resulted in the diminished expression of
cleaved-caspase 3 in irradiated keratinocyte monolayers. Taken together with a
similar effect observed in DNA fragmentation, as measured using the TUNEL assay,
this indicates that fibroblast-secreted factors may protect against UVB-induced
keratinocyte damage by inhibiting apoptosis.
4.3.4 Fibroblast co-culture and the removal of UVB-induced DNA dimers in
irradiated keratinocytes
In the previous section, it was demonstrated that keratinocyte viability and the
protection from apoptosis was enhanced by fibroblast co-culture before UVB
irradiation. The balance between cell survival and the initiation of cell death
pathways is known to be heavily influenced by the formation of DNA damage via
various environmental genotoxic agents, including UVR (Christmann et al., 2003;
Roos and Kaina, 2006). In the case of sub-lethal doses of UVB in particular, the
106 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
action of repair systems such as the NER, together with pro-survival signals, allow
for DNA damage to be effectively removed prior to the progression of the cell cycle
(Ahmed and Setlow, 1993). However, in the event of severe and unrepairable
damage to the cell’s genetic material, apoptosis, as part of the cellular stress
response, serves as a safety net to prevent oncogenic transformation (Christmann et
al., 2003). Hence, the removal of UVB-induced DNA dimers (in the form of CPDs)
was analysed in keratinocyte-fibroblast co-cultures to determine whether the pro-
survival effect observed in the previous section was related to an activation of DNA
repair mechanisms.
Using immunofluorescence, the presence of UVB-induced CPDs was
visualised in irradiated keratinocyte monolayers (Figure 4.6). The ratios of CPD-
positive keratinocytes within the keratinocyte population were determined using
image analysis, as detailed in 2.2.9. These keratinocyte cultures were maintained
under the different co-culture conditions as detailed previously (Figure 4.1).
Immediately after exposure, all keratinocyte nuclei in irradiated monolayers were
found to be positive for the presence of CPDs (Figure 4.6A). Keratinocytes cultured
on Transwell® inserts were probed for the presence of CPDs again at 24 hours post-
irradiation to monitor differences in the rate of CPD repair by keratinocyte DNA
repair mechanisms (Figure 4.6 B to D). The majority of UVB-induced CPDs have
been shown to be repaired during the initial 24 hours subsequent to UVB exposure in
vitro (Riou et al., 2004). Keratinocytes grown in SFM only appeared to contain the
greatest number of CPD-positive nuclei and the highest intensity of immuno-
reactivity detected at 24 hours post-irradiation, as compared to those from fibroblast
co-cultures (Figure 4.6B, Figure 4.7). At this time point, it was observed that
63.5±26.6% of the total keratinocyte population were still positive for CPDs.
Keratinocytes grown in the presence of fibroblasts before UVB-exposure were
demonstrated to have a significantly lower number of cells harbouring CPDs, as
compared to cells co-cultured after irradiation (21.5 ± 4.9% and 49.9 ± 9.9%
respectively) (Figure 4.7). This result strongly suggests that dermal fibroblasts not
only play an important role in protecting against UVB-induced cell death in
irradiated keratinocytes in vitro, but may also contribute to stimulation of DNA
repair pathways upon genotoxic insult.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 107
Figure 4.6. Repair of CPD in irradiated keratinocyte monolayers in different culture conditions
The presence of CPD DNA dimers in irradiated Transwell® insert monolayers exposed to a single dose of 4
MED UVB were detected using immunofluorescence. Immediately following UVB exposure, the majority of
keratinocyte nuclei were positive for CPD photolesions, as shown in the representative image (A). 24 hours after
irradiation, keratinocyte monolayers cultured in SFM only (B), in fibroblast co-culture prior to irradiation (C) and
in co-culture post-irradiation (D) were probed for the presence of unrepaired CPDs. Images are representative of
experimental triplicates from experiments conducted on three independent skin cell donors. The scale bar
represents 50 µm.
A
B
DAPI CPD Merged
0 hr
(all treatments)
24 hr
SFM
D
24 hr
+ Fib (after)
C
24 hr
+ Fib (before)
108 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
Figure 4.7. Fibroblasts enhance repair in irradiated keratinocyte monolayers
The presence of UVB-induced CPDs was detected in irradiated keratinocyte monolayers grown in different
Transwell® co-culture conditions. Keratinocyte nuclei with DNA dimers were visualised using
immunofluorescence (as shown in Figure 4.6) and quantified using image analysis software. The percentages of
unrepaired CPD-positive keratinocyte nuclei at 24 hours under different culture conditions were normalised to
those observed at 0 hours post-irradiation. The results shown are representative of experimental triplicates
conducted on three experiments, each using independent skin cell donors. Asterisks represents statistical
significance at p<0.05. Error bars indicate SEM values.
Pe
rce
nta
ge
of C
PD
+ k
era
tino
cyte
s
UV 0 h
r
SFM +
UV
+ Fib
(bef
ore U
V)
+ Fib
(afte
r UV)
0
20
40
60
80
100 *
*
24 hr
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 109
Aim 2: Development and characterisation of a 3D HSE model to study
fibroblast-keratinocyte interactions during the UVB response.
In the previous section, a Transwell® co-culture technique was used to
demonstrate the effect of dermal fibroblasts on modulating keratinocyte survival and
repair following UVB-induced damage. The next aim was to investigate this
paracrine interaction during the acute UVB response using a 3D tissue-engineered
HSE-KCF model. This organotypic model consists of primary human dermal
fibroblasts incorporated into the DED component, with a stratified keratinocyte
epidermal layer. It was hypothesized that the HSE-KCF could be utilised to further
investigate the fibroblast-induced changes to keratinocyte photoresponses
(previously reported in 4.3.2, 4.3.3 and 4.3.4) in a 3D physiologically-relevant tissue
model.
4.3.5 Development and characterisation of the HSE-KCF skin model
An array of skin equivalent models composed of different combinations of
dermal and epidermal cell populations have been described as in vitro models for
studying paracrine interactions between these two distinct skin regions (Topol et al.,
1986; Hudon et al., 2003; Ng and Ikeda, 2011). As detailed in Chapter 3, the HSE-
KC model adopted by our laboratory has been characterised for its use in
photobiology studies. Fundamentally, this reconstituted skin model retains key
physiological elements of native skin known to influence the epidermal UVB
response, and shows similar markers of UVB-induced damage and subsequent
regeneration upon irradiation. Herein, I report the adaptation of this model to include
fibroblasts within the HSE-DED, in order to create an in vitro platform for studying
epidermal-dermal interactions during the UVB response.
Firstly, the protocol for seeding primary dermal fibroblasts and epidermal
keratinocytes on the DED scaffold required for the generation of the HSE-KC was
optimised. Dermal fibroblasts are known to possess the ability to migrate into the
collagen network of the dermis, a process vital for wound healing (Lin et al., 2005).
Accordingly, in vitro skin models incorporating fibroblast-populated collagen dermal
scaffolds have been extensively used (Rhee and Grinnell, 2007; Rhee, 2009; Ahn,
2010). However, the technique of reconstituting an ex vivo DED scaffold with
primary dermal fibroblasts has been relatively less well-documented (Lee and Cho,
110 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
2005). Therefore, for this study, various fibroblast-seeding methods were initially
analysed histologically to ensure successful incorporation of these cells at the
papillary dermal-epidermal junction.
Dermal fibroblasts were seeded onto either the reticular or the papillary surface
of the DED using different protocols to optimise the fibroblast-repopulation of this
scaffold (Figure 4.8 A to D). Firstly, fibroblasts were seeded onto the reticular DED
surface three days prior to the addition of keratinocytes on the papillary side (Figure
4.8A). The histological assessment of the resulting skin constructs revealed that
fibroblasts appeared to be concentrated around the deeper reticular layers of the
dermis. In these composites, it appeared that fibroblasts did not successfully migrate
superficially towards the epidermal-dermal junction (Figure 4.8A and B). It has been
previously demonstrated that papillary fibroblasts play a more significant role in
influencing epidermal behaviour through factors secreted across the BM at the
epidermal-dermal junction (Sorrell, 2004; Sorrell et al., 2007). In view of this, the
incorporation of fibroblasts into the papillary region of the DED was further
optimised.
Figure 4.8. Optimisation of fibroblast seeding in the construction of HSE-KCF
Different protocols for seeding fibroblasts into HSE-KCF composites were analysed histologically. Fibroblasts
were seeded on either the reticular surface (A and B) or the papillary surface (C and D) of the DED during HSE
construction, and samples analysed after 9 days of culture at the air-liquid interface. When fibroblasts were
seeded onto the reticular surface three days prior to keratinocyte seeding on the papillary surface, histology of
HSE sections shows fibroblasts (indicated by arrows) remained localised to the reticular region of the DED (A).
The epidermis appeared to be well-organised, though no fibroblasts appeared to be present at the dermal-
epidermal junction (B). Fibroblasts were seeded together with keratinocytes onto the papillary surface of the DED
(C). The histology of this composite shows a poorly-organised epidermis with a few dermal fibroblasts present
around the papillary surface of the DED. Fibroblasts seeded on the papillary surface 48 hours prior to the addition
of keratinocytes onto the same surface (as described in section 2.2.3) revealed a well-organised and stratified
epidermis, with fibroblasts present in the papillary region of the DED (D). Images from histological analyses are
representative of experimental triplicates conducted on three independent skin donors. The scale bar represents 50
µm.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 111
Next, fibroblasts were seeded onto the papillary surface of the DED in order to
promote fibroblast infiltration into the papillary regions of the dermal component
(Figure 4.8C and D). Due to keratinocytes also being seeded on this surface of the
DED for the formation of the epidermis, two fibroblast and keratinocyte cell seeding
protocols were tested. The simultaneous addition of both cell types onto the papillary
surface showed histological evidence pointing towards cellular infiltration into the
papillary region of the DED (Figure 4.8 C). However, the histology of the constructs
produced using this protocol also showed a poorly-organised epidermal layer (Figure
4.8 C). Indeed, the separation of the epidermal layer from the supporting DED
scaffold was strongly indicative of poor basal keratinocyte attachment onto the
papillary surface of the scaffold. Furthermore, a well-defined differentiating gradient
of keratinocytes was not observed in these constructs. The failure of basal
keratinocytes to adequately attach to the DED indicated a potential degradation of
the BM by fibroblasts during their migration into the dermis.
To overcome this effect, fibroblasts were then seeded onto the papillary surface
three days prior to the subsequent addition of keratinocytes (Figure 4.8D). Using this
technique, it was hypothesized that migrating fibroblasts would be able to
sufficiently remodel the BM at the dermal-epidermal junction prior to the addition of
the keratinocytes, which would facilitate their attachment, proliferation and
differentiation, as reported previously (Smola et al., 1998; Ralston et al., 1999).
Histological sections from HSE-KCF constructed using this protocol showed a well-
organised, stratified epidermis, together with evidence of cellular migration into the
papillary regions of the DED (Figure 4.8D). Further characterisation of these
migrated cells was subsequently conducted to confirm the presence of fibroblasts
within the dermal component. This method was then employed for all successive
experiments utilising the HSE-KCF skin composites.
From the histological analysis of different fibroblast seeding protocols and
their associated impact on epidermogenesis, it can be suggested that BM remodelling
by fibroblasts may be a critical process during HSE-KCF formation. Hence, we
analysed components of the BM layer in the HSE-KCF composites using
immunohistochemistry to detect the expression of collagen IV in these skin
constructs (Figure 4.9). When compared to ex vivo native skin controls,
immunohistochemical analysis revealed similar patterns of collagen IV expression,
112 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
in particular at the dermal-epidermal junction and lining other elements of the dermis
such as blood vessels (Figure 4.9). The deposition of collagen IV at the BM zone by
infiltrating fibroblasts during the maturation of the HSE-KCF indicates that this
process may be integral in influencing formation of the HSE-KCF epidermis. Indeed,
several studies have demonstrated that collagen IV facilitates keratinocyte
attachment, proliferation and stratification in the developing epidermis both in vitro
and in vivo (Aumailley and Timpl, 1986; Krejci et al., 1991; Tinois et al., 1991).
Figure 4.9. Collagen IV expression in HSE-KCF and native skin
Collagen IV, a major component of the BM, was detected in tissue sections of HSE-KCF (A) and native skin (B)
using immunohistochemistry. Images from immunohistochemical analyses are representative of experimental
triplicates conducted on three independent skin donors. The scale bar represents 50 µm.
Next, the nature of the cells that repopulated the papillary region of the DED in
the HSE-KCF was characterised. Vimentin is ubiquitously expressed by
mesenchymal cells as an intermediate filament and serves primarily to maintain
cellular integrity (Satelli and Li, 2011). Hence, vimentin has been established as a
cellular marker of dermal fibroblasts in the characterisation of in vitro skin models
(El Ghalbzouri et al., 2002). Immunofluorescently-labelled tissue sections of ex vivo
native skin, HSE-KC and HSE-KCF reveal significantly higher levels of vimentin
expression in tissues containing fibroblasts in the dermal component (Figure 4.10).
The HSE-KCF showed similar vimentin expression levels to native skin which
appeared to be absent in the HSE-KC sections comprised of an acellular dermal
scaffold. This result provides further evidence for the successful incorporation of
fibroblasts into the DED using the HSE-KCF construction protocol described here.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 113
Figure 4.10. Expression of vimentin in native skin and HSE composites
Sections of native skin (A), HSE-KC (B) and HSE-KCF (C) were probed with an immunofluorescent antibody to
the mesenchymal marker, vimentin (red fluorescence). The sections were counterstained with the DAPI nuclear
marker (blue fluorescence). Merged images show the localisation of vimentin within the dermis of native skin
and HSE-KCF tissues, but with a significantly lower staining intensity observed in the HSE-KC. Images from
immunofluorescent analyses are representative of experimental triplicates conducted on three independent skin
donors. The scale bar represents 50 µm.
Following the seeding of fibroblasts and keratinocytes onto the DED using the
abovementioned optimised protocol, the HSE-KCF constructs were maintained in
culture conditions prior to UVB irradiation as described in 2.2.3 and 2.2.5. Hence,
the histology of the mature HSE-KCF sampled at different time-points in culture
conditions was analysed (Figure 4.11). Initially, a stratified epidermis with well-
defined stratum corneum was observed, nine days after keratinocyte seeding and
subsequent culture at the air-liquid interface (Figure 4.11 A). Over the subsequent
five day period, a gradual thickening of the stratified keratinocyte layer, together
with the stratum corneum (stained in dark pink), was observed (Figure 4.11B to F).
The presence of fibroblasts in the DED was also detected, with haematoxylin-stained
DAPI Vimentin Merged
A
B
C
114 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
cell nuclei evident in the papillary regions. However, the numbers of migrated
fibroblasts within the dermis appeared to be variable between composites. Overall,
the histology of the HSE-KCF mirrored that of the HSE-KC model described
previously (Figure 3.1), and was thus utilised as an in vitro model for studying
keratinocyte-fibroblast interactions during the UVB response.
Figure 4.11. Histology of HSE-KCF in culture
Haematoxylin and eosin stained tissue sections of the HSE-KCF depict the histology of the HSE-KCF over five
days in culture. The HSE-KCF constructs were cultured at the air-liquid interface for nine days to promote the
formation and stratification of the epidermis. At this stage, the HSE-KCFs were found to have a differentiated
epidermis with a well-defined stratum corneum (A). In subsequent experiments, UVB treatments were applied at
this time point. Over the next five days in culture conditions (B to F), the HSE-KCFs showed a gradual
thickening of the stratified keratinocyte layer and the stratum corneum. Images from the histological analyses are
representative of experimental triplicates conducted on three independent skin donors. The scale bar represents 50
µm.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 115
4.3.6 Characterisation of the UVB response in HSE-KCF
In the previous section, the HSE-KCF, a 3D organotypic model incorporating
cell-based dermal and epidermal components, was successfully established.
Henceforth, the potential fibroblast-induced modulation of keratinocyte responses to
UVB in the HSE-KCF model, as compared to those previously detailed in the HSE-
KC, were investigated. Specifically, the formation of characteristic UVB-damage
markers including CPD photolesions, apoptotic sunburn cells and the expression of
proinflammatory cytokines, were studied in the irradiated HSE-KCF constructs.
Additionally, the effect of fibroblasts in the HSE-KCF constructs on epidermal repair
and regeneration was also investigated.
The histological analysis of sections obtained from UVB-exposed HSE-KCF
constructs over a five day period post-irradiation is shown in Figure 4.12. During this
timeframe a gradual thickening of the stratum corneum is observed in the epidermis
of the irradiated HSE-KCFs (Figure 4.12A to F). At Day 2 after UVB treatment, a
decrease in the thickness of the viable keratinocyte layer in the epidermis is also
apparent, which shows a gradual regeneration from Day 3 onwards (Figure 4.12D to
F). Sections of the HSE-KCF sacrificed at Day 5 after UVB exposure show a
thickened stratum corneum with evidence of keratinocyte nuclei also present in this
epidermal layer. This may be attributed to the accelerated differentiation of
keratinocytes as part of the previously-described epidermal regenerative process
(Liu, 1996; Del Bino et al., 2004). Due to the absence of the natural sloughing of the
superficial layers of the epidermis in the in vitro HSE-KCF model, rapidly
differentiating cells may therefore become incorporated into the stratum corneum
during the post-irradiation blistering process. Overall, the histology of the UVB-
exposed HSE-KCF appeared to be aligned with previous experiments conducted
using the HSE-KC (Figure 3.2). Hence, further analyses were conducted to
characterise UVB responses in the HSE-KCF.
116 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
Figure 4.12. Histology of irradiated HSE-KCF
Images A to F represent tissue sections of irradiated HSE-KCF exposed to a single dose of 2 MED UVB and
sacrificed over a five-day period in culture. A gradual thickening of the stratum corneum layer (in dark pink) is
observed from Day 0 (A) to Day 5 (F) post-exposure. Images from the histological analyses are representative of
experimental triplicates conducted on three independent skin donors. The scale bar represents 50 µm.
Expression of proinflammatory cytokines by irradiated HSE-KCFs
The upregulation of IL-6 and IL-8 synthesis in keratinocytes following
erythemal doses of UVB has been linked to both local and systemic inflammatory
reactions commonly observed during the sunburn response (Kondo et al., 1993;
Petit-Frere et al., 1998). This response has also been previously reported using the
HSE-KC photobiology model (Figure 3.7). As a key marker of the epidermal
sunburn response, the production of IL-6 and IL-8 was also investigated in the
irradiated HSE-KCF culture model as reported herein.
The concentrations of these proinflammatory cytokines present in the cell
culture supernatants from irradiated HSE-KCF were analysed using an ELISA
quantification method, as described in 2.2.13 (Figure 4.13). Overall, relatively higher
levels of both IL-6 and IL-8 were present in cell culture medium samples obtained
from HSE-KCFs as compared to those previously reported from HSE-KC cultures
(Figure 3.7).
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 117
Figure 4.13. Quantification of IL-6 and IL-8 concentrations in HSE-KCF culture supernatants
Serum-free culture medium samples were obtained from HSE-KCF cultures at the 0 hour and 24 hour time points after 2 MED UVB irradiation. Quantification of IL-6 and IL-8 levels was
achieved using an ELISA immunoassay. Data represents the average quantities of IL-6 and IL-8 detected at the two time points (mean±SEM), in experimental triplicates from two experiments
conducted on two different skin donors, expressed in pg/mL. All measurements were normalised to total protein levels in culture supernatants. Asterisks represent statistical signifance (p<0.05)
in the difference from matched samples at t=0 hr. Error bars indicate the SEM values.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 118
For example, mean concentrations of 67.0 ± 12.1 pg/mL IL-6 were detected in
medium from non-irradiated HSE-KCF cultures as compared to 2.37 ± 0.32 pg/mL
in those from HSE-KC cultures. In culture medium samples from the HSE-KCF
obtained 24 hours post-irradiation, both IL-6 and IL-8 showed significant increases
in concentration as compared to matched samples at the 0 hour time-point. In these
samples, a 12.7-fold and 10.5-fold increase in IL-6 and IL-8 concentrations,
respectively, were detected. These results suggest that the presence of fibroblasts in
the HSE-KCF may enhance the concentrations of these proinflammatory cytokines in
both non-irradiated and irradiated tissues.
Repair of CPDs in the irradiated HSE-KCF
The generation of nucleic CPDs is a cardinal feature of UVB-induced cell
damage and is generally used as a marker of cutaneous UVB damage in vivo
(Snellman et al., 2003; Courdavault et al., 2005; ten Berge et al., 2011).
Furthermore, the accumulation of genetic mutations associated with the formation of
these DNA lesions is strongly associated with the onset of skin cancers (Ravanat et
al., 2001). The generation and repair of CPDs in the HSE-KC photobiology model
was previously reported in this study (3.3.4, 3.3.8). Hence, these processes were
investigated using the HSE-KCF constructs to identify the impact of paracrine
signals from fibroblasts on the removal of UVB-induced photolesions on
keratinocytes.
Using immunohistochemistry, keratinocytes harbouring CPD damage were
positively identified in irradiated HSE-KCF (Figure 4.14). Papillary fibroblasts
situated near the BM zone border were also found to harbour nucleic CPD (Figure
4.14B). Immuno-labelled sections viewed at a higher magnification show the clear
localisation of CPDs within the nuclei of basal and suprabasal keratinocytes within
the epidermis of irradiated HSE-KCF (Figure 4.14 C).
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 119
Figure 4.14. UVB-induced formation of CPDs in HSE-KCF
UVB-induced CPDs were detected in HSE-KCF at 24 hours after UVB treatment using immunohistochemistry as
previously described. Non-irradiated control HSEs (A), HSE-KCF exposed to 2 MED of UVB (B) and irradiated
HSE-KCF at a higher magnification (C) are shown above. The presence of CPDs, indicated by brown immuno-
reactivity, was observed predominantly within the nuclei of basal and suprabasal cells of the epidermis and in
papillary fibroblasts near the dermal-epidermal junction. Images from the immunohistochemical analyses are
representative of experimental triplicates conducted on three independent skin donors. Black and red scale bars
represent 50 µm.
The presence of unrepaired CPDs in the HSE-KCF constructs was quantified
using image analysis as described previously (2.2.7 and 2.2.9). The percentages of
CPD-positive cells present in the HSE-KCF epidermis after UVB-exposure were
determined and compared to those previously obtained from HSE-KC cultures
(Figure 4.15). The initial numbers of keratinocytes containing CPDs immediately
after UVB irradiation did not differ significantly between the HSE-KC and HSE-
KCF. This suggests that that the presence of fibroblasts in the skin model did not
greatly influence the capacity for UVB to form photolesions in the HSE-KCF
epidermis. At 24 hours post-exposure, however, a significant drop in the proportion
of CPD-positive keratinocytes was observed in the HSE-KCF as compared to that in
the HSE-KC. This marked decrease in DNA-damaged keratinocytes relative to that
in the keratinocyte-only skin model continued at Day 2 and Day 3 after the UVB
treatment. At Days 4 and 5, no further difference was observed in the proportion of
CPD-positive cells between the two skin models. Importantly, the results obtained in
the investigation of CPD repair in the irradiated HSE-KCF mirror the responses
observed in the irradiated 2D co-culture models described in 4.3.4. The enhancement
of CPD removal in irradiated keratinocytes with the presence of fibroblasts in both
the cell monolayer and skin equivalent culture systems suggest that paracrine
120 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
signalling from the dermis may be critical in the activation and/or efficiency of DNA
repair mechanisms in epidermal cells.
Figure 4.15. Rate of CPD repair in irradiated HSE-KC and HSE-KCF
The proportions of CPD-positive keratinocytes in the epidermis of the irradiated HSE-KC and HSE-KCF culture
models were analysed using immunohistochemical detection followed by image analysis. The percentage of
CPD-positive cells per field (normalised to the total number of epidermal cells) are expressed over a period of 5
days post-irradiation. Data represents the average results from three independent experiments (each with
experimental triplicates) using cells obtained from three separate donors. Asterisks represent statistical
significance (p<0.05). The error bars represent SEM values.
Epidermal regeneration in the HSE-KCF
A complex balance of the induction of cell death and cycle arrest and the
opposing effects of cell survival and proliferation occur in UV-exposed skin (Ziegler,
1994; Young, 2006). While the precise regulators of this balance have yet to be fully
defined, post-irradiation epidermal hyperplasia is thought to be a consequence of the
activation of a variety of growth factor receptors and the actions of cytokines
(Rosette and Karin, 1996; Kuhn et al., 1999; Peus et al., 2000). These include
fibroblast-produced factors such as EGF, IGF-I and IL-1, which are thought to play a
role in maintaining the barrier function of the epidermis following UVB damage
(Rosette and Karin, 1996).
The Ki-67 protein is a cellular proliferation marker as it has been shown to be
expressed within the dividing cell’s nucleus during all active phases of the cell cycle
and mitosis (Heenen et al., 1998; Scholzen and Gerdes, 2000; Burcombe et al.,
Days post-irradiation
Perc
enta
ge o
f C
PD
+ k
era
tinocyte
s
0 1 2 3 4 5
0
50
100
150
HSE-KC
HSE-KCF
*
*
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 121
2006). In the previous Chapter, shifts in the proportion of proliferating keratinocytes
within the HSE-KC model following UVB treatment were described (3.3.9). Herein,
the levels of keratinocyte proliferation in the HSE-KCF were analysed, to determine
the impact of fibroblasts on epidermal regeneration post-irradiation.
The HSE-KCF composites were exposed to either a 2 MED UVB or a mock
irradiation treatment before being returned to culture conditions and were
subsequently sacrificed daily during a five-day post-irradiation period (Figure 4.16).
It has previously been reported that the epidermis of in vitro skin models undergoes a
process similar to re-epithelialisation in vivo, in which a combination of basal
keratinocyte proliferation and differentiation occurs in the newly-formed epidermis
(Moll et al., 1998). Accordingly, non-irradiated control HSE-KCF samples revealed
keratinocytes expressing Ki-67 throughout this five-day period, with peak levels
observed at Day 2. In the UVB-exposed HSE-KCF constructs, however, a significant
decrease in the proportion of Ki-67-positive cells was observed at Day 1 and 2 after
UVB treatment, as compared to those in non-irradiated controls. From Day 3 to 5
after UVB irradiation, a rise in proliferating Ki-67-expressing keratinocytes was
observed, suggesting the active renewal of the UV-damaged epidermis during this
time. Interestingly, when compared to the levels of post-irradiation proliferation
previously observed in the HSE-KC (Figure 3.10), the HSE-KCF constructs showed
a significant reduction in Ki-67-expressing cells at Day 1 compared to matched non-
irradiated controls. In vivo, the majority of UVB-induced photolesions in
keratinocytes have been shown to be repaired within 24 hours of irradiation, before
which cell cycle checkpoints prevent the replication of unrepaired cells (Suzuki et
al., 1978; Xu et al., 2000). This suggests that fibroblast-secreted factors in the HSE-
KCF may aid in the regulation of the cell cycle in irradiated keratinocytes, by
delaying cell proliferation prior to complete DNA repair.
122 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
Figure 4.16. Effect of UVB on keratinocyte proliferation in the HSE-KCF
The number of proliferative keratinocytes were analysed via the detection of Ki-67 using immunohistochemistry.
Results shown are expressed as the average number of Ki-67-positive cells detected in ten microscope fields per
tissue section. Data represents the average results from three independent experiments using cells obtained from
three separate donors. Asterisks represent statistical significance (p<0.05). Error bars represent SEM values.
UVB-induced apoptosis in HSE-KCF
Most DNA photolesions, such as CPDs are efficiently removed by the NER
system, which acts as the first line of defence against permanent genetic alterations at
risk of being propagated to daughter cells (Sancar, 1995). Exposure to UVB also acts
as a physiological trigger for the induction of apoptosis which mainly occurs in the
proliferative basal layer in the epidermis (Qin et al., 2002). This complex process
involves the integration of multiple signals including direct DNA damage, cell
surface death receptors and oxidative stress (Lippens et al., 2009). The balance
between cell survival and death is thought to be influenced by the dose of UVB
applied, intracellular signalling, as well as various survival and death factors in the
keratinocyte microenvironment (Akunda et al., 2007; Claerhout et al., 2007; Lewis
and Spandau, 2008).
In this Chapter, the modulation of keratinocyte proliferation and the removal of
CPDs as a result of the presence of fibroblasts in the HSE-KCF skin culture model
(4.3.6) are reported. Next, changes to UVB-induced keratinocyte apoptosis
influenced by the fibroblast-populated DED as compared to the acellular DED
scaffold were investigated. To study this, the TUNEL assay, previously employed to
Days post-irradiation
Ave
rage
Ki-6
7+ c
ells
/fiel
d
0 1 2 3 4 5
0
20
40
600 MED
2 MED
**
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 123
visualise sunburn cells in irradiated HSE-KC, was utilised (3.3.4). The presence of
TUNEL-positive apoptotic cells was detected in the irradiated HSE-KCF 24 hours
after UVB-exposure, predominantly in the epidermal layer (Figure 4.17A).
The presence of apoptotic cells in the HSE-KC and HSE-KCF was then
quantified over a five-day period following UVB treatment (Figure 4.17B). In the
HSE-KC, the formation of sunburn cells was detected as early as 6 hours post-
irradiation. The numbers of TUNEL-positive cells in these skin composites
subsequently increased, reaching a peak at around 24 hours post-irradiation. In the
HSE-KCF, a similar induction in keratinocyte apoptosis was observed from 6 hours
following UVB exposure, in which the incidence of sunburn cell formation was
found to be highest around the 12 to 24 hour time points. The presence of TUNEL-
positive cells in the HSE-KCF significantly decreased from Days 1 to 5 post-
irradiation. Interestingly, at 6 and 24 hours proceeding UVB treatment, a
significantly lower ratio of apoptotic keratinocytes was observed in the HSE-KCF as
opposed to that of the HSE-KC.
Overall, the presence of fibroblasts in these skin equivalent models results in
the inhibition of apoptosis in the first 24 hours after UVB irradiation. This
enhancement of cell survival after UVB exposure as a result of fibroblast co-culture
has also been observed in 2D Transwell® models, as described in 4.3.2 and 4.3.3.
This fibroblast-mediated enhancement of epithelial cell survival has been described
in a variety of in vitro studies (Tseng et al., 1996; Houchen et al., 1999). Notably,
however, the results from this study report for the first time the use of this HSE-KCF
skin model to study the effects of fibroblast-produced factors on the responses of
irradiated human keratinocytes in a biologically-similar epidermis.
124 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
Figure 4.17. Keratinocyte apoptosis in irradiated HSE-KC and HSE-KCF following UVB irradiation.
Apoptotic keratinocytes were detected in HSE-KCF, 24 hours following a single exposure of 2 MED UVB (A).
In the first column, blue fluorescence (DAPI) indicates keratinocytes in the epidermis and fibroblasts in the
dermal component. The second column shows TUNEL positive cells, indicated by the presence of green
fluorescence. The white dotted line indicates the epidermal-dermal junction in the skin equivalents. Yellow lines
indicate the scale of 50 µm. The presence of apoptotic keratinocytes post-UVB irradiation were detected and
counted using the TUNEL assay (B). The average number of TUNEL-positive cells per field (normalised to the
total number of basal epidermal cells) are expressed over time (in hours) post-irradiation. Keratinocytes in the
HSE-KCF show a decreased incidence of UVB-induced apoptosis, with significantly fewer TUNEL-positive cells
being observed in these composites at 6 and 24 hours following a single dose of 2 MED UVB. The presence of
apoptotic keratinocytes was predominantly detected between 6 and 48 hours post-irradiation. In non-irradiated
controls of both HSE constructs, no significant numbers of TUNEL-positive cells were detected (data not shown).
Data represents the average results from three independent experiments (each with samples in triplicate) using
cells obtained from three separate donors. Asterisks represent statistical significance (p<0.05). Error bars
represent SEM values.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 125
Aim 3: Identification of molecular mediators associated with fibroblast-
induced modulation of keratinocyte responses to UVB
4.3.7 Mechanism of fibroblast-induced keratinocyte survival post-irradiation
To further characterise the mechanism behind fibroblast-induced keratinocyte
survival, we used antibody arrays pre-bound with antibodies against central players
in the major apoptotic pathways. These included the total and phosphorylated Bcl-2-
associated death promoter (Bad), cleaved poly (ADP-ribose) polymerase (PARP) and
cleaved caspase-3. To determine the effect of fibroblasts on the expression of these
factors in keratinocytes, HSE-KC and HSE-KCF were exposed to a single dose of 2
MED UVB. At various time points in the first 24 hours post-irradiation, the
epidermis was separated from the composites and keratinocytes lysed, as described
in 2.2.20. These cell lysates were then probed for the above-mentioned apoptotic
markers using a sandwich ELISA-based assay as per the protocol outlined in 4.2.13.
Bad is an intracellular pro-apoptotic protein belonging to the Bcl-2 family
(Reed, 1998). The formation of a complex between Bad, Bcl-2 and Bcl-xL has been
shown to enhance the release of cytochrome c from the mitochondria, thus initiating
apoptosis via the action of Bax and Bak proteins (Gajewski and Thompson, 1996).
Phosphorylation of Bad at serine 112, on the other hand, inhibits binding to the Bcl-
2/Bcl-x complex, which, in the absence of Bad, produces anti-apoptotic effects,
through blocking cytochrome-c release from mitochondria (Hengartner, 1997b). A
number of cell survival factors, such as EGF, IGF-I, IL-3 and TGF-β1 induce the
phosphorylation of Bad at serine residues 112 and 136, which both have been shown
to be instrumental in signalling cell survival (Zha et al., 1996; Schurmann et al.,
2000; Zhu et al., 2002).
The relative expression of Bad in irradiated HSE-KC and HSE-KCF
composites was therefore analysed to detect potential differences in the presence of
this central apoptosis-regulator following UVB exposure. Figure 4.18 (A) shows
relative expression levels of Bad in irradiated and control HSE composites. No
significant differences in total Bad levels were observed in mock-irradiated controls,
as well as UVB-treated HSE constructs at the 3 and 6 hour post-irradiation time-
points. At 24 hours following their exposure to UVB, however, there were
significantly lower levels (p<0.01) of Bad present in the HSE-KCF, as compared to
126 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
that of the HSE-KC. This indicates that the inhibition of keratinocyte apoptosis
observed in HSE-KCF (Figure 4.17) may be attributed to decreased levels of Bad as
a result of fibroblast-secreted factors.
To further confirm this, the relative amounts of phosphorylated Bad (at serine
112) were also measured in these samples. Results from Figure 4.18 (B) show similar
relative quantities of phospho-Bad in HSE composites with and without fibroblasts in
the mock-irradiated samples, as well as those sacrificed 3 hours after UVB exposure.
However, at the later time-points, the levels of phospho-Bad in the irradiated HSE-
KC appeared to decline, while those of the HSE-KCF increased with time. This
resulted in significantly higher levels of phospho-Bad in keratinocyte cell lysates
from the HSE-KCF, as compared to those in the HSE-KC (p<0.05). Since the
phosphorylation of Bad at this particular residue is critical for maintaining cell
survival, the elevated phospho-Bad observed in the HSE-KCF indicates that this may
be a mechanism by which fibroblasts exert anti-apoptotic effects in irradiated
keratinocytes.
Figure 4.18. Relative expression of Bad and phosphorylated-Bad in irradiated HSE-KC and HSE-KCF
The relative levels of total Bad (A) and phosphorylated-Bad (B) apoptotic proteins in irradiated and non-
irradiated controls of HSE-KC and HSE-KCF were measured using a sandwich ELISA antibody assay. HSE-KC
and HSE-KCF were exposed to a single dose of 2 MED UVB or to a mock irradiation in the control samples. The
HSE composites were sacrificed at 3, 6 and 24 hours post-irradiation and epidermal cell lysates obtained. Equal
quantities of total keratinocyte lysates (based on protein concentration) were added to 96-well plates pre-bound
with antibodies against human Bad (A) or Bad phosphorylated at serine 112 (B). Measured absorbances indicate
relative levels of target antigens in cell lysates samples. Statistical significance of p<0.05 is represented by ‘*’,
while p<0.01 is denoted by ‘#’. The results represent mean values from experiments performed in triplicate, each
using samples obtained from independent skin donors. Error bars represent SEM values.
Time post-irradiation
Ab
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m)
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A B
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 127
The nuclear enzyme PARP has been shown to strongly bind to DNA strand
breaks occurring as a result of environmental stress, thus being an important
mediator of DNA repair (Satoh and Lindahl, 1992). The cleavage of PARP into the
89 and 24 kDa fragments by caspases, such as caspase-3, has been shown to inhibit
DNA repair and facilitate cellular apoptosis (Boulares et al., 1999). Using an ELISA
targeted against the large (89 kDa) fragment of cleaved PARP, I aimed to identify
differences in the relative levels of this protein fragment between HSE-KC and HSE-
KCF as a measure of apoptotic activity. In Figure 4.19, the relative levels of cleaved
PARP detected in epidermal cell lysates from these two composite types are shown.
At the 3, 6 and 24 hour time points after irradiation, there appears to be a slight
increase in the detected cleaved PARP fragment in the irradiated HSE-KC, as
compared to the unexposed samples. Matched samples from the irradiated HSE-
KCF, however, did not show a significant deviation from the non-irradiated controls.
Also, while there was a trend towards relatively lower levels of the PARP fragment
over all these time-points in the HSE-KCF in relation to the HSE-KC samples, this
difference did not prove to be statistically significant.
As previously described, caspase-3 is cleaved downstream of caspase-8,9 and
10, thus catalysing the cleavage of several essential cellular proteins (Porter and
Janicke, 1999). This caspase belongs to the family of downstream or executioner
caspases and along with caspases 6 and 7 is widely thought to be responsible for the
actual destruction of the cell during apoptosis (Slee et al., 2001). The PARP enzyme
is a substrate for cleavage (but not exclusively) by caspase-3, along with other
proteins critical to the apoptotic cascade such as α-fodrin and the DNA fragmentation
factor (Jänicke et al., 1998a).
128 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
Figure 4.19. Relative expressions of cleaved PARP in irradiated HSE-KC and HSE-KCF
The relative levels of cleaved PARP were detected in irradiated and non-irradiated controls of HSE-KC and HSE-
KCF and quantified using a sandwich ELISA antibody assay. The HSE-KC and HSE-KCF constructs were
exposed to a single dose of 2 MED UVB (coloured bars), or a sham irradiation treatment (black bars). The HSE
composites were then sacrificed at 3, 6 and 24 hours post-irradiation and epidermal cell lysates obtained. Equal
quantities of total keratinocyte lysates (based on protein concentration) were added to 96-well plates pre-bound
with antibodies against the 89 kDa fragment of human cleaved PARP. Measured absorbances indicate relative
levels of target antigens in cell lysates samples. The results represent mean values from experiments performed in
triplicate, each using samples obtained from independent skin donors. Error bars represent the calculated SEM.
Previously, it was demonstrated that fibroblast-keratinocyte co-culture prior to
UVB irradiation diminished levels of cleaved caspase-3 in keratinocytes (Figure 4.5).
Keratinocyte cell lysates from UVB-exposed HSE-KC and HSE-KCF were also
probed for the presence of this caspase using an ELISA. Figure 4.20 shows the
relative levels of cleaved caspase-3 detected in these composites from control
samples, as well as irradiated HSEs, sampled at 3, 6 and 24 hours post-irradiation. In
the HSE-KCF samples, no appreciable differences in the levels of cleaved caspase-3
were detected in the first 24 hours, as compared to the non-irradiated controls. The
samples obtained from the HSE-KC, however, showed a steady incline from the
levels detected in the sham-irradiated samples, which peaked at 24 hours post-UVB
exposure. At this time point, the difference between the cleaved caspase-3 levels in
the HSE-KC and the HSE-KCF was found to be statistically significant (p<0.05).
Time post-irradiation
Ab
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Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 129
Figure 4.20. Relative expressions of cleaved caspase-3 in irradiated HSE-KC and HSE-KCF
The relative levels of cleaved caspase-3 were detected and quantified in irradiated and non-irradiated controls of
HSE-KC and HSE-KCF using a sandwich ELISA antibody assay. The HSE-KC and HSE-KCF constructs were
exposed to a single dose of 2 MED UVB (coloured bars), or a sham irradiation treatment (black bars). The HSE
composites were then sacrificed at 3, 6 and 24 hours post-irradiation, followed by the extraction of epidermal cell
lysates from these skin equivalents. Equal quantities of total keratinocyte lysates (based on protein concentration)
were added to 96-well plates pre-bound with antibodies against the large fragment (17/19 kDa) of activated
caspase-3, formed as a result of cleavage adjacent to the Asp175 site. Measured absorbances indicate relative
levels of target antigens in cell lysates samples. Statistical significance of p<0.05 is represented by ‘*’. The
results represent mean values from experiments performed in triplicate, each using samples obtained from
independent skin donors. Error bars represent the calculated SEM.
Time post-irradiation
Ab
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450 n
m)
Contr
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hr6
hr
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Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 130
4.3.8 Fibroblast-induced regulation of p53 in irradiated keratinocytes
The p53 tumour suppressor protein has long been established as being an
integral component in starting and propagating cellular photoresponses via the
activation of DNA repair, growth arrest and apoptotic pathways (Benchimol et al.,
1982; Ananthaswamy and Kanjilal, 1996; Lozano and Elledge, 2000; Caulin, 2007;
Cui, 2007). In vitro, this 53 kDa phospho-nucleoprotein has been shown to be
expressed in keratinocytes in a UVB dose and time-dependent manner, with its
expression levels known to be influenced by factors such as the surrounding ECM
and the differentiation status of the cell (Cotton and Spandau, 1997; Kumar et al.,
1999). Upon UVB-induced damage, p53 proteins are stabilised and activated via a
phosphorylation-mediated disruption of the inactive MDM2-p53 complex and
subsequently translocate to the nucleus (Hall et al., 1993; Matsumura and
Ananthaswamy, 2002). Depending on the cell and environmental stressor type,
increases and reductions in p53 mRNA and protein levels have been observed
experimentally following cellular insult (Reich and Levine, 1984; Fritsche et al.,
1993).
Based on the modulation of many known p53-mediated UVB response
processes between HSE-KC and HSE-KCF, the next aim of this study was to
investigate changes to p53 expression on both gene and protein levels, as well as its
activation via phosphorylation in these irradiated skin constructs. Firstly, p53
expression analysis was performed on irradiated and control HSE composites using
qRT-PCR. The mRNA levels of p53 in the HSE-KC, expressed relative to samples
from the HSE-KCF constructs, are represented in Figure 4.21. The expression of this
gene in the non-irradiated controls and at the 3 hour post-irradiation time point
showed no significant difference between the two HSE types. Interestingly, however,
at the 6 and 24 hour time points, an average of about a 500 and 280-fold upregulation
in p53 expression, respectively, was observed in the HSE-KC relative to the HSE-
KCF. This significant alteration in p53 expression levels in the irradiated skin
composites lacking dermal fibroblasts strongly suggests a marked shift in the p53-
mediated response to UVB in these constructs.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 131
Figure 4.21. Relative expression of p53 in HSE-KC and HSE-KCF after UVB irradiation.
The relative post-irradiation gene expression of p53 in HSE-KC constructs was determined using qRT-PCR,
normalised to matched data observed in the HSE-KCF. Data shows changes in p53 expression in the non-
irradiated control skin composites, as well as from HSE-KC and HSE-KCF harvested at 3, 6 and 24 hours after
their exposure to a single dose of 2 MED UVB. A significant upregulation in p53 expression in HSE-KC was
found to take place at the 6 and 24 hour time points, as compared to that in the HSE-KCF samples. At these time
points, an average of about a 500-fold and 280-fold upregulation of p53 was observed at the 6 and 24 hour time
points, respectively. The dotted line indicates a 1.8-fold difference to the control. The results represent mean
values from experiments performed in triplicate, each using triplicate samples obtained from independent skin
donors. Asterisks represent statistical significance (p<0.05). Error bars represent SEM values.
Following the discovery of altered p53 mRNA levels in the HSE-KC as
compared to the HSE-KCF using qRT-PCR, the next aim was to validate this result
using protein quantification methods. Firstly, the total amount of p53 was measured
in epidermal cell lysates from these two HSE types using a sandwich ELISA (with a
pre-bound polyclonal antibody against human p53) and Western blot (using a
monoclonal antibody specific for an epitope between the N-terminal amino acids 37
and 45 in p53) (Figure 4.22 A and B). From the p53 ELISA, it was observed that the
total p53 detected in HSE-KC keratinocyte lysates appeared to be raised slightly at
all the post-irradiation time-points (as compared to that in the controls). In the HSE-
KCF, however, an elevation in relative p53 levels from the non-irradiated control
Time post-irradiation (hr)
Re
lativ
e g
ene
exp
ressio
np
53
Control 3 6 240.1
1
10
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1000
HSE-KCFHSE-KC
**
132 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
was seen at 3 hours after UVB exposure, which progressively diminished at the 6 and
24 hour marks. At the 6 and 24 hour post-irradiation time points, the HSE-KC
composites showed significantly raised p53 levels in relation to those in the HSE-
KCF (p<0.05, p<0.01, respectively). The Western blot using a monoclonal p53
antibody showed similar levels of p53 at 3 and 6 hour post-irradiation and between
the two irradiated skin constructs (Figure 4.22B). However, in contrast to the ELISA
results, the HSE-KCF showed slightly elevated p53 levels, as compared to that of
HSE-KC, at 24 hours and in non-irradiated controls.
The phosphorylation of p53 at key serine sites is thought to be a critical process
in its stabilisation and subsequent activation (Jimenez et al., 1999; Ljungman, 2000).
Therefore, the relative proportions of phospho-p53 were analysed between the HSE-
KC and HSE-KCF cell lysates using an ELISA (pre-bound with an antibody against
p53 phosphorylated at serine 15) (Figure 4.22C). The levels of phospho-p53 were
noted as being relatively similar between the irradiated HSE-KC and HSE-KCF
samples at the 3 and 6 hour points. At 24 hours, a drop in phospho-p53 was seen in
both skin equivalents, with significantly higher levels seen in the HSE-KCF.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 133
Figure 4.22. Relative levels of p53 protein in irradiated HSE-KC and HSE-KCF composites
The relative protein expression of total p53 in irradiated HSE-KC and HSE-KCF from epidermal cell lysates was assayed using an ELISA (A) and validated using Western blot analysis (B).
Samples were sacrificed at different time points post-irradiation and epidermal cell lysates obtained. Equal quantities of total keratinocyte lysates from irradiated and control (non-irradiated)
HSE constructs (based on protein concentration) were probed with antibodies against human p53. Activated p53 was analysed using an ELISA assay for p53 phosphorylated at serine 15 (C). In
the ELISA assays, measured absorbances indicate relative levels of target antigens in cell lysates samples. Statistical significance of p<0.05 is represented by ‘*’, while p<0.01 is denoted by ‘#’.
The results represent mean values from experiments performed in triplicate, each using triplicate samples obtained from independent skin donors. Error bars represent SEM values.
Time post-irradiation
Ab
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m)
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hr6
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r
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*
Time post-irradiation
Ab
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r
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#
Time post-irradiation
Ab
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Time post-irradiation
Ab
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Total p53 Phospho-p53A C
B
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 135
4.4 DISCUSSION
A wide range of developmental, homeostatic and disease processes in skin that
are influenced by fibroblast-keratinocyte interactions have been identified, ranging
from wound healing to psoriasis (Schultz et al., 1987; Priestley and Lord, 1990; Wu
et al., 1996; Maas-Szabowski et al., 2000; Ghahary and Ghaffari, 2007). The full
significance of these epithelial-mesenchymal interactions in the cutaneous UV-
response, however, still requires further investigation. The study reported herein
describes for the first time the use of two in vitro systems to model these paracrine
interactions experimentally. The Transwell® co-culture technique and the 3D HSE-
KCF skin model were successfully utilised in the identification of key fibroblast-
mediated influences on keratinocyte responses to UVB in the acute phase.
Importantly, dermal fibroblasts were shown in this Chapter to influence keratinocyte
survival, apoptosis and the repair of UVB-induced DNA damage in these models, all
of which are processes playing central roles in photocarcinogenesis (Black et al.,
1997; Claerhout et al., 2006). Furthermore, the presence of fibroblasts in the
keratinocyte culture system modulated the activation of p53, a central regulator of
the normal cellular response to UVB exposure. Overall, these results strongly suggest
that fibroblast-secreted factors may play a critical role in regulating the epidermal
photoresponse and determining cellular fate following UVB irradiation.
In vitro models for studying dermal-epidermal interactions
Studies into the cellular processes surrounding the epidermal response to UVB
have employed numerous experimental models, from organotypic skin constructs to
elegant transgenic mouse models (Nakazawa, 1998; Duval et al., 2003; Noonan et
al., 2003; Duval et al., 2012). Indeed, such investigations have shed light on a
multitude of biological pathways involved in acute and chronic photodamage and the
risk of oncogenic transformation in the epidermis. Nonetheless, physiologically-
relevant in vitro models with the flexibility to incorporate various primary human
skin cell types are still limited in the study of paracrine interactions occurring during
the UV response. Conventional 2D primary keratinocyte monolayer models may not
be suitable for this application as they are commonly cultured together with a mouse
fibroblast feeder layer, thus diverging from physiological conditions in vivo, in which
these cell populations exist in different skin regions, separated by the BM
(Rheinwald, 1975; Alitalo et al., 1982).
136 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
To address this limitation of traditional keratinocyte cell culture methods, the
application of a two-tiered culture system was employed to study cellular interactions
between human keratinocytes and fibroblasts. Indeed, emerging technologies such as
this Transwell® co-culture system can provide a versatile tool for investigating
paracrine interactions between two distinct cell populations, with several advantages
over other experimental skin models. Firstly, the permeable membrane separating
these two cell monolayers allows for the easy visualisation of the cells seeded on its
surface using various imaging techniques (Kandyba et al., 2008). Thus, the need for
time-consuming histological and immunohistochemical techniques required for
whole tissue samples is negated using such models. Next, the detachable cell culture
inserts in this system can be easily moved for the purpose of UV irradiation, or to be
transferred to different culture or treatment condition. Moreover, keratinocyte
monolayers on the membrane inserts can be cultured at the air-liquid interface to
promote the formation of a stratified epidermis, which more closely mimics the in
vivo situation (Fartasch and Ponec, 1994; Parnigotto et al., 1998; Ng and Ikeda,
2011). Limitations associated with some skin equivalent models, such as the
requirement for relatively larger amounts of donor skin tissue, the longer processing
and culture times and bigger volumes of cell culture medium, are reduced using the
Transwell® culture system. Importantly, keratinocytes cultured in this system have
previously been shown to possess cellular properties that paralleled the in vivo
situation in terms of their morphology, proliferative capacity and differentiation
patterns (Kandyba et al., 2008).
Previous studies have proposed that dermal fibroblasts may be central in
influencing the activation of UV stress responses in keratinocytes (Lewis et al.,
2008; Kovacs et al., 2009; Lewis et al., 2010). However, the nature of these
interactions, in terms of whether fibroblasts exert a protective effect on keratinocytes
prior to UVB insult, or aid in triggering post-irradiation repair mechanisms, is still
unclear. The Transwell® culture system utilized in this study provided a highly-
advantageous tool for examining these effects, with the potential to easily manipulate
keratinocyte culture conditions prior to and after irradiation. Significantly, it was
observed that fibroblast co-culture with keratinocytes before UVB treatment was
most effective at protecting against apoptosis and the overall reduction in
keratinocyte viability (Figure 4.2B, Figure 4.4 Figure 4.5). This observation suggests
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 137
that the activation status of keratinocytes by fibroblast-secreted factors at the time of
UVB exposure may be critical in determining the efficacy of downstream survival
post-irradiation. Notably, a similar effect has been observed in related in vivo studies,
with growth factors produced by fibroblasts shown to protect cells of the salivary
gland epithelium from radiation-induced apoptosis (Grundmann et al., 2010). In
addition to this pro-survival effect, a significant enhancement of CPD photolesion
repair was observed in keratinocyte monolayers cultured in fibroblast conditioned
medium before being exposed to UVB (Figure 4.7). The stimulation and activation
of DNA repair processes following genotoxic insult via the action of secreted factors
has also been demonstrated experimentally in a variety of other tissue types (Wu et
al., 1998; Huang and Harari, 2000; Kanamoto et al., 2002). Besides epithelial-
mesenchymal signals that can augment repair responses, studies demonstrate that α-
MSH (acting via autocrine and paracrine mechanisms) also greatly improves the
removal of DNA damage in UV-exposed keratinocytes (Dong et al., 2010). Overall,
the data obtained in this study further supports these previously-documented studies
showing a clear link between mesenchymal and epithelial cell populations during
regeneration.
Skin equivalent models have been widely used in a diverse range of cutaneous
biology studies, as an alternative to 2D cell culture and animal models (Nakazawa et
al., 1997; Bernerd and Asselineau, 2008; Egles et al., 2010). Fundamentally, such
tissue-engineered organotypic skin models have been shown to more accurately
represent structural and physiological aspects of native skin over submerged
monolayer cultures (Prunieras et al., 1983; Rosdy and Clauss, 1990). In the previous
Chapter, the use of a HSE-KC 3D skin photobiology model was validated and was
shown to retain several important morphological and biological features of human
skin. In Chapter 4, this tissue-engineered skin construct was further developed
through the addition of primary dermal fibroblasts into the DED component of this
model. Hence, it was hypothesized that this HSE-KCF model could then be applied
to studying how dermal-epidermal interactions influence key keratinocyte UVB
responses in the epidermis.
Studies into the optimal method for HSE-KCF construction reveal fibroblasts
seeded onto the papillary surface of the DED, followed by the subsequent addition of
primary keratinocytes, achieves the best result in terms of fibroblast migration into
138 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
the DED and epidermal morphogenesis (Figure 4.8). This is further supported by the
expression of the mesenchymal tissue marker, vimentin, in the DED of HSE-KCF
constructs (Figure 4.10). Histological analysis suggests that fibroblasts may migrate
into the dermal compartment by remodelling the BM layer. This represents a process
occurring in vivo during wound-healing, in which activated fibroblasts migrate into
the wound bed while producing an abundance of ECM factors and proteases required
for remodelling the healing tissue (Abraham et al., 2007). Additionally, it was
observed that upon maturation of the HSE-KCF by culture at the air-liquid interface,
the BM zone at the epidermal-dermal junction was found to be comparable with that
of native skin immunohistochemically (Figure 4.9). Indeed, it has been shown that
certain sub-populations of fibroblasts work in conjunction with epidermal cells to
assemble the BM during skin regeneration (Marinkovich et al., 1993). However,
despite fibroblast infiltration into the DED, inconsistencies associated with the
incorporation of cells into the HSE-KCF remained an issue (Figure 4.11). This may
be due to individual variations in the ex vivo DED tissue between skin donors, or
differences in the sub-type of isolated fibroblasts, which may account for a
diminished migratory capacity. To counteract this effect, alternative approaches such
as the use of a DED scaffold in conjunction with a fibroblast-populated synthetic
collagen layer can be employed in future to increase consistency of the dermal layer
within in vitro skin equivalents (Lee et al., 2000; Lee and Cho, 2005; Rhee, 2009;
Ahn, 2010). However, dermal contraction and tissue instability has been reported
with the use of synthetic collagen scaffolds (Bell et al., 1979). Alternatively, a
centrifugal fibroblast seeding technique as described by El Ghalbzouri and
colleagues (2002) may aid in enhancing the reproducibility of fibroblast
incorporation into the DED. Moreover, further characterisation of the fibroblasts
within the DED needs to be conducted, as the dermis in vivo is known to contain a
heterogeneous population of fibroblastic cells (Sorrell, 2004; Sorrell et al., 2007).
The HSE-KCF represents a novel and versatile tool for mapping epithelial-
mesenchymal interactions during the UVB response, with the potential to be
improved via the incorporation of a range of cutaneous cell populations. Importantly,
the HSE-KCF can be utilized as an in vitro experimental model to be used as an
alternative to in vivo studies in human volunteers or animal models (Quan et al.,
2004). Potentially, for example, primary skin cells cultured from a diverse range of
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 139
donors, such as those with specific skin pathologies or an increased risk of cancer
development can be incorporated into this model to identify pathways leading to
photocarcinogenesis. Likewise, various other radiation sources, such as solar-
simulated UVR, can be used to gain a more accurate picture of how environmental
sources of radiation impact the skin. Pigment-producing melanocytes are paramount
in the protection of keratinocytes against the damaging effects of UVB radiation
(Yaar and Gilchrest, 1991; Yamaguchi et al., 2007). Thereupon, the addition of
epidermal melanocytes into the HSE-KCF model in future would produce a more
robust skin model for understanding the mechanisms involved in the keratinocyte
photoresponse. To date, numerous pigmented organotypic skin models have been
described, with mixed results in terms of their congruence to the in vivo situation
(Bertaux et al., 1988; Todd et al., 1993; Nakazawa et al., 1997; Lee et al., 2007;
Duval et al., 2012). However, the intricate biology of the epidermal melanocyte unit
and the complex regulation of melanocyte proliferation and activity still represents
an ongoing challenge to model in vitro (Halaban, 2000; Czajkowski, 2011).
Conceivably, paracrine signals from fibroblast and keratinocyte populations, as well
as the preservation of essential elements of the BM in the melanocyte
microenvironment, may be favourable in the future development of a pigmented
HSE-KCF skin model (Scott and Haake, 1991).
Enhancement of keratinocyte survival post-irradiation by fibroblasts
The analysis of irradiated HSE-KCF composites reported herein revealed
several important modes by which fibroblasts are capable of enhancing cellular
survival and the regeneration of the epidermis after UVB exposure. In parallel with
the data obtained from 2D co-culture studies, keratinocytes in the UVB-treated HSE-
KCF constructs were observed to be more resistant to apoptosis and showed
accelerated rates of CPD removal compared to those in the HSE-KC (Figure 4.17
and Figure 4.15). The inhibition of cell death pathways in the epidermis HSE-KCF
was most pronounced during the first 24 hours following irradiation, a critical period
in which the majority of many types of photolesions are repaired by DNA repair
machinery (Ruven et al., 1993; You et al., 2001; Zheng et al., 2001; Snellman et al.,
2003). In similar HSE-KC constructs lacking fibroblasts, however, both significant
increases in TUNEL-positive epidermal cells, as well as the proportion of CPD-
positive keratinocytes, were observed during this time period. This further reinforces
140 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
the importance of epidermal-dermal interactions in maintaining the functionality and
integrity of the epidermis in the acute phase following UVB exposure.
Sunburn as a result of UVR exposure is a characteristic clinical response to
erythemal doses of UVB (Young, 2006). Direct DNA damage in the epidermis is
thought to be an important activator of this UVR-induced inflammation, with the
production of cytokines being pivotal in initiating this response (Enk et al., 1995;
Krutmann and Grewe, 1995; Brink et al., 2000a; Grandjean-Laquerriere et al., 2003).
The elevated expression of these cytokines in response to UVB has also been
observed both in vivo and in various skin models in vitro (Urbanski et al., 1990;
Kondo et al., 1993; de Vos et al., 1994; Bechetoille et al., 2007; Yoshizumi et al.,
2008).
In Chapter 3, it was reported that UVB radiation initiated the upregulated
expression of the inflammatory cytokines IL-6 and IL-8 in the HSE-KC model
(Figure 3.7). Herein, it was observed from the quantification of these cytokines in the
cell culture culture medium from HSE-KCF constructs, that UVB treatment
significantly increased the levels of both IL-6 and IL-8 (Figure 4.13). Notably, the
detected levels of both these cytokines were also relatively higher than those detected
from the UVB-treated HSE-KC. This may be in line with studies that have shown
paracrine signals from the epidermis stimulate the secretion of IL-6 and IL-8 by
dermal fibroblasts (Boxman et al., 1996). Indeed, responses in keratinocytes to
external stimuli have been shown to induce the production of pro-inflammatory
cytokines in fibroblasts via the action of IL-1α, which would contribute to the
elevated levels of these factors in the HSE-KCF system (Boxman et al., 1996).
While the current HSE-KCF skin model lacks vasculature, which is also a key
factor in the sunburn response, ongoing work in our laboratory towards incorporating
microvascular endothelial cells into this skin construct would enable future
investigations into these processes. Other organotypic skin models have also been
previously described, consisting of endothelial cells and dermal fibroblasts cultured
in synthetic collagen scaffolds (Hudon et al., 2003). Moreover, elements of the
cutaneous immune system, such as Langerhans cells, have also been shown to play a
role in orchestrating the inflammatory response following UVB exposure (Simon et
al., 1991; Denfeld et al., 1998). Accordingly, immunocompetent in vitro skin
equivalent models can be employed in future to better understand the relationship
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 141
between the innate immune system and the regulation of epidermal responses to
UVB radiation (Régnier et al., 1997; Bechetoille et al., 2007; Laubach et al., 2011).
Apoptosis is a critical event underlying homeostatic maintenance as well as
acting as a safety net against oncogenic transformation (Fadeel et al., 1999).
Therefore, it is not surprising that the elimination of damaged cells via apoptosis is a
fundamental mechanism by which the skin is protected against the carcinogenic
potential of UVR (Lippens et al., 2009). Both intrinsic pathways, such as the
initiation of apoptosis by DNA damage or the generation of ROS, and extrinsic
pathways, by death receptor signalling, contribute to UVB-induced keratinocyte cell
death (Chow and Tron, 2005). What still remains relatively obscure are the
extracellular and intercellular factors that can influence the apoptotic program,
together with how these elements may contribute to the formation of premalignant
cells. Using 2D and 3D co-culture techniques, HSE-KC and HSE-KCF skin models,
an association between the presence of fibroblasts and the inhibition of keratinocyte
apoptosis in response to UVB was reported (4.3.3 and 4.3.6). In view of this, the
expressions of key members of the UVB-induced apoptotic cascade were
investigated in order to further dissect the mechanism by which fibroblasts conferred
their protective effects upon keratinocytes.
The Bcl-2 related protein, Bad, is a key promoter of cellular apoptosis by
dimerising and thus inactivating the prosurvival proteins Bcl-2 and Bcl-xL (Kelekar
et al., 1997). The phosphorylation of Bad at Serine 112 and 136 has been correlated
with its interactions with 14-3-3 proteins, thus promoting cell survival (Gajewski and
Thompson, 1996). The involvement of Bad in regulating apoptotic and cell survival
responses to UVB-irradiation was therefore studied in the HSE-KC and HSE-KCF
models (Figure 4.18). In line with the inhibition of keratinocyte apoptosis observed
previously in irradiated HSE-KCF composites (Figure 4.17), this study showed an
associated reduced expression of Bad post-irradiation in the HSE-KCF, compared to
that of the HSE-KC (Figure 4.18A). Both the upregulation of Bad expression, as well
as its phosphorylation, act as regulators of the post-irradiation apoptotic program and
have been well-documented in vitro (She et al., 2002; Yang et al., 2006). In view of
this, the levels of phosphorylated Bad were also quantified in the epidermal extracts
from the irradiated HSE constructs (Figure 4.18B). The significantly higher
proportions of phospho-Bad at the 6 and 24 hour post-irradiation time points in the
142 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
HSE-KCF indicate that this mechanism may be pivotal in enhancing the keratinocyte
survival effect brought about by fibroblast-secreted mediators. Interestingly, the
activation of the Akt, MAPK and PI3-K pathways by fibroblast-synthesized growth
factors have all been implicated in controlling the phosphorylation status of Bad
(Datta et al., 1997; Peso et al., 1997; Kulik and Weber, 1998; Shimamura et al.,
2003). Of these fibroblast growth factors, IGF-I in particular has been shown to
promote its prosurvival effects via inducing Bad phosphorylation in a variety of cell
types (Kulik, 1997; Kulik and Weber, 1998; Fang et al., 1999; Raile et al., 2003;
Fernandez et al., 2004). Therefore, the regulation of Bad activity by fibroblast-
produced factors may represent a critical step in orchestrating keratinocyte responses
to UVB radiation.
The cleavage of the nuclear enzyme PARP is known to be another key
mechanism through which DNA-damaged keratinocytes are eliminated following
UVB exposure (Aragane et al., 1998; Shimizu et al., 1999). Under physiological
conditions, PARP functions to repair damaged DNA, and in conjunction with the
action of several DNA repair mechanisms maintains the integrity of the genome
(Benjamin and Gill, 1980). In cells harbouring severe DNA damage, the cleavage of
PARP by the action of various caspases is believed to be a hallmark feature in UVB-
induced apoptosis (Tewari et al., 1995; Kim et al., 2000). Using the HSE-DED
models, we reported elevated levels of cleaved PARP as a result of UVB irradiation
in the HSE-KC composites compared to that observed in the HSE-KCF (Figure
4.19). However, no significant differences were observed between these composites
at the various sampling time points. Potentially, this could be a result of the post-
irradiation sampling time points chosen. Indeed, other in vitro studies have shown
changes to UVB-induced levels of cleaved PARP in keratinocytes to occur as early
as 1.5 hours following irradiation (Han et al., 2011). Hence, future studies should
investigate the earlier cellular events occurring in response to UVB radiation.
The activation of caspases is known to be yet another essential step in the
execution of cells damaged by UVR (Mancini et al., 1998; Denning et al., 2002;
Sitailo et al., 2002). The cleavage and subsequent activation of caspase-3 is therefore
an important marker of UVB-induced apoptosis (Cejudo-Marin et al., 2012). In the
irradiated 3D skin constructs, samples from the HSE-KC were found to have
considerably elevated levels of cleaved caspase-3 at the 24 hour time point (Figure
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 143
4.20). Furthermore, the presence of fibroblasts was also shown to cause a diminished
expression of cleaved caspase-3 in irradiated keratinocytes cultured in a cell
monolayer format (Figure 4.5). Paracrine interactions between the dermis and
epidermis have indeed been shown to be involved in the activation of such caspases
under both pathological and physiological conditions (Funayama et al., 2003; Lewis
et al., 2010). The modulation of caspase-3 activation through the action of
fibroblasts, therefore, represents yet another mechanism by which the dermis may
influence epidermal cell survival in response to UVB exposure. The roles of other
members of the caspase family in maintaining epidermal function after acute
photodamage also requires further investigation. For example, caspase-14 has been
implicated in regulating terminal differentiation in irradiated keratinocytes to inhibit
replication while maintaining the barrier function of the epidermal layer (Denecker et
al., 2008). Similarly, caspase-9 has been demonstrated to play a fundamental role
upstream in the caspase cascade in activating apoptosis in UVB-damaged cells
(Sitailo et al., 2002).
Fibroblast-keratinocyte interactions influencing p53 activity during the acute
UVB response
In the past 30 years, p53 has been one of the most widely-studied tumour
suppressor genes due to its central role in numerous cell biology and cancer
development pathways (Leppard and Crawford, 1983; Levine et al., 1991;
Blagosklonny, 2000). Importantly, p53 exerts its affects as the ‘guardian of the
genome’ by regulating aspects of DNA repair mechanism, cell-cycle progression and
various apoptotic pathways (Lane, 1992). Under homeostatic conditions, p53 levels
are downregulated via the action of inhibitor proteins such as Mdm2, which promote
the degradation of p53 through ubiquitin proteolytic pathways (Kubbutat et al., 1997;
Kubbutat et al., 1998). Consequently, p53 has a rapid turnover rate and has been
detected at low levels in epidermal cells (Bottger et al., 1997). Cellular insult by
UVB radiation results in rapid elevation in p53 levels, along with enhanced stability,
half-life and DNA-binding function of the protein via post-translational
modifications and phosphorylation (Gu and Roeder, 1997). Indeed, in vitro studies in
mouse keratinocyte cultures have demonstrated that this UVB-induced increase in
p53 protein and post-transcriptional modification levels is associated with relatively
unchanged mRNA levels (Liu et al., 1994).
144 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
The expression and activity of p53 in irradiated HSE-KC and HSE-KCF skin
constructs was investigated herein, to identify potential differences to UVB-induced
p53 activity from fibroblast-secreted factors (4.3.8). From this study, I report that
p53 mRNA levels were shown to be differentially upregulated in the HSE-KC
constructs at 6 and 24 hours after UVB treatment, as compared to matched samples
from the HSE-KCF (Figure 4.21). At these time points, the p53 protein quantified
using the polyclonal antibody-based ELISA showed significantly elevated levels, in
relation to the HSE-KCF (Figure 4.22A). On the other hand, p53 detected using a
monoclonal antibody in the Western blot analysis showed a slightly diminished HSE-
KC p53 band in contrast to that of the HSE-KCF (Figure 4.22C). Collectively, this
suggests that while UVB exposure triggers the increased expression of p53 in the
HSE-KC, this may not be associated with a matched enhancement of p53 protein
stability, thus making it susceptible to degradation and a short half-life. This would
account for the detection of fragments (a product of proteolysis) by the
abovementioned polyclonal antibody. Furthermore, at 24 hours post-irradiation, the
HSE-KCF samples had proportionately higher phosphorylated p53 as opposed to that
in the HSE-KC (Figure 4.22C). This further supports the theory that dermal
fibroblasts may play a role in enhancing p53 activation through improving its
stability and phosphorylation status. Indeed, it has been shown previously that
fibroblasts can directly affect UVB-induced p53 activation in keratinocytes in vitro
through the epidermal microenvironment (Will, 2000). Intriguingly, recent evidence
has also suggested that fibroblast-produced growth factors can aid in p53-dependent
keratinocyte senescence, which allows for the inhibition of apoptosis in irradiated
cells, while maintaining cellular metabolism (Handayaningsih et al., 2012). Taken
together, this implies that fibroblast interactions with keratinocytes may influence the
cell death and survival balance via p53-dependent actions upon UVB exposure.
Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response 145
Conclusions and significance
The apoptotic, cell survival and repair programs all play substantial roles in the
removal of UV-damaged cells and the maintenance of the epidermal barrier function
(Cotton and Spandau, 1997; Bernstein et al., 2002; Assefa et al., 2005; Garner and
Raj, 2008). In light of this, it is clear that dysregulation or deviations from these
protective mechanisms may have serious long-term consequences in the epidermis.
Indeed, theories implicating the reduced activity of dermal fibroblasts in the onset of
skin cancers in aging skin have been proposed (Gillman et al., 1955; Lewis et al.,
2008; Lewis et al., 2010). These studies have demonstrated that diminishing levels of
fibroblast-secreted factors directly impact on DNA repair and uncontrolled
proliferation in irradiated keratinocytes, thus contributing to NMSC development.
The need to more fully understand the complexities of these dermal-epidermal
interactions in the acute and chronic response to UV-irradiation is therefore apparent.
In order to achieve this, improved in vitro systems for testing the biological
effects of UVB in keratinocytes and the paracrine interactions regulating these
processes need to be developed and characterised. In Australia, commercially
manufactured 3D skin culture models such as EpiSkin® (L’Oreal, Paris, France) and
EpiDerm™ (MatTek, Massachusetts, USA) are not readily available due to
quarantine laws, international transport logistics and cost issues (Poumay et al.,
2004). Moreover, while rigorous standards of protective strategies to minimise
photodamage have been generated to reduce the incidences of skin cancers in the
Australian public, the availability of effective therapeutic agents with minimal side
effects to counteract skin photodamage are still lacking (Reichrath, 2003; Shih et al.,
2009). Current gold standard therapeutics for photodamaged skin include
fluorouracil, imiquimod, photodynamic therapy and microdermabrasion, which
induce epidermal damage to activate skin regeneration processes (Orringer et al.,
2008; Karimipour et al., 2009; Sachs et al., 2009; Firnhaber, 2012). Thus, the
availability of improved in vitro skin models for photobiology, such as the HSE-KCF
described herein, may assist in the generation of novel and improved therapeutics
and preventative strategies against the harmful effects of UVR exposure.
Furthermore, increasing our understanding of the complex biological processes
surrounding the acute epidermal photoresponse and the links between the
dysregulation of these events during photocarcinogenesis is an essential next step.
146 Chapter 4: Fibroblast-keratinocyte interactions and the acute UVB response
Results from the study described in this Chapter further substantiate the role of
dermal fibroblasts in modulating keratinocyte responses to UVB, using the novel
application of Transwell® co-culture and HSE-KCF skin models. Importantly, the
presence of fibroblasts in these culture systems was shown to significantly enhance
keratinocyte viability, augment the expression of proinflammatory cytokines, inhibit
apoptosis and improve the rate of UVB-induced DNA photolesion repair. Moreover,
the activation of key members of the apoptotic cascade, including Bad, PARP and
caspase-3, were also shown to be affected by fibroblast-secreted factors.
Significantly, the study conducted herein reports the modulation of the functional
status of p53, a tumour suppressor gene central in the protective response against
UV-damage, by the presence of fibroblast-secreted factors. Collectively, these results
provide further evidence towards the associations between epidermal and
mesenchymal cells during the UVB response, indicating a strong correlation between
paracrine signals from the dermis and their influence on keratinocyte repair
responses during epidermal regeneration (Saiag et al., 1985; Priestley and Lord,
1990; Werner and Smola, 2001).
In Chapter 5, the influence of IGF-I, a key fibroblast-secreted factor involved
in epithelial-mesenchymal crosstalk, on the processes of keratinocyte survival, repair
and proliferation was further explored. Various soluble factors synthesized by
fibroblasts have been shown to induce proliferative and reparative effects in
keratinocytes, including KGF and IL-1β (Maas-Szabowski et al., 1999; Witte and
Kao, 2005). Of these, the IGF system, with its potent ability to promote cell survival
and proliferation, has been implicated in directing epithelial growth and stress
responses (Ishihara et al., 2000). Hence, the involvement of IGF-I in modulating the
UVB response in keratinocytes was further investigated using the in vitro skin
models developed and described herein.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 147
Chapter 5: Investigating the role of IGF-I in
the keratinocyte UVB response
5.1 INTRODUCTION
The maintenance of epidermal integrity and the removal of damaged cells
following UVR exposure are complex, multi-factorial processes that are governed by
a network of autocrine and paracrine mechanisms (Aaronson et al., 1990; Baba et al.,
2005; Benitah, 2007). In the previous Chapter a tissue-engineered skin equivalent
model, incorporating both primary dermal fibroblasts and keratinocytes, was used to
demonstrate the importance of dermal-epidermal crosstalk in the orchestration of
UVB responses in the epidermis. Importantly, this data indicated that mesenchymal-
secreted factors are vital in maintaining the cell death-survival balance in the
irradiated epidermis by inhibiting apoptosis and promoting the removal of UVB-
induced DNA lesions. The mechanisms of apoptosis inhibition were also explored in
this study, with key members of the UVB-induced keratinocyte apoptotic cascade,
including Bad, caspase-3 and PARP, shown to be influenced by the presence of
dermal fibroblasts in the HSE-KCF. Hence, the next aim of this project was to
further investigate the cell survival and repair aspects of this keratinocyte-fibroblast
interaction during the UVB response, using the previously characterised in vitro
models described in Chapters 3 and 4. In particular, the IGF system, a central
mediator of epithelial-mesenchymal paracrine interactions, was studied to determine
its role in promoting keratinocyte survival following UVB exposure.
The activation of UVB-induced apoptosis is a critical step in preventing the
accumulation of mutated keratinocytes with the potential of developing into NMSCs
(Brash et al., 1996). Keratinocyte survival following UVB exposure, on the other
hand, is important in maintaining the integrity of the epidermal barrier and normal
skin homeostasis (Blanpain and Fuchs, 2009; Sotiropoulou and Blanpain, 2012).
However, the precise factors which decide the fate of UV-damaged keratinocytes
have yet to be mapped in detail. Broadly, it is known that the dose of UVB applied
and the balance of anti- and pro-apoptotic factors in the epidermal microenvironment
significantly influence this death-survival balance (Claerhout et al., 2006).
148 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
Numerous complex pro-survival signals in keratinocytes activate anti-apoptotic
pathways following UVB irradiation, with Bcl-2, survivin, NFκB and the PI3K/Akt
pathways among the main players involved in this process (Grossman et al., 2001;
Assefa et al., 2003; Wang et al., 2003; Beak et al., 2004). The activation of these
survival pathways acts to dampen apoptotic signals by interfering with the
mitochondrial death pathway and promoting the repair of damaged DNA (Assefa et
al., 2003). In the case of Bcl-2, for example, its overexpression potently blocks the
activation and translocation of the apoptotic protein Bax to the mitochondria, thus
blocking cytochrome c release and the subsequent activation of caspases in response
to UVB exposure (Van Laethem et al., 2004). Similarly, the activation of the
PI3K/Akt pathway by growth factors is central in promoting keratinocyte survival;
this is achieved in part by blocking the activities of caspase-9 and -3 (Decraene et al.,
2004; Claerhout et al., 2007). One such growth factor implicated in promoting cell
survival following UVB exposure via the PI3K/Akt pathway is IGF-I (Kulik, 1997).
The IGF system (as reviewed in 1.5) encompasses a broad network of growth
factor, ECM and cellular interactions, which in turn influence cellular functions and
responses via numerous endocrine, paracrine and autocrine mechanisms (Annunziata
et al., 2011). The activation of IGF receptors (by the peptide ligands IGF-I, IGF-II
and insulin) is known to play a significant role in normal growth and development,
and has also been implicated in elevating the risk and progression of various cancers
(Wood, 1995; Samani, 2007). As part of these diverse biological actions, IGF-IR-
mediated effects include short-term metabolic effects, such as the stimulation of
glucose uptake, along with long-term effects, including the regulation of cell cycle
progression, differentiation, apoptosis and DNA synthesis (Humbel, 1990).
In the skin, IGF-I is produced predominantly by dermal fibroblasts and acts in
a paracrine manner by subsequently stimulating basal keratinocytes, hence forming a
fundamental pathway in the dermal-epidermal crosstalk (Flier et al., 1986; Philpott et
al., 1994; Rudman, 1997). Physiologically, this interaction has been shown by
members of our research group as being central in orchestrating tissue repair and
remodelling during wound healing, as well as in other studies for the maintenance of
epidermal homeostasis (Edmondson et al., 2003; Hyde et al., 2004; Upton et al.,
2008; Kricker et al., 2010). While the involvement of the IGF-I system in the
cutaneous UVB response has been relatively less well-studied, in vitro and in vivo
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 149
data points towards a possible role for this growth factor system in the regulation of
cell survival pathways (Kulik, 1997; Kuhn et al., 1999; Lewis et al., 2008; Lewis et
al., 2010). Following cellular stress, downstream pathways triggered by the IGF-I
peptide ligand binding to IGF-IR have been shown to promote cell survival by
inhibiting intracellular apoptotic factors and enhancing cell survival signals (Figure
1.7) (Assmann, 2009). In terms of the photoresponse in keratinocytes, this
augmentation of cell survival has also been demonstrated experimentally to coincide
with the facilitation of DNA repair and the temporary arrest of the cell cycle (Héron-
Milhavet et al., 2001; Decraene, 2002). Furthermore, the activity of p53, a key
mediator in cell survival and repair processes post-irradiation, has been shown to be
strongly influenced by the functional status of the IGF-IR (Lewis, 2008).
With the IGF system playing central roles in many homeostatic and stress
responses in skin, it is not surprising that emerging evidence points towards its
potential role in skin cancer development. Indeed, in vitro studies have shown that
blocking IGF-IR activation directly impacts on the ability of keratinocytes to
undergo cell cycle arrest and adequate DNA repair processes (Lewis et al., 2010).
Furthermore, diabetic patients undergoing insulin therapy have been shown to
demonstrate lower rates of NMSC development, which is potentially correlated with
enhanced IGF-IR activation by elevated insulin levels in these individuals (Chuang et
al., 2005). Nonetheless, further investigations need to be conducted into the complex
network of IGF-mediated pathways and their role in epidermal responses to UVB.
As outlined in Chapter 4, the data obtained indicated a clear link between
fibroblast-synthesized factors and their importance in regulating major features of the
acute responses of keratinocytes to UVB exposure. The overriding aim of the studies
described in this current Chapter was therefore to investigate the role of IGF-I as a
key player of fibroblast-keratinocyte crosstalk during the acute UVB response. In
particular, the involvement of IGF-I in influencing critical elements of the
keratinocyte photoresponse, namely cell survival, cell cycle regulation, apoptosis,
DNA repair and the DNA damage response were explored. It was hypothesized that,
as a prominent pathway in epithelial-mesenchymal communication that is known to
modulate significant aspects of epidermal growth and regeneration, the IGF system
may contribute to the orchestration of keratinocyte responses to UVB damage.
150 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
The aims of this Chapter were therefore to:
1) Detect and quantify the presence of IGF-I in the Transwell® fibroblast-
keratinocyte co-culture model;
2) Investigate how IGF-I influences cell survival, cell cycle progression,
apoptosis and DNA repair in irradiated keratinocytes; and to
3) Study the role of IGF-I in the DNA damage response in UVB-exposed
keratinocytes.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 151
5.2 MATERIALS AND METHODS
5.2.1 Isolation and culture of primary human skin cells
The procedures for the isolation and culture of primary human keratinocytes
and fibroblasts from ex vivo skin tissues are detailed in 2.2.2.
5.2.2 Transwell® co-culture of keratinocytes and fibroblasts
The co-culture of primary keratinocytes and fibroblasts using Transwell® cell
culture systems is described in 2.2.4.
5.2.3 Irradiation of keratinocyte monolayers
Keratinocyte monolayers grown on the insert membranes of Transwell® cell
culture systems were irradiated with a single dose of UVB as outlined in 2.2.5.
5.2.4 Quantification of IGF-I in cell culture supernatants
The concentration of IGF-I was quantitatively determined in cell culture
supernatants from Transwell® and HSE cultures using a pre-bound sandwich ELISA
kit (Quantikine© human IGF-I immunoassay, R&D Systems, MN, USA) as per the
manufacturer’s protocols. Briefly, serum-free cell culture supernatants were obtained
from confluent co-cultures and keratinocyte monolayers after three days under
culture conditions. Samples were pooled from cells obtained from three different
patient donors. Complete keratinocyte growth medium, FGM, was also tested as a
positive control. The volume of supernatants added to the pre-bound ELISA plates
were normalised to the cell density. IGF-I standards with known concentrations were
prepared and incubated, along with the culture medium samples, in the plates
containing a mouse monoclonal antibody against human IGF-I for two hours at 4°C.
Following a thorough wash, an IGF-I conjugate was added to all wells and these
were incubated for a further one hour at 4°C. Finally, a substrate solution was added
for 30 minutes, followed by a stop solution. The resulting absorbances of the wells
were read at 450 nm using a microplate reader.
5.2.5 Testing the effects of exogenous IGF-I on irradiated keratinocytes
In studies investigating the effects of exogenous IGF-I added into the cell
culture medium of irradiated keratinocytes, the following experimental set up was
utilised, as shown in Figure 5.1. Recombinant receptor grade human IGF-I (GroPep,
Adelaide, Australia) was used in these studies.
152 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
Figure 5.1. Experimental set up for testing the effects of IGF-I on irradiated keratinocytes
Human keratinocytes were cultured in the presence of fibroblasts as described in 5.2.2. Upon reaching
approximately 80% confluency, the keratinocytes were transferred to new culture wells (without fibroblasts) and
serum-starved in SFM. Following this, specific IGF-I concentrations in SFM or SFM only were added to the
keratinocyte monolayers 30 minutes prior to irradiation with UVB. Cell culture medium in both the upper and
lower chambers was then replaced with SFM and the keratinocytes returned to culture conditions of 37°C and 5%
CO2.
5.2.6 Testing the effects of fibroblast conditioned medium on irradiated
keratinocytes
To test the effects of fibroblast conditioned medium (CM) on keratinocyte
responses to UVB, the experimental set up detailed in Figure 5.2 was used.
Fibroblast CM collected from 5 x 105 primary dermal fibroblasts at Passage 2,
cultured for 72 hours in SFM was utilised. This fibroblast CM (from fibroblasts
cultured from the same skin tissue donor) was used in all experiments requiring the
use of CM.
Figure 5.2. Experimental set up for testing the effects of fibroblast conditioned medium on irradiated
keratinocytes
Human keratinocytes were cultured in the presence of fibroblasts as described in 5.2.2. Upon reaching
approximately 80% confluency, the keratinocytes were transferred to new culture wells (without fibroblasts) and
serum-starved in SFM. Following this, fibroblast CM or SFM only were added to the keratinocyte monolayers 30
minutes prior to irradiation with UVB. Cell culture medium in both the upper and lower chambers was then
replaced with SFM and the keratinocytes returned to culture conditions of 37°C and 5% CO2.
5.2.7 Analysis of keratinocyte viability
Cell viability was determined using the MTS assay, as detailed in 2.2.12.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 153
5.2.8 Analysis of IGF-IR-mediated cell signalling pathways using Western
immunoblotting
Western immunoblots were performed as previously outlined in 2.2.23. For
studies on cell signalling pathways in irradiated keratinocytes, the following primary
antibodies were utilised: anti-human AKT rabbit polyclonal IgG (Cell Signaling
Technology), anti-phospho-AKT (Thr 308) rabbit monoclonal IgG (Cell Signaling
Technology), anti-p44/42 MAP kinase (ERK1/2) rabbit polyclonal IgG (Cell
Signaling Technology), anti-phospho-p44/42 MAP kinase (phospho-ERK 1/2)
(dually phosphorylated at Thr 202/Tyr 204 of ERK1, Thr 185/Tyr 187 of ERK2 and
singly phosphorylated at Tyr 204) mouse monoclonal IgG (Cell Signaling
Technology), anti-IGF-IRβ rabbit monoclonal IgG (Cell Signaling Technology),
anti-phospho-IGF-IRβ (Tyr 1135/1136) rabbit monoclonal IgG (Cell Signaling
Technology) and anti-GAPDH rabbit monoclonal antibody (Cell Signaling
Technology).
5.2.9 Cell cycle analysis by flow cytometry
The proportion of keratinocytes at different phases of the cell cycle was
determined using flow cytometric analysis of the total DNA content as described in
2.2.14 (Kurki et al., 1986; Nunez, 2001). Briefly, keratinocytes cultured in
Transwell® inserts with various cell culture medium treatments were exposed to 4
MED UVB radiation (as described in 2.2.5). After 24 hours, the medium was
removed (and kept on ice) and keratinocyte monolayers were washed once with PBS
and removed from the membrane inserts using a cell scraper and transferred to low-
bind tubes and resuspended in ice cold PBS. The cell culture medium (containing
non-adherent apoptotic cells) was then added to this cell suspension and the
keratinocytes were washed twice by centrifugation at 200 x g for 5 minutes, followed
by re-suspension in cold PBS. After the final wash, the keratinocytes were
resuspended in ice cold PBS with 2% BSA and were passed through a 70 µm nylon
mesh (BD Falcon) to minimise cell clumping. The keratinocyte suspension was then
added drop-wise to an equal volume of ice cold absolute ethanol and stored at 4°C
overnight prior to staining as detailed in 2.2.14.
5.2.10 Analysis of apoptosis in irradiated keratinocytes
The TUNEL assay was used to detect and quantify apoptosis in irradiated
keratinocyte monolayers, as previously described in 2.2.11.
154 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
5.2.11 Analysis of the formation of UVB-induced CPD photolesions in
irradiated keratinocytes
The formation of CPD DNA dimers was detected in irradiated keratinocyte
monolayers using a mouse monoclonal antibody against CPDs as described
previously in 4.2.11.
5.2.12 Blocking IGF-IR function using a neutralisation antibody
For IGF-IR neutralisation studies, the mouse monoclonal IGF-IRα blocking
antibody (1H7 clone, azide-free antibody, Santa Cruz Biotechnology, Santa Cruz,
CA) was utilised. The addition of this antibody into cell culture systems blocks the
binding of IGF-I to IGF-IR, thus inhibiting ligand-dependent IGF-IR
phosphorylation and the subsequent activation of downstream signalling pathways
(Burtrum et al., 2003; Maloney et al., 2003). For IGF-IR function blocking studies,
the culture medium from keratinocyte monolayers grown in Transwell® cell culture
plates were removed and the cells were washed once with PBS. The IGF-IRα
blocking antibody (10 µg/mL in SFM) was then added to the keratinocyte cultures
and incubated for 30 minutes at 37°C with 5% CO2. Negative controls using mouse
IgG (25 µg/mL in SFM) were also run in parallel. The medium was then removed
and IGF-I (15 ng/mL in SFM) was added to the keratinocyte cultures for 30 minutes
at 37°C with 5% CO2 prior to irradiation with UVB as described in 2.2.5.
5.2.13 Analysis of differential gene expression using qRT-PCR
The protocol for qRT-PCR performed in this study is described in 2.2.19. The
primer sequences for p53 were TAACAGTTCCTGCATGGGCGGC (5’ to 3’) and
AGGACAGGCACAAACACGCACC (3’ to 5’).
5.2.14 UVB radiation-induced DNA damage response antibody array
A sandwich ELISA technique was used to detect the presence of cellular
proteins involved in the response to UVB-induced DNA damage. Primary antibodies
against human phospho-histone H2A.X (Ser 139) (Clone 20E3, Cell Signaling
Technology), phospho-cdc25C (Ser 216) (Clone 63F9, Cell Signaling Technology),
phospho-Chk1 (Ser 345) (Clone 133D3, Cell Signaling Technology), ATRIP (Cell
Signaling Technology) and phospho-ATR (Ser 428) (Cell Signaling Technology)
were used. These primary antibodies were diluted 1:1000, with 200 µL of this diluted
antibody solution added to the appropriate wells of a 96-well plate and incubated for
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 155
1 hour at 37°C. The antibody solution was removed and the plates were washed three
times with PBS with 0.05% Tween-20 (PBS-T). Samples of keratinocyte total cell
lysates were standardised by protein concentration and with serial dilutions from 10
µg added to the pre-coated wells. The plates were incubated for 60 minutes at room
temperature before being washed three times with PBS-T. Next, 1:5000 dilutions of
the secondary detection antibody (goat anti-rabbit IgG, HRP-linked antibody, Cell
Signaling Technology) was added to each well and the plate incubated for 60
minutes at room temperature. Plates were washed as described earlier and 100 µL of
3,3', 5,5"-tetramethylbenzidine (TMB) substrate solution (Cell Signaling
Technology) was added to each well and the plate incubated for 30 minutes at room
temperature. The stop solution (Cell Signaling Technology) was then added (100
µL/well) and the resulting absorbances were read at 450 nm using a
spectrophotometer.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 157
5.3 RESULTS
5.3.1 In vitro concentration of IGF-I in 2D and 3D cultures
The secretion by IGF-I by dermal fibroblasts forms an important intermediary
of dermal-epidermal crosstalk, by stimulating a variety of processes including growth
and proliferation in keratinocytes via the activation of the IGF-IR (Nickoloff et al.,
1988; Kratz et al., 1992). In vitro, cultured human fibroblasts and melanocytes, but
not keratinocytes, have been shown to secrete IGF-I (Tavakkol et al., 1992; Tavakkol
et al., 1999). Additionally, fibroblast-secreted IGF-I has also been detected in cell
culture supernatants from 3D organotypic skin culture systems (Tavakkol et al.,
1999; Swope et al., 2001). In Chapter 4, the use of both bi-layer Transwell® 2D co-
culture and 3D HSE-KCF culture systems were employed to study epidermal-dermal
interactions during the UVB photoresponse. Henceforth, in this Chapter, the potential
role of IGF-I in mediating the keratinocyte photoresponse was investigated using the
Transwell® culture system.
Firstly, the levels of IGF-I present in cell culture supernatants obtained from
both Transwell® fibroblast-keratinocyte co-cultures and HSE-DED 3D cultures were
quantified. As shown in Figure 5.3, IGF-I was detected at significantly higher levels
(23.2 ± 4.29 ng/mL) in culture medium obtained from fibroblast-containing cultures
compared to cultures containing solely keratinocytes (0.09 ± 0.02 ng/mL). A similar
trend was observed in HSE-KCF culture medium as compared to that collected from
HSE-KC cultures, with mean concentrations of 3.12 ± 0.55 ng/mL and 0.42 ± 0.09
ng/mL IGF-I detected respectively. The concentrations of IGF-I detected in the
supernatants from the HSE-KCF cultures were lower in comparison to those from 2D
co-cultures, which may be attributed to IGF-I being bound to the ECM in the 3D
construct.
158 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
Figure 5.3. Quantification of IGF-I concentration in cell culture supernatants
The concentrations of IGF-I in Full Green’s medium (FGM) and cell culture supernatants from Transwell®
monolayer cultures consisting of keratinocytes only (KC), or keratinocyte-fibroblast co-cultures (KC+F), HSE-
KC and HSE-KCF were quantified using an ELISA protein quantification method (5.2.4). Samples in triplicate
were normalised based on the cell density, in triplicate experiments with cells cultured from three independent
tissue donors. Statistical significance of p<0.01 is represented by ‘#’, and p<0.05 by ‘*’. Error bars indicate the
SEM values.
5.3.2 Effect of IGF-I on irradiated keratinocyte viability
It was previously demonstrated that fibroblast co-culture enhanced cell
viability levels in UVB irradiated keratinocyte populations (Section 4.3.2).
Moreover, previously published studies indicate a strong correlation between the
IGF-I system and fibroblast-mediated regulation of keratinocyte survival during the
acute UVB response (Kulik, 1997; Kuhn et al., 1999). In the previous section,
fibroblast-secreted IGF-I was positively detected and quantified in the keratinocyte
culture systems utilised throughout this project. The next aims of this study were
therefore to: 1) determine the minimum concentration of exogenous IGF-I required
to significantly enhance cell survival in irradiated keratinocytes; and 2) compare
irradiated keratinocyte viabilities between this minimum effective dose of IGF-I and
fibroblast CM and 3) to determine if the IGF system is involved in fibroblast-induced
keratinocyte survival post-UVB irradiation.
Figure 5.4A shows the effect of increasing doses of exogenous IGF-I pre-
treatment on the resulting relative cell viabilities of keratinocyte populations
following UVB irradiation. Cell monolayers were pre-treated with IGF-I in SFM
Cell culture supernatant
IGF
-I c
on
c.
(ng
/mL
)
FGM K
C
KC+F
HSE-K
C
HSE-K
CF
0
10
20
30
*
#
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 159
prior to their exposure to 100 mJ/cm2 UVB radiation and returned to culture
conditions for 24 hours before viability analysis using an MTS assay. Overall,
increasing concentrations of IGF-I showed a positive effect on keratinocyte viability,
as compared to those cultured in SFM only. A dose of 15 ng/mL of IGF-I was found
to be the minimum effective dose to significantly enhance keratinocyte viability in
relation to the SFM control. Concentrations of IGF-I between 15 and 30 ng/mL also
improved keratinocyte survival post-irradiation, but did not differ greatly from
results observed using the minimum effective dose.
Figure 5.4. Effects of IGF-I treatment on the viability of irradiated keratinocyte monolayers
The effects of IGF-I on irradiated keratinocyte viability were tested using an MTS assay. Cell monolayers
cultured on Transwell® membrane inserts were pre-treated with different concentrations of IGF-I, serum-free
medium (SFM), complete keratinocyte growth medium (FGM) or fibroblast CM, 30 minutes prior to irradiation
with 100 mJ/cm2 UVB. After 24 hours, the MTS assay was performed to determine dose-response changes to
keratinocyte viability with IGF-I pre-treatment (A). The minimum concentration of IGF-I required to
significantly enhance keratinocyte viability after UVB exposure was 15 ng/mL. Pre-treatment with this IGF-I
dose was further compared with the effects of fibroblast CM on irradiated keratinocytes (B). Data is represented
as a percentage of the cell viability in non-irradiated controls (white bars). Asterisks represent statistical
significance (p<0.05) relative to results obtained with SFM. Data represents the mean values of experimental
triplicates, each with cells obtained from three individual donors. The error bars indicate the SEM values.
Next, irradiated keratinocyte survival (after 15 ng/mL IGF-I pre-treatment) was
compared to the viability of cells incubated with fibroblast CM prior to irradiation.
As shown in Figure 5.4B, both IGF-I and CM pre-treatment resulted in significantly
increased viabilities in irradiated keratinocyte monolayers, as compared to those in
Treatment
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ll v
iab
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CM
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g/mL IG
F-I
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**
A B
Treatment
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L IGF-I
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F-I
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20 n
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F-I
25 n
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F-I
30 n
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F-I
0
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* * **
160 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
SFM only controls. Furthermore, the percentage of cell survival induced by this dose
of IGF-I was comparable to that observed in the CM pre-treated samples containing
fibroblast-secreted factors. Together with the data obtained from IGF-I
concentrations detected in CM (5.3.1), this strongly suggests that IGF-I may be a key
growth factor involved in the fibroblast-directed enhancement of cell survival after
UVB exposure in keratinocytes
5.3.3 IGF-IR signalling in irradiated keratinocytes
In the previous section, IGF-I was detected in fibroblast-keratinocyte co-
cultures and the addition of exogenous IGF-I into fibroblast-free keratinocyte
cultures resulted in a similar enhancement of keratinocyte viability post-irradiation
(Section 5.3.1). Next, the activation of IGF-IR signalling pathways following UVB
irradiation in these keratinocytes was investigated. The intracellular signalling events
triggered by IGF-IR activation via IGF-I ligand binding are discussed in detail in
Section 1.5.1. Briefly, IGF-IR activation stimulates the intra-cellular phosphorylation
of signalling transducers in the PI3K and Raf-1/MEK/ERK pathways. Subsequently,
downstream cellular processes triggered by these signalling pathways include cell
survival, differentiation, growth and proliferation (Furstenberger and Senn, 2002).
In the study reported herein, the activation of the PI3K and ERK pathways, via
the phosphorylation of ERK and AKT was investigated using immunoblot analysis
(Figure 5.5). Phosphorylated-IGF-IR was undetectable in cell lysates obtained from
keratinocytes in SFM only (Figure 5.5A). However, the addition of 15 ng/mL IGF-I
significantly increased the detectable levels of phosphorylated-IGF-IR in these cell
lysate samples. It is known that human keratinocytes express the IGF-IR on the cell
surface, and respond to IGF-I synthesized by fibroblasts and melanocytes in a
paracrine manner (Tavakkol et al., 1992). Hence, it was demonstrated herein that the
addition of exogenous IGF-I to keratinocytes in the Transwell® culture system
resulted in the phosphorylation of the IGF-IR and the activation of its associated
signalling pathways.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 161
Figure 5.5. Activation of IGF-IR-mediated signalling pathways in irradiated keratinocytes
Irradiated keratinocyte monolayers pre-treated with serum-free medium (SFM) only (-IGF-I) and in the presence
of 15 ng/mL IGF-I (+IGF-I) prior to irradiation were lysed at the indicated times post-irradiation. The relative
levels of phosphorylated and total proteins shown above were determined by immunoblot analysis. The cell
lysates were also probed for GAPDH to ensure equal loading in all wells. Data shown is representative of
experiments performed in triplicate using independent skin sample donors.
The activation of the ERK pathway is believed to play an important role in
initiating cell survival and proliferation responses by the actions of various growth
factors (Chang et al., 2003). As seen in Figure 5.5B, the enhanced phosphorylation
of ERK 1/2 (p44 and p42) in keratinocytes was associated with increased phospho-
IGF-IR. However, a slight increase in the relative levels of total-ERK was also
observed in IGF-I-treated cells. Elevated levels of phosphorylated AKT were
detected in these cells, which may be the predominant signalling pathway triggered
as a result of IGF-I treatment in these cells. Activation of both these pathways has
been shown to be linked to the promotion of cell survival and apoptosis inhibition
following cellular insults (Burgering and Coffer, 1995; Franke et al., 1997). Hence,
collectively, this result confirms the activation of IGF-IR-mediated intracellular
signalling pathways as a result of the addition of IGF-I to keratinocyte culture
systems following UVB irradiation in this experimental system.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 162
5.3.4 Effect of IGF-I on UVB-induced apoptosis
It has been established that the IGF system is a cardinal factor in protecting
cells against apoptotic signals (Vincent and Feldman, 2002; Denley et al., 2005).
Mounting experimental evidence, including from research conducted in our
laboratory, has shown that IGF-I has potent effects, such as promoting cell survival
in a variety of cell types (Kashyap et al., 2011; Aburto et al., 2012; Fernández et al.,
2012). In the previous Chapter, it was demonstrated that fibroblast co-culture in both
2D and 3D tissue-engineered systems had a similar effect on UVB-irradiated
keratinocytes, with apoptosis being significantly inhibited in these cultures (4.3.3).
Hence, I aimed to investigate the effect of IGF-I on apoptosis suppression in
irradiated keratinocytes following UVB exposure, as a mechanism involved in the
dermal-epidermal crosstalk during the acute UV response.
The relative levels of UVB-induced apoptosis were measured using the
TUNEL assay in keratinocyte monolayers pre-treated with either SFM only,
fibroblast CM or IGF-I prior to irradiation (Figure 5.6). As shown previously, pre-
treating keratinocytes with fibroblast-CM significantly reduced the proportion of
apoptotic cells detected after UVB irradiation. Notably, the addition of 15 ng/mL of
IGF-I to SFM also showed a significant decrease in the ratio of TUNEL-positive
cells in the irradiated keratinocyte population. The percentage of apoptotic cells
observed in UVB-treated keratinocytes exposed to fibroblast-CM and IGF-I prior to
exposure was comparable. This result strongly suggests that IGF-I present in
fibroblast CM may be an important element in inhibiting apoptosis in UVB-exposed
keratinocytes. Hence, the IGF system may be involved in resisting extensive tissue
damage in the epidermis following physiologically-relevant doses of UVB, and
thereupon helping to maintain the important barrier function of the epidermis.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 163
Figure 5.6. Effect of IGF-I on UVB-induced apoptosis in human keratinocytes
The relative proportions of UVB-induced apoptotic keratinocytes were measured using the TUNEL assay,
following the addition of different culture medium treatments. Non-irradiated controls using all culture medium
pre-treatments showed negligible numbers of apoptotic cells. Keratinocyte monolayers were serum-starved and
pre-treated with serum-free medium (SFM), fibroblast conditioned medium (CM) or SFM containing 15 ng/mL
IGF-I, 30 minutes prior to irradiation with 100 mJ/cm2 UVB radiation. The average numbers of TUNEL-positive
cells (from experimental triplicates) are presented as a percentage of the total number of cells counted in an
average of five microscopy fields. The experiment was repeated using cells obtained from three independent skin
tissue donors. The asterisks represent statistical significance (p<0.05). Error bars represent SEM values.
5.3.5 Effects of the blocking of IGF-IR activation on cell death in irradiated
keratinocytes
In the previous sections, it was demonstrated that IGF-I influenced key
elements of the UVB response including cell survival and apoptosis. These findings
were in parallel to those in the previous Chapter, in which fibroblast-keratinocyte co-
culture prior to UVB exposure brought about similar changes to the keratinocyte
photoresponse. To further investigate whether the IGF system represents a major
component in the fibroblast-mediated inhibition of apoptosis, an IGF-IR neutralising
antibody was utilised. This function-blocking antibody is known to stop the binding
of the IGF-I ligand to the receptor, thus preventing intracellular signalling pathway
cascades and the subsequent cellular effects evoked by IGF-IR activation (Burtrum et
al., 2003; Maloney et al., 2003).
Treatment
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*
164 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
Keratinocyte-fibroblast co-cultures were pre-treated with an IGF-IR
neutralising antibody in different culture treatments prior to keratinocyte irradiation,
as described in 5.2.12. The relative ratios of apoptotic keratinocytes present in the
irradiated monolayers were analysed 24 hours after UVB treatment and the results
are represented in Figure 5.7. As shown in previous sections, control keratinocytes
pre-treated with SFM only resulted in the highest proportion of apoptotic
keratinocytes. The addition of IGF-1 and fibroblast CM pre-treatment to
keratinocytes significantly reduced the numbers of UVB-induced apoptotic cells
detected. The addition of the 1H7 neutralising antibody before CM pre-treatment
also resulted in a significant drop in the percentage of TUNEL-positive keratinocytes
following UVB irradiation.
Interestingly, however, blocking the functionality of IGF-IR did not
significantly augment the numbers of apoptotic keratinocytes after irradiation. This
indicates that alternate pathways to those regulated by IGF-IR may also play an
important role in fibroblast-mediated keratinocyte survival post-irradiation.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 165
Figure 5.7. Effect of IGF-IR blocking antibody treatment on UVB-induced apoptosis in irradiated
keratinocytes
The proportions of TUNEL-positive apoptotic keratinocytes were quantified in UVB-irradiated keratinocytes
subjected to different medium treatments prior to UVB exposure. Control samples were treated to a sham
irradiation. Irradiated cells were pre-treated with either SFM only, 15 ng/mL IGF-I, IGF-I and the 1H7 IGF-IR
neutralising antibody, fibroblast CM, CM and 1H7 or CM and an IgG control prior to exposure to 100 mJ/cm2
UVB. The average numbers of TUNEL-positive cells (from experimental triplicates) are presented as a
percentage of the total number of cells counted in an average of five microscopy fields. The experiment was
repeated using cells obtained from three independent skin tissue donors. The asterisks represent statistically
significant (p<0.05) values in comparison to samples treated with SFM only. Error bars represent SEM values.
Treatment
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*
**
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 166
5.3.6 Effect of IGF-I on the repair of UVB-induced DNA damage
As reported earlier in this thesis, fibroblast co-culture in the Transwell®
culture system has been reported to have a positive impact on the rate of CPD
removal in irradiated keratinocytes (4.3.4). These CPD photolesions are a primary
form of UVB-induced photodamage in the epidermis and have been strongly
implicated in the development of skin malignancies (Evans and Bohr, 1994; You et
al., 2000). Hence, identifying biological factors that can improve the efficiency of
CPD photodamage in UVB-exposed skin is an essential step in the generation of
more effective strategies against UV-damage. Moreover, identifying intercellular and
intracellular pathways that function to repair genotoxic damage in vivo can aid in
understanding how the dysregulation of these protective mechanisms can increase
the risk of photocarcinogenesis. Therefore, the effects of IGF-I at modulating the
removal of UVB-induced CPDs in irradiated Transwell® keratinocyte cultures were
investigated.
At 24 hours post-UVB irradiation, irradiated keratinocytes still harbouring
CPD damage were visualised using immunofluorescence and quantified (Figure 5.8).
In these experiments keratinocytes pre-treated with basal SFM prior to UVB
exposure showed the highest rate of unrepaired CPD in irradiated keratinocyte
nuclei. However, pre-treatment with fibroblast CM or SFM containing 15 ng/mL
IGF-I significantly reduced the ratio of CPD-positive keratinocyte nuclei in these
samples. Keratinocytes exposed to fibroblast CM had a lower proportion of cells
with unrepaired CPDs, as compared to those treated with IGF-I, which suggests other
fibroblast-secreted factors may be responsible for promoting DNA repair in
epidermal cells.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 167
Figure 5.8. Effect of IGF-I on the repair of UVB-induced CPDs in irradiated keratinocytes
Keratinocyte nuclei containing UVB-induced DNA dimers were visualised using immunofluorescence and
quantified using image analysis software. The presence of UVB-induced CPDs was detected in keratinocyte
monolayers pre-treated with serum-free medium (SFM), fibroblast conditioned medium (CM) or SFM containing
15 ng/mL IGF-I prior to irradiation. Data is represented as a percentage of the total number of keratinocyte
nuclei. Results shown are representative of three experiments (with samples in triplicate) conducted from three
independent skin cell donors. Asterisks represents statistical significance at p<0.05. Error bars represent SEM
values.
5.3.7 Repair of UVB-induced CPDs with IGF-IR neutralisation in irradiated
keratinocytes
The enhancement of cell survival following UVB exposure, as demonstrated in
the previous section, is associated with either cellular senescence or the arrest of the
cell cycle to allow for the repair of UVB-induced DNA damage (Lewis, 2008). The
formation of CPDs in the nuclei of UVB-exposed keratinocytes represents a major
form of DNA damage, of which the recruitment of DNA repair machinery during the
acute photoresponse is critical for removing these photolesions (Regan and Setlow,
1974; Su, 2006). Previously, it has been documented herein that both fibroblast-
secreted factors, as well as keratinocyte pre-treatment with IGF-I, prior to UVB
irradiation, accelerates the rate of CPD repair in these cells (section 5.3.6). To further
investigate the role of IGF-I at enhancing the removal of UVB-induced DNA
damage, the effect of IGF-IR neutralisation on the rate of CPD repair in irradiated
keratinocytes was studied.
Treatment
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PD
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s
Unir
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CM
IGF-I
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*
168 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
Figure 5.9 shows the proportions of unrepaired CPDs present in irradiated
keratinocytes pre-treated with different medium conditions at 24 hours post-
irradiation. Pre-treatment with 15 ng/mL IGF-I, fibroblast CM and CM with an IgG
control showed a statistically significant decrease in the proportion of unrepaired
CPDs in irradiated keratinocytes in relation to those in SFM only. The addition of the
IGF-IR function-blocking antibody resulted in significantly higher levels of CPD-
positive keratinocytes as compared to cell monolayers treated with CM and the IgG
control. This result indicates that the blocking of IGF-IR-activated pathways by the
1H7 neutralising antibody may impact the recruitment and functionality of DNA
repair machinery required for the efficient removal of CPDs. Importantly, this
provides further evidence for the importance of IGF-IR-mediated activities in the
orchestration of DNA repair, a process that is vital in suppressing potentially
oncogenic events in skin.
Figure 5.9. Effect of IGF-IR blocking antibody treatment on the formation of UVB-induced DNA
dimers in irradiated keratinocytes
The proportions of CPD-positive keratinocytes containing UVB-induced DNA dimers were quantified in
irradiated keratinocyte monolayers in different medium treatments prior to UVB exposure. Irradiated cells were
pre-treated with either SFM only, 15 ng/mL IGF-I, fibroblast CM, CM and the 1H7 IGF-IR neutralising antibody
or CM and an IgG control, prior to exposure to 100 mJ/cm2 UVB. The presence of CPDs in keratinocyte nuclei
was detected using immunofluorescence. Data is represented as a percentage of the total number of keratinocyte
nuclei. Results shown are representative of three experiments (with samples in triplicate) conducted from three
independent skin cell donors. The asterisks represent statistically significant values (p<0.05). Error bars represent
SEM values.
Treatment
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PD
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s
Non-ir
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F-I
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+ Ig
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*
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 169
5.3.8 Effect of IGF-I on p53 expression in irradiated keratinocytes
Upon the generation of UVB-induced DNA damage, cellular p53 is known to
be activated and stabilised, through the disruption of p53-MDM2 complexes, and
subsequent translocation to the nucleus (Hall et al., 1993; van Laethem et al., 2009).
During this UV stress response, p53 activation can trigger both G1 and G2/M cell
cycle arrest, thus facilitating DNA repair (Matsumura and Ananthaswamy, 2004).
The activation of IGF-IR in keratinocytes has been demonstrated to be linked to p53-
mediated cellular pathways during the UVB response, both in this thesis as well as in
previously reported studies, which include the inhibition of apoptosis and the
regulation of the cell cycle (Kuhn et al., 1999; Chaturvedi et al., 2004).
Previously, I have observed significant differences in keratinocyte p53
expression levels as well as that of total and phosphorylated intracellular p53
following UVB radiation as a result of fibroblast co-culture (4.3.8). Hence, the
relative gene and protein expression levels analysed in irradiated keratinocytes pre-
treated with fibroblast CM and IGF-I are described herein (Figure 5.10).
Keratinocyte monolayers cultured in Transwell® culture systems were exposed to
either SFM only, IGF-I or CM prior to irradiation, with RNA and protein samples
obtained at various time points after UVB exposure. In non-irradiated controls, no
significant changes in gene or protein expression levels were observed in all culture
conditions, over all the time points sampled (Figure 5.10A). At 6 and 24 hours post-
irradiation, however, a significantly decreased relative gene expression of p53 was
observed in keratinocytes pre-treated with IGF-I and CM prior to irradiation. In IGF-
I pre-treated keratinocytes, a 3.99 ± 0.97 and 8.27 ± 1.99 fold decrease in p53 mRNA
levels were detected relative to SFM-treated cells at 6 and 24 hours post-irradiation
respectively. Culture of keratinocytes in CM induced a 2.38 ± 0.97 and 6.78 ± 2.38
fold decrease in relative p53 mRNA expression at these time points.
170 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
Figure 5.10. Relative gene and protein expressions of p53 in irradiated keratinocytes
The relative gene expression of p53 in irradiated keratinocytes was analysed using qRT-PCR and normalised to
results from cells pre-treated with SFM only (A). The control results represent non-irradiated keratinocyte
samples at the 0 hour time point. Keratinocytes were pre-treated with either SFM, SFM with 15 ng/mL IGF-I or
fibroblast-CM prior to UVB irradiation, with samples obtained at 3, 6 and 24 hours post-irradiation. The dotted
line indicates a 1.8-fold difference in gene expression relative to SFM treated cells. The relative protein
expression of total p53 in cell lysates from irradiated keratinocytes was analysed using ELISA (B) to validate the
gene expression data previously described (A). The relative levels of phosphorylated p53 (Ser 15) in these
samples were also analysed (C). Statistical significance of p<0.05 is represented by ‘*’. Data are represented as
mean ± SEM (error bars) from three independent replicate experiments using samples obtained from three
independent skin donors.
Next, the relative levels of total and phosphorylated p53 protein in these
samples were analysed using an ELISA (Figure 5.10B and C). Compared to p53
levels in SFM-treated keratinocytes, cells cultured in IGF-I-containing medium prior
to irradiation showed significantly higher total p53 levels at 6 and 24 hours post-
irradiation and elevated phospho-p53 at the 3 and 6 hour time-points. Total p53 was
shown to be increased in CM-treated keratinocytes at 6 hours after UVB-exposure,
with the relative levels of the phosphorylated protein shown to be higher than SFM-
treated controls at all time-points sampled. Intracellular p53 has been shown to have
Time post-irradiation (hr)
Re
lativ
e g
ene
exp
ressio
n o
f p
53
Control 3 6 24-15
-10
-5
0
5
SFMIGF-I
*
CM
*
Time post-irradiation
Ab
so
rban
ce (
450 n
m)
Contr
ol 0 h
r3
hr6
hr
24 h
r
0.0
0.5
1.0
1.5SFM
+ IGF-I
*
CM
*
*
Time post-irradiation (hr)
Ab
so
rban
ce (
450 n
m)
Contr
ol 0 h
r3
hr6
hr
24 h
r
0.0
0.5
1.0
1.5SFM
+ IGF-I
*CM
**
A
B CTotal p53 Phospho-p53
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 171
a rapid turnover and its short half-life is a result of ubiquitin-proteasome pathways
(Maki et al., 1996). During cellular stress, downstream p53-mediated processes are
associated with the increased stabilisation via the inhibition of its binding to MDM2
and an increase in the phosphorylation of p53 at Ser15, Thr18, Ser20, Ser33 and
Ser37 (Shieh et al., 1997; Kapoor et al., 2000). The results described herein therefore
indicate that both fibroblast-secreted factors in the CM and the activation of IGF-IR
may enhance p53 via inhibiting its degradation and enhancing its phosphorylation
status.
5.3.9 Effect of IGF-I on cell cycle phase
The inhibition of apoptosis and the enhancement of cellular survival in UVB-
irradiated keratinocytes by IGF-I have previously been described (section 5.3.2).
Moreover, prior studies pertaining to downstream cellular effects resulting from the
activation of the IGF system have shown evidence towards the regulation of cell
cycle progression via Akt/PI3K mediated pathways (Chen and Rabinovitch, 1990;
Mawson et al., 2005). Thereupon, alterations to the regulation of the cell cycle in
irradiated keratinocytes as a result of IGF-I pre-treatment were investigated.
The ratios of the total keratinocyte population in the G0/G1, S and G2/M cell
cycle phases were determined using flow cytometric analysis of fluorescently-
labelled DNA content (Kurki et al., 1986; Nunez, 2001). Keratinocytes undergoing
apoptosis were also able to be detected using this technique. Figure 5.11 shows the
proportions of irradiated keratinocytes (pre-treated with different medium conditions
prior to UVB treatment) in these cell cycle phases at 24 hours post-irradiation. The
addition of exogenous IGF-I and fibroblast CM significantly decreased the
proportions of apoptotic keratinocytes following irradiation. Interestingly, significant
shifts were also observed in the irradiated keratinocytes G0/G1 and G2/M cycle phases
as a result of these treatments. Higher proportions (65.99 ± 6.32% and 57.68 ±
8.70%) of cells at the quiescent G0/G1 phase were also observed in keratinocytes
treated with 15 ng/mL IGF-I and CM respectively. In comparison, irradiated
keratinocyte monolayers cultured in SFM and FGM were observed to have a
significantly greater proportion of cells in the G2/M phases of the cell cycle. This
data points towards the possible induction of G1/S cell cycle checkpoints by IGF-I
and CM pre-treatments, which reduce the proportion of cells able to progress to the
G2/M mitotic phases.
172 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
Figure 5.11. Effect of growth factor pre-treatment on cell cycle phases in irradiated keratinocytes
The effect of IGF-I and CM pre-treatment on the cell cycle phase in irradiated keratinocytes was analysed using
flow cytometry. Cell monolayers were pre-treated with serum-free medium (SFM), Full Green’s Medium (FGM),
15 ng/mL IGF-I or fibroblast conditioned medium (CM) for 30 minutes prior to irradiation with 100 mJ/cm2
UVB. After 24 hours, keratinocytes were harvested and analysed for total DNA content by fluorescent labelling
with PI and flow cytometric analysis. The asterisks represent statistical significance (p<0.05) compared to the
SFM controls. Data is representative of experiments performed in triplicate using cells obtained from three
independent skin tissue donors. Error bars represent SEM values.
5.3.10 DNA damage response proteins
A close relationship between IGF-IR activation and the preservation of
genomic integrity in DNA-damaged cells via the initiation of DNA repair
mechanisms has been previously described in various cell models (Trojanek et al.,
2003b; Yang et al., 2005). However, as yet, the precise link between the IGF system
and the regulation of DNA repair systems to remove UVB-induced damage,
particularly in keratinocytes, is still not fully understood. In order to address this,
alterations to the activation of various proteins associated with the DNA damage
response (as discussed in 1.3.2 and 1.3.3) as a result of IGF-IR phosphorylation were
investigated. Irradiated and non-irradiated control keratinocyte monolayers pre-
treated with and without IGF-I were lysed at 1 hour post-irradiation and analysed
using an ELISA antibody array against the DNA damage response proteins ATR,
ATRIP, chk-1, cdc25C and H2A.X (Figure 5.12).
Cell cycle phase
Perc
enta
ge o
f to
tal c
ell
popula
tion
Apopto
tic 1/G0G
S M2G
0
10
20
30
40
50
60
70
80
90
100
SFM
FGM
+ IGF-I
CM
**
*
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 173
Figure 5.12. Expression of DNA damage response proteins in IGF-I-treated irradiated keratinocytes
The relative expression levels of proteins associated with the UVB-induced DNA damage response were
investigated in IGF-I pre-treated and untreated irradiated keratinocyte monolayers using a sandwich ELISA
technique. Keratinocytes were pre-treated with either SFM only or SFM containing 15 ng/mL IGF-I prior to
irradiation with 100 mJ/cm2 UVB, or a sham irradiation. Cell lysates obtained 1 hour post-irradiation were
obtained and probed with antibodies against human phospho-ATR (Ser 428), ATRIP, phospho-chk-1 (Ser 345),
phospho-cdc25C (Ser 216) and phospho-histone H2A.X (Ser 139) to determine their relative expression levels in
these samples. The asterisks represent a statistically significant difference (p<0.05) in comparison to matched
samples without IGF-I treatment. Data shown represents the mean values from two independent experiments,
using skin tissue obtained from two independent donors. Error bars represent SEM values.
The phosphorylation of ATR by UVB-induced DNA damage is a critical
initiating step in the proper function of the DNA damage checkpoint (Stokes et al.,
2007). The activation of ATR is responsible for such diverse activities as cell cycle
arrest, regulating DNA repair activities and upregulating the transcription of other
DNA damage response genes. These responses are activated via the ATR-mediated
phosphorylation of downstream targets including chk-1 and p53 (Zhao and Piwnica-
Worms, 2001; Smith et al., 2010). In this study, UVB irradiation was shown to
increase the relative levels of phospho-ATR at serine 428 in both IGF-I treated and
untreated keratinocyte monolayer samples alike. Interestingly however, there was a
Treatment
Ab
so
rban
ce (
450 n
m)
- UVB
+ UVB
- UVB
+ UVB
0.0
0.2
0.4
0.6
0.8
1.0
- IGF-I + IGF-I
ATR
ATRIP
chk-1
cdc25C
H2A.X*
*
*
174 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
significant increase in the relative level of phospho-ATR detected in irradiated
keratinocytes pre-treated with IGF-I prior to UVB exposure as compared to those
treated with SFM controls. This suggests that the activation of IGF-IR signalling
cascades may interact with factors regulating the DNA damage response such as
ATR. Additionally, this may also indicate that downstream phospho-ATR-mediated
DNA damage response elements may also be modulated as a result of IGF-IR
activation.
One such substrate of phospho-ATR, ATRIP, has been shown to form a
complex with ATR at the sites of DNA damage and is critical for regulating ATR-
mediated DNA repair and cell cycle checkpoint activities (Cortez et al., 2001).
Importantly, key functions of ATRIP include the regulation of ATR stability,
localisation and kinase activation (Nam and Cortez, 2011). In the UVB-exposed
keratinocytes, the levels of ATRIP remained relatively unchanged as compared to
those from non-irradiated samples under both IGF-I treated and untreated conditions.
The signalling cascade resulting from the ATR-mediated phosphorylation of
chk-1 is essential for promoting cell survival and DNA repair, as the principal
effector of the DNA damage and replication checkpoints (Smith et al., 2010).
Notably, this ATR/Chk1-dependent checkpoint signalling cascade also regulates the
activity and function of p53 by enhancing its stability and ability to inhibit apoptosis
(Adimoolam and Ford, 2003). In line with the increased levels of phosphorylated
ATR detected in irradiated keratinocytes pre-treated with IGF-I, significantly
elevated levels of phospho-chk-1 were also found in these cells.
Another downstream process associated with chk-1 activation is the
phosphorylation of the protein phosphatase cdc25C, a regulator of cellular mitosis
(Peng et al., 1997). Both IGF-I treated and untreated keratinocytes showed elevated
levels of phospho-cdc25C in cell lysates in response to UVB radiation. Interestingly,
there was a significant reduction in the proportion of phospho-cdc25C in cells
exposed to IGF-I prior to irradiation. However, studies on cell lysate samples
obtained at more post-irradiation time-points need to be conducted, with previously
published data showing peak levels of phospho-cdc25C being detected between 6
and 9 hours after DNA damage (Gutierrez et al., 2010).
Histone H2A.X is activated via phosphorylation at serine 139, thus forming a
central component in several DNA repair mechanisms, such as those initiated
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 175
following double-strand breaks (Rogakou et al., 1998). UVB-irradiated cells have
been shown to have elevated levels of phosphorylated H2A.X, a downstream result
of ATM and ATR-dependent pathways, and can be used as a marker for analysing
the efficacy of DNA repair systems in irradiated skin (Albino et al., 2004; Barnes et
al., 2010). Exposure to UVB radiation significantly increased the relative levels of
phospho-H2A.X in keratinocytes. However, pre-treatment with IGF-I had no
apparent effect on the levels of this histone detected in the cell lysates of irradiated
keratinocytes.
In conclusion, irradiated keratinocytes pre-treated with IGF-I demonstrated an
enhanced DNA damage response. In these cells, significantly higher levels of
phosphorylated ATR, chk-1 and cdc25C were observed following UVB irradiation.
This enhanced DNA damage response may therefore contribute to the IGF-I-
mediated modulation of downstream effects such as accelerated DNA repair,
increased cell survival and the activation of p53 described in the previous sections.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 176
5.4 DISCUSSION
Paracrine signalling via the ligand-induced activation of receptor tyrosine
kinases represents an important paradigm of mesenchymal-epithelial interactions
(Birchmeier and Birchmeier, 1993). For example, the KGF, HGF/SF and IGF
systems have been well-established as modulators of several important processes in
epithelial tissues including proliferation, differentiation and migration (Aaronson et
al., 1990; Gartner et al., 1992; Parrott et al., 1994; Stewart, 1996; Ohmichi et al.,
1998; Maas-Szabowski et al., 1999). Besides playing central roles in developmental
and homeostatic programs within the epidermis, fibroblast-keratinocyte signalling
has also been shown to be critical for epithelial regeneration during regenerative
processes, such as wound-healing (Werner et al., 2007). The experimental data
reported in Chapter 4 of this thesis have further highlighted the importance of
dermal-epidermal crosstalk in orchestrating keratinocyte responses to UVB-induced
damage. Indeed, the presence of fibroblasts in 2D and 3D in vitro models was
reported herein to significantly inhibit UVB-induced keratinocyte apoptosis, enhance
cell viability and the repair of CPD photolesions and regulate the activation of key
tumour suppressor proteins, such as p53. Thereupon, the next objective of this
project was to investigate the role of the IGF system as part of the fibroblast-
mediated modulation on the abovementioned keratinocyte photoresponse.
Keratinocytes are well-equipped to resist oncogenic transformation resulting
from chronic exposure to physiologically-relevant doses of environmental UVB
through the activation of cell-cycle arrest and complex DNA repair mechanisms prior
to resuming normal cell replication (Liu et al., 1983; Bykov et al., 1999). High levels
of UVB, on the other hand, result in irradiated keratinocytes undergoing programmed
cell death in response to the extensive genotoxic stress (Zhuang et al., 2000). The
degree of genomic damage sustained by the UVB-exposed cell, the generation of
ROS and the activation of death receptors by UVB have all been named as major
factors in determining cellular fate post-irradiation (Kulms et al., 2002). However,
other regulatory intercellular factors capable of influencing this balance between cell
death and survival in vivo are only partially understood to date. Hence, a clearer
understanding of the molecular mechanisms surrounding these processes is of prime
concern in developing targeted strategies against photocarcinogenesis.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 177
IGF-I is a major cell survival factor present in most tissues in vivo, and is
known to activate the PI3K/AKT signalling pathway with downstream effects
including the promotion of cell proliferation and regulation of cell cycle progression
for both tissue homeostasis and regeneration (Baserga et al., 1997; Cohen, 2006).
Moreover, this system has also been implicated as a key element in the multistep
development of skin cancer in mouse models (Wilker et al., 2005). Accordingly, the
study described in this Chapter was aimed at uncovering the role of IGF-I in
mediating the effects of fibroblast co-culture on keratinocyte photoresponses, using
the Transwell® culture system. Overall, results obtained in this Chapter indicate
IGF-I is a main effector in the regulation of keratinocyte survival, cell cycle
regulation and the DNA damage response after UVB treatment.
Dermal fibroblasts secrete IGF-I, which activates and regulates a variety of
IGF-IR-expressing keratinocyte responses in a paracrine manner (Tavakkol et al.,
1992). Several studies have demonstrated the importance of IGF-IR activation in
maintaining the epidermal equilibrium, primarily via the stimulation of proliferation
and inhibition of differentiation in keratinocytes (DiGiovanni et al., 2000;
Sadagurski et al., 2006). Moreover, elevated levels of IGF-I have been associated
with skin hyperplasia and mutations resulting in the downregulation of IGF-IR result
in a thinner, disrupted epidermis in transgenic mouse models (Liu et al., 1993;
Kanter-Lewensohn et al., 2000a; Kanter-Lewensohn et al., 2000b). Notably, in vivo
studies into the function of IGF-IR signalling in skin have been largely limited due to
IGF-IR-null mice having low survival rates as well as epidermal cells obtained from
IGF-IR-knockout mice possessing poor cell viabilities and proliferative capacities for
in vitro culture (Baker et al., 1993; Baserga et al., 1997). Hence, alternative cell-
based approaches are required to dissect the role of the IGF-IR signalling in the
cellular responses to UVB irradiation.
Application of the Transwell® photobiology model to investigating the effect
of IGF-I photoresponses
The 2D Transwell® model adopted in our study possessed certain advantages
over more complex 3D tissue-based skin models. For example, in the Transwell®
system, keratinocyte monolayers were cultured on permeable membranes physically
separated from fibroblasts in the lower chamber. This allowed for the detachment of
fibroblast-free keratinocyte cultures for both the UVB irradiation protocol as well as
178 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
for administering IGF-I and control treatments in SFM. Primary keratinocytes have
been shown to demonstrate poor levels of cell attachment and proliferation in culture
conditions in SFM lacking essential growth factor supplementation, and in the
absence of ECM factors in the in vitro system (Gilchrest et al., 1982; Hirobe, 1994).
Coating of cell culture vessels with ECM components such as collagen I and
fibronectin has been reported to enhance keratinocyte attachment levels in SFM;
however, these factors are also able to independently influence keratinocyte
responses to UVB (Gilchrest et al., 1980; Wang et al., 2012).
Additionally, in the HSE-DED models, the formation of a stratified keratinised
epidermal layer and the 3D nature of the tissue construct may result in
inconsistencies to the penetration of exogenous IGF-I added to cell culture medium
to proliferating basal keratinocytes in these systems. Moreover, interactions between
IGF-I, IGFBPs and ECM proteins such as vitronectin in both the in vivo and in vitro
tissue context have been shown to regulate the bioavailability and functionality of
this growth factor system on keratinocytes (Upton et al., 1999; Hyde et al., 2004).
Hence, future studies pertaining to the role of IGF-I in the photoresponse can take
advantage of organotypic culture models, such as the HSE-DED, in order to examine
these processes in a biologically-relevant system.
Lastly, previously published studies investigating the effects of growth factors
including EGF, KGF, IGF-I and other paracrine factors on keratinocyte cultures
commonly make use of bovine serum albumin or FCS in the cell culture medium
required to support these cells (Bhora et al., 1995; Andreadis et al., 2001; Lee et al.,
2012). With the undefined nature of FCS possibly contributing to experimental
variability in primary keratinocyte responses to exogenous growth factors, the ability
to culture keratinocyte responses in SFM using the Transwell® model as described
herein represents a significant advance.
IGF-I-mediated effects on cell survival in irradiated primary keratinocytes
In both the Transwell® and HSE-KCF skin culture systems described in this
Chapter, IGF-I was detected in cell culture supernatants, with relatively higher levels
detected in the 2D co-culture model. This may be a result of the abovementioned
interactions between IGF-I and ECM elements which may limit the secretion of IGF-
I into the cell culture medium in the HSE-KCF cultures. The physiological
concentration range of cutaneous IGF-I in vivo has yet to be conclusively
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 179
determined. However, IGF-I concentrations in blood plasma have been reported to
range between 20 to 100 nM (153.8 to 769.2 ng/mL) (Daughaday and Rotwein,
1989). On studies investigating the minimum effective dose of exogenous IGF-I
required to significantly enhance keratinocyte viability post-irradiation as described
herein, this concentration was found to be 15 ng/mL (Figure 5.4). Collectively, these
results are in parallel with other studies showing levels of IGF-I between 5 and 50
ng/mL as having potent effects not only on keratinocyte proliferation, but also their
resistance to UVB-induced damage (Kuhn et al., 1999; Claerhout et al., 2007).
The regulation of cellular fate, in particular the processes of cell survival,
proliferation and terminal differentiation by the actions of IGF-I have been widely
reported (Stewart and Rotwein, 1996; Sandhu et al., 2002). Largely due to the
practical limitations described previously, relatively few experimental studies
utilising primary human-derived cells and tissues to investigate these IGF-I mediated
processes during the acute UVB response have been conducted. Nonetheless, the
regulation of keratinocyte survival following UV-insult by IGF-I has also been
implicated as being a key aspect of dermal-epidermal interactions during the acute
photoresponse, with the dysregulation of these pathways potentially being involved
in photocarcinogenesis (Lewis et al., 2008; Lewis et al., 2010).
The novel application of the Transwell® culture system to study the impact of
both exogenous IGF-I and human fibroblast-derived secreted factors on primary
keratinocyte photoresponses as described in this Chapter has been previously
unreported. Herein, I report both the increase in post-irradiation keratinocyte cell
viability as well as the inhibition of UVB-induced apoptosis using this in vitro
model. A significant improvement in keratinocyte viability was observed after UVB-
exposure with the pre-treatment of cells with 15 ng/mL of IGF-I, as well as with
fibroblast-CM prior to irradiation. Notably, at these concentrations of exogenous
IGF-I added to the keratinocyte monolayers, the enhancement of cell viability after
UVB exposure was also higher than those observed in cells pre-treated with FGM.
Importantly, FCS-containing cell culture medium contains various undefined factors
shown to inhibit cellular apoptosis via the activation of MAPK pathways (Jung et al.,
2000). Moreover, FGM contains insulin, which shares structural and functional
homology with IGF-I and at high concentrations can cross-react and activate the
IGF-IR (Siddle et al., 2001; Jamali et al., 2003).
180 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
In Chapter 4, it was demonstrated that fibroblast co-culture with keratinocytes
prior to UVB exposure decreases the levels of apoptotic cells observed post-
irradiation in both 2D and 3D cell culture contexts. Similarly, a significant inhibition
of keratinocyte apoptosis was reported here, in monolayers pre-treated with both CM
and IGF-I. Interestingly, the addition of an IGF-IR neutralising antibody into the
keratinocyte culture systems prior to irradiation resulted in the significant blocking of
IGF-I-mediated inhibition of apoptosis in cells treated with IGF-I, but not in those
treated with fibroblast CM. This suggests that other fibroblast-secreted factors
besides IGF-I may also provide alternative pathways for preventing keratinocyte
apoptosis after UVB exposure.
The human epidermis is equipped to withstand environmental damage through
sophisticated protective and repair mechanisms in keratinocytes, while importantly,
maintaining its barrier function (Slominski and Pawelek, 1998; Yamaguchi et al.,
2006). To achieve this, the promotion of basal proliferative keratinocyte cell survival
following UVB irradiation is a vital process in maintaining the regenerative potential
of the epidermis (Boehnke et al., 2012). This has been demonstrated in the studies
reported herein; indicating IGF-IR activation and other fibroblast-mediated pathways
promote keratinocyte survival in UVB-exposed cells. This cell survival is associated
with either the activation of cell cycle damage checkpoints and DNA repair or
terminal differentiation (Gandarillas, 1999, 2000).
The cellular signalling pathways surrounding keratinocyte differentiation
following UVB insult, however, remain poorly understood. IGF-IR signalling has
been shown to be critical in regulating keratinocyte differentiation in both normal
and psoriatic skin (Wertheimer et al., 2000; Lerman et al., 2011). Moreover, the
activation of IGF-IR and the subsequent activation of PI3K, ERK 1/2 and mTOR
have previously been shown as important inhibitors of keratinocyte differentiation
post-irradiation (Sadagurski et al., 2006). In future, the role of the IGF-I in activating
differentiation pathways in irradiated keratinocytes can be investigated, taking
advantage of the in vitro models described herein.
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 181
IGF-IR signalling and the control of cell cycle progression in UVB-treated
keratinocytes
Besides stimulating proliferation, the activation of IGF-IR and its downstream
signalling pathways have been demonstrated to regulate the progression of the cell
cycle in a variety of cell types (Chen et al., 1995; Sun et al., 1999; Stull et al., 2002).
The activation of cell cycle checkpoints in response to genotoxic stressors is
necessary for the maintenance of genomic integrity and preventing the propagation
of UV-damaged cells (Stokes et al., 2007). Cells harbouring unrepaired DNA
damage which bypass cell cycle checkpoints either undergo apoptosis, or if not
removed, can accumulate genetic changes, thus leading to cancer development
(Hartwell and Kastan, 1994). Upon exposure to UVB radiation, cells have been
shown to activate both the G1/S and G2/M cell cycle checkpoints (Petrocelli and
Slingerland, 2000).
Here, the association between IGF-IR activation and the regulation of
keratinocyte cell cycle phases using a Transwell® culture system has been described.
Importantly, both IGF-I and CM pre-treatment was shown to significantly enhance
the proportion of irradiated keratinocytes in the G0/G1 phase. The p53-dependent
initiation of cell cycle arrest at G1 has been extensively studied in cells exposed to
ionizing radiation (Wade Harper et al., 1993; Lakin and Jackson, 1999). However,
relatively less is known about this response in UV-irradiated cells, with conflicting
results into the mechanisms behind G1 arrest in these DNA-damaged cells having
been described, which may be cell type dependent. While p53-mediated pathways
are not critical for initiating G1 arrest in UVB-treated dermal fibroblasts in vitro, the
increased expression and activation of intranuclear p53 has been shown to be
essential for triggering this checkpoint in UVB-irradiated keratinocytes (Gujuluva et
al., 1994; Loignon et al., 1997).
In the study of p53 expression and activation by IGF-I and CM pre-treated
keratinocytes prior to UVB-irradiation described herein, it was demonstrated that the
abovementioned culture conditions significantly enhanced both the expression and
activation of p53, as compared to cells exposed to SFM only. This increase in the
p53 protein level was also associated with a relative downregulation in p53 gene
expression in IGF-I and CM treated keratinocytes, which points towards post-
transcriptional modification of the p53 protein or the inhibition of p53 degradation
182 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
pathways in these cells. Accordingly, the elevation of activated intracellular p53
levels in cells exposed to IGF-I and fibroblast-secreted factors may be associated
with the activation of G1 cell cycle arrest response in keratinocytes described
previously.
In both fibroblast-CM and IGF-I pre-treated irradiated keratinocytes described
in this Chapter, a significant reduction in the proportion of cells at the G2/M phases
was also observed. Complex cellular DNA damage surveillance mechanisms
function to regulate the progression of the cell cycle into this critical mitotic phase
(Morgan and Kastan, 1997). Studies on UVB-irradiated keratinocytes have revealed
an association between the functional status of p53 and the ability of cells to undergo
G2/M arrest, with p53-defective irradiated keratinocytes observed to have elevated
levels of cell cycle arrest at this checkpoint (Athar et al., 2000). Thus, the enhanced
activation of p53 by IGF-IR activation in keratinocytes described herein may
contribute to the p53-mediated inhibition of G2/M arrest in IGF-I pre-treated
keratinocytes. Moreover, the increased chk-1 phosphorylation observed in irradiated
keratinocytes reported herein has also been linked to the inhibition of G2 to M
transition via the downstream activation of Cdc25C (Sanchez et al., 1997; Xiao et
al., 2003).
IGF-IR activation and the DNA damage response in UVB-irradiated
keratinocytes
Keratinocytes respond to UVB-induced DNA damage by activating complex
DNA damage response signal-transduction pathways, which serve to prevent the
propagation of heritable mutations during cell division (Zhou and Elledge, 2000).
Various interacting pathways consisting of an array of DNA damage sensors,
transducers and effectors operate in concert to execute the downstream cellular
responses, such as cell cycle arrest, DNA repair and apoptosis (Stokes et al., 2007;
Liu et al., 2009). The molecular mediators responsible for sensing DNA damage in
mammalian cells have yet to be comprehensively defined to date. However,
transducers of these pathways have been well-characterised, of which ATM and
ATR are known to be the main players involved in propagating downstream signals
in response to DNA damage (Elledge, 1996).
The detection of CPDs in irradiated keratinocyte monolayers and skin
substitutes has been utilised commonly as a measure of photogenotoxicity and the
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 183
repair of UVB-induced DNA damage (Marrot et al., 2010; Wölfle et al., 2011).
Herein, the enhancement of CPD repair in irradiated keratinocytes pre-treated with
IGF-I and fibroblast CM was reported (Figure 5.8). Furthermore, blocking the
activation of the IGF-IR using neutralising antibodies in both IGF-I and CM-treated
keratinocyte cultures reversed this promotion of CPD repair in UVB-exposed cells
(Figure 5.9). These results illustrate the significance of fibroblast-secreted factors,
including IGF-I, on driving the repair of DNA photolesions in keratinocytes.
Importantly, the accumulation of unrepaired nuclear CPDs has been identified as the
principal cause of NMSC development (Jans et al., 2005). Moreover, p53 hot spot
mutations from human NMSC tumours strongly correlate with incomplete CPD
repair in these tissues (Drouin and Therrien, 1997). Hence the fibroblast-mediated
acceleration of CPD repair may have important implications in the nature of
photocarcinogenesis.
In UVB-exposed cells, the activation of ATR has been shown to be an
initiating step in the recruitment of downstream members of the DNA damage
response program (Smith et al., 2010). In the study reported herein, a significant
enhancement of phosphorylated ATR and chk-1 were detected in UVB-irradiated
keratinocytes pre-treated with IGF-I. The role of IGF-IR activation in the ATR-
mediated DNA repair response has been a relatively unexplored area. However,
emerging experimental evidence indicates a connection between IGF-I and the
cellular maintenance of genomic stability (Yang et al., 2005; Hinkal and Donehower,
2008). For example, it has been demonstrated that IGF-IR signalling enhances
neuronal and kidney cell survival via the stimulation of ATM (Yang and Kastan,
2000). Moreover, this activated phospho-ATM was also shown to upregulate the
transcription of IGF-IR, which may also act as a positive regulator of cell survival
and DNA repair after irradiation (Peretz et al., 2001). Additionally, the functionality
of the DNA repair protein Rad51 has been associated with the enhanced activity of
the IGF-IR signalling molecule, IRS-1 following the formation of double-strand
DNA breaks by ionizing radiation (Trojanek et al., 2003a). Downstream from ATR
activation, ATRIP has been demonstrated to regulate many aspects of ATR-mediated
DNA repair and cell cycle regulation, though the precise biological activity of ATR-
ATRIP complexes is still not completely understood (Cortez et al., 2001). In the
Transwell® keratinocyte system reported herein, the activation of IGF-IR did not
184 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
significantly alter the levels of ATRIP in keratinocytes after UVB irradiation. The
activation of downstream pathways following ATR phosphorylation, however, have
been observed to also occur in an ATRIP-independent manner (Ball et al., 2005).
Hence, the regulation of ATR activity in irradiated keratinocytes by IGF-I requires
further investigation.
Phospho-ATR propagates DNA damage response signals via the activation of
chk-1, a process which has been shown to be a principal trigger of DNA damage and
replication checkpoints after genomic damage (Liu et al., 2000; Zhao and Piwnica-
Worms, 2001). The analysis of DNA damage pathways activated in irradiated
keratinocytes described herein showed an elevation in the levels of phospho-chk-1 in
cells pre-treated with IGF-I. The activation of the ATR/chk-1 pathway has
previously been described as having a protective effect on irradiated keratinocytes by
inhibiting apoptosis and enhancing p53 stability (Adimoolam and Ford, 2003;
Blasina et al., 2008; Lu et al., 2008; Heffernan et al., 2009). The phosphorylation of
the protein phosphatase cdc25C at Serine 216, an important mitotic regulator, is
another downstream event from chk-1 activation (Peng et al., 1997). Phospho-
cdc25C creates a binding site for 14-3-3 proteins, thus forming a complex which
blocks cellular entry into mitosis at the G2/M checkpoint following DNA damage
(Furnari et al., 1999; Kumagai and Dunphy, 1999). In IGF-I treated irradiated
keratinocytes, a significant reduction in the levels of phospho-cdc25C was observed
as compared to irradiated cells in SFM only. However, studies on cell lysate samples
obtained at more post-irradiation time-points need to be conducted, with previously
published data showing peak levels of phospho-cdc25C being detected between 6
and 9 hours after the generation of DNA damage (Gutierrez et al., 2010).
Theoretically, the ATR/chk-1-mediated activation of DNA damage response
pathways and cell cycle checkpoints in the keratinocytes would enhance the repair of
UVB-induced DNA damage in these cells. However, recent studies suggest a
potential link between the inhibition of ATR/chk-1 pathways and the seemingly
contradictory effect of NMSC suppression (Heffernan et al., 2009; Kawasumi et al.,
2011). These studies have demonstrated the potential for caffeine as a therapeutic
against NMSC development by blocking ATR/chk-1-regulated cell survival and
promoting apoptosis in keratinocytes harbouring p53 mutations (Kerzendorfer and
O'Driscoll, 2009). These findings, along with the IGF-I-mediated enhancement of
Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response 185
ATR/chk-1 pathways reported herein illustrate the complexity of keratinocyte
photoresponses and epidermal photocarcinogenesis, for which identifying potential
therapeutic targets remains challenging.
Conclusion and significance
The body of work presented in this Chapter describes for the first time, the
effects of both fibroblast-secreted factors and recombinant IGF-I on keratinocyte
responses to UVB radiation, using a Transwell® in vitro model. The addition of IGF-
I to keratinocyte cultures and the subsequent activation of the IGF-IR resulted in
significant shifts in cell cycle progression, the inhibition of apoptosis, the stimulation
of DNA repair, as well as the modulation of p53 expression and activity.
Furthermore, blocking of IGF-IR activity in co-cultured fibroblasts significantly
reduced the fibroblast-induced inhibition of apoptosis and enhancement of CPD
repair in irradiated keratinocytes. Notably, IGF-IR activation was also shown to play
a role in the activation of members of the DNA damage response following UVB
irradiation, such as ATR, chk-1 and cdc25.
Studies on the influence of IGF-IR activation and its downstream signalling
pathways on UVB responses in primary human keratinocytes have thus far been
limited (Héron-Milhavet et al., 2001; Trojanek et al., 2003a; Thumiger et al., 2005;
Lewis et al., 2008). The requirement of a fibroblast feeder layer and complex cell
culture medium using undefined elements such as FCS for supporting the in vitro
culture of human keratinocytes has restricted investigations into the specific
pathways involved in orchestrating the protective UVB photoresponse. The
Transwell® photobiology utilised in this study describes the cultivation of primary
keratinocytes in a defined SFM, with and without the presence of human dermal
fibroblast co-culture for analysing the cellular effects associated with IGF-I pre-
treatment on cell survival, repair and apoptosis post-irradiation. Importantly, this in
vitro model represents a highly advantageous platform from which other molecular
mediators critical to the epidermal response to UVB exposure can be characterised in
future. Potentially, the testing of novel photoprotectants and therapeutics against
cellular UVB-damage can also be conducted using this technique in combination
with 3D organotypic skin models. Additionally, this technique of keratinocyte
culture can also be modified to promote the stratification of keratinocytes, thus
186 Chapter 5: Investigating the role of IGF-I in the keratinocyte UVB response
forming a 3D Transwell® keratinocyte culture model which more closely
recapitulates the epidermis in vivo (Kandyba et al., 2008).
Importantly, the results reported herein demonstrate the close association
between fibroblast-secreted factors, in particular IGF-I, on the regulation of normal
protective responses of keratinocytes to the damaging effects of UVB radiation.
Hence, the dysregulation of normal fibroblast function in skin may therefore impact
negatively upon the protective and regenerative potential of proliferative
keratinocytes within the epidermis. Indeed, emerging epidemiological and
experimental evidence points towards a correlation between diminishing fibroblast
functionality in photodamaged or aging skin and an increased risk of NMSC
development (Chuang et al., 2005; Lewis et al., 2010). Accordingly, with the
importance of IGF-I in facilitating DNA repair and cell cycle regulation in irradiated
keratinocytes as demonstrated in this Chapter, this pathway may have the potential to
be targeted in the development of novel therapeutics to minimise photodamage and
the risk of photocarcinogenesis. Researchers within our laboratory have characterised
a novel growth factor complex, VitroGro® ECM (Tissue Therapies, Brisbane,
Australia), consisting of ECM, IGFBP and IGF-I components, which has been
proven to accelerate re-epithelialisation and epidermal regeneration following skin
damage. With the potent photoprotective effects of IGF-I observed in UVB-treated
human keratinocytes reported herein, such therapies may conceivably be applied to
treating and protecting against skin sun damage.
Chapter 6: General discussion 187
Chapter 6: General discussion
Australia experiences some of the highest levels of solar UVR globally and as
a consequence, it is estimated that 2 in 3 Australians will be diagnosed with a form of
skin cancer by the age of 70 (Gies, 1998; Staples et al., 2006). Skin cancers represent
the biggest cancer-related financial burden on the Australian health care system,
costing an estimated $300 million annually (Australian Institute of Health and
Welfare, 2005). In particular, NMSCs represent the most commonly diagnosed skin
malignancy in Australia, with an estimated 430,000 new cases diagnosed in 2008
(Australian Institute of Health and Welfare, 2008). Moreover, recent studies have
indicated that rapidly rising incidences of NMSCs also represent a global health
problem (Lomas et al., 2012). Hence, there is an urgent need to develop more
effective preventative and therapeutic strategies to address the incidences of UVR-
induced skin cancers. Associations between unprotected sun exposure and
photocarcinogenesis have been well-established over 40 years of photobiology
research (Epstein, 1978). However, major knowledge gaps still exist in our
understanding of the skin’s intrinsic responses to UVR and in particular, how these
cellular processes are disrupted during skin cancer development and progression. In
part, the lack of a complete, accessible and biologically-relevant experimental model
to investigate the human photoresponse has impeded the progress of photobiology
research thus far.
In view of this, the overriding aim of this project was to develop and
characterise novel in vitro approaches to study human keratinocyte responses to
UVB. Importantly, some of the major limitations associated with several currently-
used photobiology models, including small animal and conventional cell culture
systems were addressed in the development of the experimental skin models
described herein. The data obtained from the studies reported in this dissertation, as
presented in Chapters 3 to 5 have described the novel application of fibroblast-
keratinocyte Transwell® co-cultures and 3D tissue-engineered skin equivalent
constructs as primary cell-based platforms for the analysis of epidermal responses to
UVB.
188 Chapter 6: General discussion
Using these experimental strategies, the most significant outcomes of this
project are:
1. Two 3D organotypic skin equivalent culture models comprised of a
stratified epidermal layer cultured on an acellular or fibroblast-
populated dermal scaffold, termed the HSE-KC and HSE-KCF
respectively, have been successfully characterised in terms of their
physiological similarity to native skin.
2. Significantly, I have reported for the first time, the validation of these
reconstituted skin composites as in vitro photobiology models, with
several archetypal markers of UVB skin damage shown to be
experimentally-induced in these skin composites. Moreover, it was
demonstrated that keratinocytes within the HSE-KC and HSE-KCF
retained the ability to repair UVB-induced damage and undergo a
process of epidermal regeneration following irradiation.
3. This thesis describes a novel strategy for investigating epidermal-
dermal interactions during the acute UVB response by utilising the 3D
HSE-KCF skin culture model and the novel Transwell® fibroblast-
keratinocyte co-culture system. Notably, the influence of paracrine
signalling from dermal fibroblasts on the inhibition of keratinocyte
apoptosis, the acceleration of DNA photolesion repair and the
enhancement of p53 activation and epidermal regeneration following
UVB irradiation in these in vitro photobiology systems were described.
4. The IGF system, in particular the IGF-I-mediated activation of IGF-IR,
was reported to be an important pathway of fibroblast-keratinocyte
communication during the UVB response. Importantly, I have
described in this thesis that the suppression of apoptosis, accelerated
repair of CPD DNA photolesions, modulation of cell cycle progression
and the expression and activation of p53, as well as the enhanced
activation of DNA damage response proteins in IGF-I-treated irradiated
keratinocytes. Notably, this represents the first study of the effects of
IGF-I on primary human keratinocyte responses to UVB using the
Transwell® serum-free medium culture system.
Chapter 6: General discussion 189
Novel in vitro approaches for studying keratinocyte responses to UVB
Commonly utilised biological models for studying the cutaneous responses to
UVR include cell cultures, animal models, as well as a variety of organotypic skin
substitutes (Fernandez et al., 2012b). While these experimental systems have been
advantageous in broadening our knowledge on many aspects of the keratinocyte
photoresponse, these techniques possess inherent limitations which need to be
addressed. Conventional submerged monolayer keratinocyte cultures, for example,
deviate considerably from their physiological similarity to native skin, both in terms
of their structural and biological properties (Mazzoleni et al., 2009). Similarly, small
animal models differ from humans in terms of their cutaneous biology, hence do not
accurately model the human epidermal photoresponse (Szabo et al., 1982; Menon,
2002). Consequently, increasingly robust in vitro skin substitute models
incorporating natural and synthetic tissue scaffolds have been developed to study
cutaneous photoresponses experimentally. Nevertheless, factors such as
reproducibility, accessibility, cost, ethical considerations and biological similarity to
native human skin have hampered their widespread usage in photobiology research
(Marrot et al., 2010; Fernandez et al., 2012b).
To address some of the abovementioned drawbacks of current photobiology
models, the HSE-KC and HSE-KCF skin cultures utilised in this project were chosen
for their ability to recapitulate the basic architecture of human skin, in particular the
organisation of the epidermis. As described in Chapter 3, the HSE-KC was observed
to show several parallels to ex vivo skin in relation to not only the overall structure
and organisation of the epidermis, but also the expression of key epidermal
differentiation markers, including keratins 1, 2e, 10, 11, 14 and filaggrin. Also, in
contrast to some monolayer and skin substitute culture models, the use of the skin
tissue-derived DED scaffold in the HSE-KC was reported to retain elements of the
basement membrane, such as collagen IV (Hinterhuber et al., 2002). Therefore, the
HSE-KC captures aspects of the epidermal microenvironment more faithfully, which
has been shown to impact on cell surface receptor expression, proliferative capacity,
the synthesis of ECM components and importantly, the cellular responses to external
stimuli such as UVR (Bissell et al., 1982; Lin and Bissell, 1993; Smalley et al.,
2006).
190 Chapter 6: General discussion
UVB is regarded as the most damaging of the UVR wavelengths, in particular
due to its detrimental effects on the stratum basale of the epidermis, especially DNA
damage through the formation of nuclear photolesions and ROS (Brash et al., 1996;
Gailani et al., 1996). Analysis of the irradiated HSE-KC and HSE-KCF constructs
revealed the generation of several key characteristic UVB-induced damage markers,
including the formation of CPD photolesions, apoptotic sunburn cells, p53
expression, as well as increased synthesis of inflammatory cytokines. Importantly,
besides mirroring these aspects of UVB-induced epidermal damage, these skin
equivalent composites were also observed to retain their capacity to repair
photodamage and activate regenerative responses post-irradiation. In the future, other
markers of UV-damage, such as the generation of reactive oxygen species can also
be analysed in these HSE-DED photobiology models, and hold potential for testing
novel antioxidant therapies to counteract this damage (Hanson and Clegg, 2002;
Wertz et al., 2004; Vostalova et al., 2010).
The successful characterisation of the UVB response in the HSE-KC and HSE-
KCF 3D skin models described herein has several significant implications in the field
of photobiology research. Firstly, it represents a novel strategy in the study of UV-
damage, repair and regeneration within a physiologically-relevant tissue
microenvironment. Critically, the formation of a well-organised and stratified
epidermis is advantageous over conventional monolayer cultures in a variety of
applications (Bernerd and Asselineau, 1998, 2008). This reconstituted skin model has
the potential to be used to test topically-applied photoprotectants and sunscreens
against UVB radiation-induced skin damage (Bernerd et al., 2000; Lejeune et al.,
2008; Marionnet et al., 2011; Bernerd et al., 2012). Next, the use of these skin
reconstructs can reduce the requirement for animal and human participant models,
many of which are associated with extensive ethical considerations and limitations.
Currently, there is a global shift towards restricting the use of experimental animal
models for testing and developing skin treatments and cosmetic products (Welss et
al., 2004). In view of this, the HSE-DED may represent a cost effective, easily
accessible and biologically compatible substitute for the use of animals in the testing
and development of novel and improved sunscreens and treatments for sun damage.
Besides analysing the UVB-induced alterations to the epidermis, the HSE-KCF
(consisting of both an epidermal and fibroblast-populated dermal component) can
Chapter 6: General discussion 191
also be employed to study various dermal modifications as a result of UVA
exposure. This wavelength penetrates into the dermis and damages fibroblasts, thus
causing ECM modifications associated with photoaging, including the degradation of
collagen (Kligman, 1996; Bernerd and Asselineau, 1998; Wlaschek et al., 2001;
Svobodova et al., 2006; Amano, 2009). While organotypic skin models incorporating
synthetic dermal scaffolds have recently been used as photoaging models, the
application of the HSE-KCF may be more beneficial and physiologically-relevant
due to its use of an ex vivo dermis (Sok et al., 2008).
The epidermal component of the HSE-KC consists solely of keratinocytes at
different stages of differentiation. To more closely mimic the anatomy of the native
epidermis, other epidermal cell types including melanocytes and Langerhans cells
can be incorporated into the HSE model in future. Pigmented HSE models have
previously been employed to study events in the epidermal photoresponse, such as
melanogenesis (Topol et al., 1986; Archambault et al., 1995; Liu et al., 2011).
However, the isolation and culture of human melanocytes in vitro have had mixed
results in terms of preserving the original morphological, genetic and functional
aspects of these cells (Halaban et al., 1986; Halaban, 2000; Czajkowski, 2011). Skin
equivalents for photobiology with an integrated immune component have also been
previously described, wherein Langerhans and dendritic cells obtained from blood-
derived monocytes have been seeded into the HSE epidermis (Bechetoille et al.,
2007). Hence, combining these techniques to create improved HSE-DED models
represents a possible strategy towards creating experimental skin models that more
closely mimic the in vivo situation.
The HSE-DED skin construct utilised primary human keratinocytes seeded
onto an ex vivo dermal scaffold. This model may therefore represent a highly
advantageous in vitro platform for studying various aspects of photobiology in
human cells while bypassing the limitations associated with exposing volunteers to
UVR. Using this technique, primary keratinocytes can be obtained and cultured from
a specific subset of individuals to study the epidermal responses to UVB in, for
example, psoriatic or geriatric skin. However, the responses of primary keratinocytes
to UVR are highly dependent on factors such as the phototype of the skin donor, the
anatomical location of the tissue of origin, the age and gender of the donor, as well as
pre-existing medical conditions (Thomas-Ahner et al., 2007; Lewis et al., 2010;
192 Chapter 6: General discussion
Weatherhead et al., 2011; Smit et al., 2012). Additionally, primary cells and tissues
have a limited lifespan and capacity to scale up to the large cell numbers that are
often required to generate multiple constructs required for statistical analyses, thus
creating significant intra- and inter-laboratory variations (Brohem et al., 2011). The
use of immortalized cell lines such as HaCaT cells in organotypic skin models can
aid in addressing the reproducibility and consistency for biological, clinical and
industrial research applications (Boelsma et al., 1999). On the other hand,
reconstructed epidermal composites comprised of HaCaT keratinocytes have been
demonstrated to possess poor tissue organisation and cellular morphology
histologically, accompanied with a marked alteration in the tissue architecture
(Schoop et al., 1999). Moreover, HaCaT cells possess p53 mutations which can
significantly alter their responses to UVB compared to normal human keratinocytes
(Lehman et al., 1993; Henseleit et al., 1997). In this thesis, I report significant
histological and immunohistochemical similarities between the HSE-DED constructs
and native skin, particularly in terms of the structure and organisation of the
epidermis. Hence, performing sufficient experimental replicates and applying
parameters to the choice of skin tissue donors may aid in limiting inter-sample
variability, thus creating a robust and representative biological skin model.
While reconstructed skin models such as the HSE-DED are beneficial for their
similarity to native skin, the complexity of these systems may limit their use for
certain experimental applications. For instance, investigating the effects of the
addition of specific treatments to the cell culture medium is limited due to the
waterproof nature of the stratum corneum and the densely packed collagen fibres of
the DED, which may limit the permeability of substances to the basal keratinocytes.
The use of keratinocyte monolayer cultures can overcome such limitations. However,
the requirement for an irradiated mouse fibroblast feeder layer and complex,
undefined cell culture medium in routine primary keratinocyte culture methods pose
a problem for: 1) studying the effects of specific growth factors, particularly at low
concentrations; and 2) investigating fibroblast-keratinocyte paracrine interactions in
primary cells (Rheinwald, 1975). Herein, the use of a Transwell® co-culture system
which possesses several novel features over conventional keratinocyte culture
methods is described. Firstly, this culture technique supports the cultivation of
fibroblast-free keratinocyte monolayers in serum-free medium conditions, thus
Chapter 6: General discussion 193
negating the requirement of lethally-irradiated mouse fibroblasts as a feeder layer.
Instead, primary dermal fibroblasts are used to support keratinocyte cultures, which
can then be used for investigating paracrine interactions between these two
physically-separated cell populations. Secondly, upon the establishment of these
primary keratinocyte cultures, they can then be transferred to fibroblast-free, serum-
free conditions for studying the effects of specific treatments on these cells. Members
of our research group have also previously described alternative methods for the
serum-free cultivation of primary keratinocytes and fibroblasts using recombinant
proteins or lethally-irradiated human fibroblasts, which in future, may also be used in
conjunction with the Transwell® model described herein (Mujaj et al., 2010).
Additionally, a recent study has also described the use of non-irradiated human
fibroblasts to support the growth of keratinocyte cultures for clinical purposes (Jubin
et al., 2011). Accordingly, the integration of these culture techniques, together with
the Transwell® and HSE-DED models described in this thesis, may aid in the
creation of improved experimental platforms for a variety of keratinocyte research
applications.
Investigating the role of dermal-epidermal paracrine interactions during the
UVB response
Human keratinocytes possess sophisticated protective and repair mechanisms
to resist oncogenic transformation by the damaging effects of UVB exposure
(D'Errico et al., 2007). However, factors within the cutaneous microenvironment that
influence these processes are still not completely understood. The studies undertaken
in this thesis describe for the first time, using novel in vitro approaches, the
modulation of keratinocyte responses to UVB through the action of fibroblast-
secreted factors. Indeed, the presence of dermal fibroblasts in both 2D and 3D cell
culture systems resulted in the significant inhibition of UVB-induced apoptosis, the
augmentation of DNA repair processes, as well as enhanced p53 activation in
irradiated keratinocytes.
Fibroblast-keratinocyte interactions and the effect of fibroblast-secreted factors
on epidermal regeneration have been extensively studied, predominantly in wound-
healing (Werner and Smola, 2001; Raja et al., 2007; Werner et al., 2007). Through
the secretion of various cytokines, growth factors and ECM components, fibroblasts
are able to regulate numerous keratinocyte processes, including survival,
194 Chapter 6: General discussion
proliferation and differentiation, all of which are also central in the epidermal UV
response (Ichihashi et al., 2003; Wong et al., 2007; Nolte et al., 2008). To date, there
has been a strong research focus in relation to the biology of melanocyte responses
and cutaneous immune responses to UV exposure and the risk of melanoma
development (Schwarz, 2008; Zaidi et al., 2012). However, relatively less is
understood about the mechanisms behind fibroblast-keratinocyte crosstalk during the
UVB response and importantly, the initiation, development and progression of
NMSC. The effect of dermal-epidermal paracrine interactions on keratinocyte
responsiveness to UVB has been observed in a variety of in vitro systems (Kuhn et
al., 1999; Will, 2000; Lewis et al., 2010). Notably, it was established via the studies
conducted in this project that fibroblasts had a protective effect on irradiated
keratinocytes, with cells in fibroblast co-culture conditions prior to UVB insult
showing enhanced cell survival during the post-irradiation period.
The inhibition of keratinocyte apoptosis and the promotion of epidermal
regeneration following UVB exposure by dermal fibroblasts, suggest that these cells
may play an important role in maintaining the barrier function of the epidermis after
UV-damage. Preventing widespread keratinocyte death via UVB-induced apoptosis,
as well as inducing proliferation and differentiation during the epidermal
regenerative phase, have been shown to be critical processes in ensuring the
epidermis retains its functionality after environmental stress (Smith and Rees, 1994;
Bernerd et al., 2001b; Del Bino et al., 2004). Also, chronic sun exposure has been
shown to negatively impact on dermal fibroblasts, with altered gene and protein
expression levels and ROS production observed in cells derived from photodamaged
skin (Gilchrest, 1980; Tyrrell and Pidoux, 1989; Ma et al., 2001). Hence, besides
UVB-induced damage to keratinocytes following exposure, the deleterious effects on
dermal fibroblasts may also contribute to shifts in epithelial-mesenchymal crosstalk
and the keratinocyte death-survival balance (van Laethem et al., 2005; van Laethem
et al., 2009).
Another notable result from this study was the influence of fibroblasts on the
expression and activation of the tumour suppressor protein, p53 in UVB-irradiated
keratinocytes. Indeed, p53 has been demonstrated to play a cardinal role in the
development of a wide range of cancer types, with p53 mutations being a key
biomarker of susceptibility in most skin cancers (Rivlin et al., 2011). Despite a
Chapter 6: General discussion 195
wealth of experimental evidence on the functional significance and biological
activity of p53 being accumulated using various in vivo and in vitro techniques,
much is still unknown regarding the modulation of p53 function and the regulation of
its activity in human keratinocytes (Jonason et al., 1996; Li et al., 1996; Li et al.,
1998). With its central cellular functions, including apoptosis regulation, cell cycle
control and the activation of DNA repair mechanisms, it is evident that the
dysregulation of p53-mediated processes is axial in the initiation of skin cancers.
Besides UVB-induced mutations, post-translational modifications of p53, including
phosphorylation, influence its localisation, stability, DNA binding affinity and
transcriptional activity, and consequently its overall activity (Vousden, 2000;
Vousden and Woude, 2000). In line with this, the results from our study show the
presence of fibroblasts reduced relative levels of p53 mRNA and total protein in
irradiated keratinocytes, while maintaining higher levels of phosphorylated p53 in
these cells, suggesting enhanced protein stability and half-life. With the balance of
p53 stabilisation and destabilisation being critical for DNA repair and the regulation
of the cell cycle, this indicates that the mediation of these processes by fibroblasts
may impact on critical aspects of photocarcinogenesis in keratinocytes (Maki and
Howley, 1997; Geyer et al., 2000). Henceforth, further investigations need to be
conducted to identify the precise mechanisms surrounding the regulation of p53
activity by fibroblast-secreted factors, which can be carried out by taking advantage
of novel in vitro platforms such as the HSE-KCF.
Recent theories suggest that the dysregulation of critical keratinocyte post-
irradiation repair processes may be linked to the diminished functionality of dermal
fibroblasts, such as in aged and photodamaged skin (Lewis et al., 2010). Indeed, the
majority of skin malignancies are detected in individuals over the age of 60, which
illustrates a strong correlation between skin cancer development and advancing age
(Krtolica et al., 2001). It has also been proposed that one of the first epidermal
changes occurring during NMSC development is the replication of keratinocytes
harbouring unrepaired UVB-damaged DNA, which could establish permanent
chromosomal mutations, hence creating a subpopulation of initiated carcinogenic
cells (Parrinello et al., 2005). Dermal-epidermal crosstalk as a pathway for
maintaining appropriate keratinocyte responses to UVB and suppressing UVB-
induced carcinogenesis is a relatively new and unexplored paradigm of non-
196 Chapter 6: General discussion
melanoma skin carcinogenesis (Lewis, 2008; Lewis et al., 2010). Indeed, the
discovery that fibroblast-secreted factors, such as IGF-I, govern appropriate
keratinocyte responses to UVB, including the balance between cell survival and
apoptosis, is consequential in terms of the development of therapeutic interventions
against NMSC development (Chuang et al., 2005; Lewis, 2008; Lewis et al., 2008).
Collectively, these results illustrate the importance of dermal-epidermal
interactions in orchestrating the repair and regeneration of the epidermis following
UVB exposure and have underlying implications towards the dysregulation of these
processes during photocarcinogenesis. Importantly, while the data obtained from the
studies described here have elucidated some of the fibroblast-mediated functional
outcomes in UVB-exposed keratinocytes, the next step in this research would
involve characterising the specific molecular mediators and pathways involved in
producing these effects.
The role of IGF-I as a fibroblast-mediated regulator of keratinocyte UVB
responses
The IGF system represents a well-studied channel through which fibroblasts
regulate many aspects of epidermal physiology (Haase et al., 2003; Van Lonkhuyzen
et al., 2007; Kricker et al., 2010). Moreover, a major research focus of our laboratory
has been the development of novel therapeutic innovations for skin regeneration by
taking advantage of the potent effects of IGF-I on epidermal repair (Van Lonkhuyzen
et al., 2007; Upton et al., 2008; Kricker et al., 2010; Upton et al., 2011; Xie et al.,
2011). Henceforth, an aim of this thesis was to investigate the role of IGF-I in
modulating keratinocyte survival and repair responses following UVB-induced
damage. Significantly, it was reported herein that physiologically-relevant
concentrations of IGF-I may play central roles in determining keratinocyte cell fate
by influencing apoptosis, cell survival, cell cycle progression, p53 activation and the
DNA damage response in these cells after irradiation. Moreover, these responses
have not previously been reported in primary human keratinocytes cultivated in
serum-free culture conditions.
Using the Transwell® culture system, the improvement of keratinocyte
viability and the inhibition of apoptosis in IGF-I and fibroblast CM-treated cells after
UVB irradiation was reported. The induction of cellular apoptosis in UVB-damaged
cells has to be tightly-regulated in the epidermal context. On one hand, this process is
Chapter 6: General discussion 197
critical for removing DNA-damaged cells and hence preventing the propagation of
cells possibly harbouring mutations (Zutterman et al., 2010). However, cell death
particularly among basal proliferating keratinocytes must also be limited in order to
retain the barrier function of the epidermis and to ensure the capacity for self-renewal
(Regnier et al., 1986). It has been previously demonstrated that basal keratinocytes
are more susceptible to UVB-induced apoptosis as opposed to terminally
differentiated spinous keratinocytes situated in the suprabasal compartment
(Chaturvedi et al., 1999; Qin et al., 2002; Chaturvedi et al., 2004). Interestingly,
IGF-I signalling is confined to the stratum basale of the epidermis, with both IGF-I
and IGFBP-mediated effects previously described as being capable of influencing
basal keratinocyte responses to UVB (Hollowood, 2000; Williams, 2000;
Handayaningsih et al., 2012). For instance, IGFBP-3 has been shown to affect
keratinocyte survival, differentiation and proliferation in an IGF-I-independent
manner following UVB-treatment (Edmondson et al., 2005; Thumiger et al., 2005).
The higher sensitivity of basal keratinocytes to UVB and the regulation of post-
irradiation cellular fate by IGF-I signalling may therefore be a crucial cutaneous
mechanism for resisting NMSC development.
Significantly, the studies reported in Chapter 5 of this thesis have described the
IGF-I-mediated augmentation of DNA damage response elements following UVB
irradiation, as well as the acceleration of CPD DNA photolesion removal in IGF-I-
treated keratinocytes. Together, this implies that IGF-I signalling may be a critical
element in maintaining the genomic integrity of basal keratinocytes, thus impeding
the initiation of photocarcinogenesis. The involvement of IGF-I in supporting DNA
repair following cellular exposure to radiation has previously been documented
(Héron-Milhavet et al., 2001; Trojanek et al., 2003b; Yang et al., 2005). Moreover,
the analysis of protein networks upregulated as a result of DNA damage reveals
several intersections between the DNA damage response and PI3K-AKT signalling
pathways (Matsuoka et al., 2007). Additionally, the association between IGF-IR
activation and increased p53 activity was reported both in this project, as well as in
other previously-described in vitro systems (Lewis, 2008; Handayaningsih et al.,
2012).
With increasing evidence mounting and implicating the involvement of IGF-IR
signalling pathways in mediating epidermal responses to UVB, and potentially the
198 Chapter 6: General discussion
initiation of NMSC, the IGF system may be targeted as a therapeutic or preventative
intervention against photodamage in skin. At present, the gold standards of clinical
strategies used to treat photodamaged skin, such as the use of retinoids, lasers and
surgery show limited effectiveness at reversing sun-damage and are associated with
severe side effects (Samuel et al., 2005). Moreover, these therapies generally target
the effects of UVA-induced damage to the dermis for cosmetic purposes (Kang et al.,
2001). Importantly, various chemopreventative strategies against the development of
NMSCs have proven to be unsuccessful thus far, with the use of sunscreens and
screening programs as a preventative measure the only clinical recommendation to
date (Choudhury et al., 2012; Wu and Stern, 2012). Thereupon, the development of
innovative therapeutics targeting the protective properties of the IGF system on UV-
exposed keratinocytes may aid in limiting or counteracting the damaging effects of
UVB. For instance, a novel growth factor complex, VitroGro®, consisting of ECM,
IGFBP and IGF-I components, developed by members of our research group has
been shown to enhance re-epithelialisation and epidermal regeneration in the wound-
healing context (Hollier et al., 2005; Dawson et al., 2006; Upton et al., 2011).
Conceivably, similar treatments may be applied to address the lack of suitable
clinical interventions against NMSC development.
Future directions
The cell-based experimental platforms described in this thesis have
successfully been employed to study factors influencing human keratinocyte
responses to UVB radiation. Following on from the studies presented herein, other
aspects of the epidermal photoresponse can be investigated using these in vitro
models. While the UVB radiation wavelength has been extensively documented as
contributing to biological and clinical aspects of skin photodamage, the role of UVA
in these responses can also be investigated using the models described herein.
Indeed, UVA is known to play major roles in skin immunosuppression, photoaging
and carcinogenesis (Lowe et al., 1995; Seite et al., 2010). Moreover, UVA accounts
for the majority of the solar UV irradiance present on the terrestrial surface. Hence,
strategies for photoprotection against the harmful effects of both UVA and UVB
radiation can be identified and tested using the in vitro platforms described in this
thesis (Moyal, 2012). For example, the HSE-DED constructs and Transwell®
Chapter 6: General discussion 199
keratinocyte cultures can be irradiated with solar simulated UVR, to more accurately
represent cutaneous solar UVR exposure (Kostyuk et al., 2012).
While IGF-I has been identified as an important modulator of the keratinocyte
UVB photoresponse, other fibroblast-secreted factors which may also function to
protect the epidermis from UVB radiation-induced damage can be explored. For
instance, EGF and KGF have also been shown to play a role in regulating post-
irradiation processes (Xu et al., 2006; Lotti et al., 2007; Kovacs et al., 2009). These
and other growth factors and ECM elements can be explored in future and potentially
be taken advantage of in the development of therapeutic and protective clinical
therapies against cutaneous photodamage. Moreover, modulating keratinocyte
expression of genes involved in the IGF-I pathway may be yet another approach to
investigating the mechanisms of IGF-induced photoprotection.
Chapter 6: General discussion 201
Conclusion
In summary, this PhD project has characterised and validated novel 2D
Transwell® and 3D HSE-DED in vitro skin cell culture photobiology models for
investigating keratinocyte responses to UVB. Notably, as an improvement to many
currently-utilised photobiology models, the HSE-KC and HSE-KCF organotypic skin
cultures described herein were found to show many biological and physiological
parallels to native human skin. Moreover, analysis of UVB-exposed HSE-DED
constructs demonstrated the generation of key markers of epidermal UVB damage
and the capacity for repair and regeneration under culture conditions. Significantly,
the HSE-KCF and Transwell® co-culture systems were employed to describe for the
first time the complex interactions between primary human keratinocytes and dermal
fibroblasts during the acute UVB response using these in vitro models. Fibroblasts
were shown to exert a protective effect on irradiated epidermal keratinocytes and
significantly modulated a range of cellular responses, including cell survival,
apoptosis, DNA repair, p53 activation and epidermal regeneration. Furthermore, the
activation of IGF-IR signalling pathways by IGF-I ligand binding was reported to be
a major pathway of the fibroblast-mediated protection of keratinocytes. Significantly,
IGF-I treatment improved keratinocyte viability, the activation of DNA damage
response pathways, repair of DNA damage and regulated cell cycle progression and
apoptosis, potentially via p53-mediated actions. Taken together, this project has
characterised the use of physiologically-relevant and novel platforms for studying
keratinocyte photoresponses experimentally, with a wide-range of potential research
applications. Additionally, the importance of dermal-epidermal interactions, in
particular via IGF-I, in protecting against UVB-damage and in orchestrating the
repair responses reported herein has identified potential targets for therapeutic and
protective strategies against epidermal photodamage.
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