Intrinsic regulation of differentiation markers in human epidermis, hard palate and buccal mucosa

Intrinsic regulation of di�erentiation markers in humanepidermis, hard palate and buccal mucosa

Susan Gibbs*, Maria Ponec

Department of Dermatology, Leiden University Medical Centre, Wassenaarseweg 72, Bld 3, Sylvius Laboratory, 2333 AL Leiden,

The Netherlands

Received 8 December 1998; received in revised form 20 July 1999; accepted 13 August 1999

Abstract

Di�erent epithelia show extensive variation in di�erentiation. Epidermis and epithelium from the hard palate are

both typical examples of orthokeratinized epithelia whereas buccal mucosa is an example of a non-keratinizedepithelium. Each of these tissues can be distinguished morphologically and also by the expression of a number ofstructural proteins. Tissue explants derived from epidermis, hard palate or buccal mucosa were cultured at the air±liquid interface on collagen gels containing human dermal ®broblasts. Reconstructed epithelia that retained many of

the morphological and immunohistochemical characteristics of the original tissue were formed. Cultures derivedfrom epidermis and the hard palate both had a well-de®ned stratum basale, stratum spinosum, stratum granulosumand stratum corneum whereas cultures derived from buccal mucosa had no stratum granulosum or corneum and the

cells retained their nuclei. Signi®cantly more living cell layers were observed in both types of epithelia obtained fromthe mouth than in epidermis. The speci®c localization of proliferation and di�erentiation markers (Ki67, loricrin,involucrin, SPRR2, SPRR3 and keratin 10) closely resembled that of the tissue from which the cultures were

derived. As identical three-dimensional culture models were used here, it is concluded that the di�erences observedbetween these epithelia were due to intrinsic properties of the keratinocytes. # 2000 Elsevier Science Ltd. All rightsreserved.

Keywords: Reconstructed epithelium; Tissue explant

1. Introduction

Epithelia from di�erent regions of the body show

extensive variation in their di�erentiation. The end

product of epithelial di�erentiation is the formation of

a protective barrier against the environment. An epi-

thelium must form a barrier that will limit the loss of

body ¯uids and at the same time it must prevent the

entry of toxic substances, bacteria and viruses.

Di�erent types of epithelia have therefore evolved to

provide the optimal form of protection for their

speci®c location. For example, epidermis is dry, con-

stantly exposed to changing humidities and tempera-

tures lower than 378C whereas oral epithelium is

exposed to heavy abrasion, 100% humidity and mainly

a temperature of 378C. Epidermis and epithelium from

hard palate are both typical examples of orthokerati-

Archives of Oral Biology 45 (2000) 149±158

0003-9969/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

PII: S0003-9969(99 )00116-8

www.elsevier.com/locate/archoralbio

* Corresponding author. Tel.: +31-71-5271917; fax: +31-

71-5271910.

E-mail address: [email protected] (S.

Gibbs).

Abbreviations: DMEM, Dulbecco's modi®ed Eagle med-

ium; PBS, phosphate-bu�ered saline; SPRR, small proline-

rich protein.

nized epithelia whereas buccal mucosa is an exampleof non-keratinized epithelium. Each of these tissues

can be distinguished morphologically and also by thedi�erences in expression of a number of structural pro-

teins (Brysk et al., 1995; Hohl et al., 1993, 1995;Mackenzie et al., 1991; Smack et al., 1994).

Our aim here was to determine whether, in variousepithelia, the tissue architecture and the expression of a

number of keratinocyte di�erentiation markers (keratin10, loricrin, involucrin, SPRR2, SPRR3) and prolifer-

ation markers (Ki67) can be reproduced in vitro andwhether these proteins are regulated by intrinsic or

extrinsic factors. Therefore, tissue explants derived fromepidermis, hard palate and buccal mucosa were used to

reconstruct epithelia using the skin equivalent modelsystem as described by Saiag et al. (1985). In this model

small, full-thickness punch biopsies are placed epithelialside upwards on to a ®broblast-populated collagen gel

and cultured at the air±liquid interface until the epi-thelial outgrowth covers the gel. With this approach,

extensive mechanical and chemical manipulations withenzymes that result in the death of an important pro-

portion of the cells can be avoided. Furthermore, thereconstructed tissue can be grown from a very small tis-

sue biopsy (2 mm2) whereas traditional culture methodsrequire a larger amount of the original tissue. The

explants are small, easily detectable and can easily beremoved to avoid any ambiguity in the results.

2. Materials and methods

2.1. Morphology and immunohistochemistry

Epidermis was obtained from healthy patientsundergoing surgical corrections; hard palate and buc-cal mucosa were obtained as 4-mm punch biopsiesfrom two healthy volunteers. The biopsies were divided

into two parts: one piece was used for culture (2 mm2)and the other piece was washed in PBS, ®xed in 4%formaldehyde, dehydrated and embedded in para�n.

Sections (5 mm) were cut, depara�nized and rehy-drated in preparation for morphological or immuno-histochemical analysis.

For morphological observations, sections werestained with haematoxylin and eosin. For immunohis-tochemical analysis, the avidin±biotin±peroxidase com-plex method was used essentially as described by the

suppliers (strepABComplex/HRP; DAKO code no. K377). Antigen retrieval for Ki67 was as described byGibbs et al. (1996). Antigen for keratin 10 was

retrieved by incubating the sections for 25 min atroom temperature in 0.01 M sodium citrate, pH 6.0,that had immediately before been preheated to boiling.

After washing in PBS, the sections were incubated for15 min at room temperature in 0.04 mg/ml pepsin in0.2 M HCl.

Fig. 1. Morphology of native tissue. Di�erences in tissue morphology between human epidermis (A), hard palate (B) and buccal

mucosa (C).

S. Gibbs, M. Ponec / Archives of Oral Biology 45 (2000) 149±158150

2.2. Antibodies

Antibodies to human SPRR2 and SPRR3 were agift from Dr. D. Hohl, Lausanne (Hohl et al., 1995);antibodies to human involucrin (SY5) were a gift from

Dr. F. Watt, London (Hudson et al., 1992); antibodiesto human loricrin were a gift from Dr. D. Roop(Mehrel et al., 1990). Anti-Ki67 was purchased from

DAKO (Glostrup, Denmark) and antikeratin 10 fromICN Pharmaceuticals (California, USA).

2.3. Cell culture

Dermal ®broblasts were obtained from healthy

patients undergoing surgical corrections and culturedas described by Ponec et al. (1977). Fibroblast-popu-lated collagen gels (containing 1 � 105 cells/ml) wereprepared as described by Smola et al. (1993).

Reconstructed epithelium was generated by placing atissue biopsy (approx. 2 mm2), with the epithelial sideupwards, on to a ®broblast-populated collagen gel

(approx. 2 cm2). The explants were cultured immedi-ately at the air±liquid interface at 378C and 100%humidity until the epithelium had expanded over the

gel (2±3 weeks). The medium used was a mixture ofDMEM and Ham's F12 (3:1) supplemented with 5%HyClone calf serum, 0.4 mg/ml hydrocortisone, 5 mg/

ml insulin, and 1 mM isoproterenol. Great care wastaken to cut an entire cross-section of the culture for

histological and immunohistochemical analysis. Theexplant was easy to identify due to the presence of adermal matrix.

3. Results

3.1. Morphological characteristics of reconstructedepithelia

Epidermis (Fig. 1A) and hard palate epithelium(Fig. 1B) are both typical examples of orthokeratinized

epithelium. They both displayed a stratum basale, spi-nosum, granulosum and corneum. Nuclei were lostfrom the terminally di�erentiating cells when the cellsreached the stratum corneum. In contrast, epithelium

from buccal mucosa (an example of non-keratinizedepithelium) (Fig. 1C) had no stratum corneum or gran-ulosum. It consisted of three strata (basal, ®lamento-

sum and distendum) and the terminally di�erentiatedcells of the stratum distendum retained their nuclei.Signi®cantly more living cell layers were observed in

both types of epithelia obtained from the mouth thanin the epidermis. This observation correlated with theproliferation status of the di�erent epithelia, where it

Fig. 2. Tissue-speci®c expression of proliferation and di�erentiation markers. Sections derived from epidermis (A), hard palate (B)

and buccal mucosa (C) stained immunohistochemically using antibodies directed against human Ki67, loricrin, involucrin, SPRR2,

SPRR3 and keratin 10.

S. Gibbs, M. Ponec / Archives of Oral Biology 45 (2000) 149±158 151

can be seen that Ki67 was more frequently expressed

in epithelia derived from the mouth than in epidermis

(Fig. 2; Table 1).

When epithelia from epidermis, hard palate and buc-

cal mucosa (Fig. 3) were reconstructed on a ®broblast-

populated collagen gel, the cultured epithelia retained

many of the morphological characteristics of the orig-

inal tissue. Cultures derived from epidermis and hard

palate both had a well-de®ned stratum basale, spino-

sum, granulosum and corneum whereas cultures de-

rived from buccal mucosa had no stratum granulosum

or corneum and the cells retained their nuclei. A thick

upper layer of nuclei containing cells was formed in

cultured buccal mucosa, which presumably accumu-

lates due to the lack of constant abrasion that occurs

in the mouth. Whether or not a stratum ®lamentosum

Fig. 2 (continued)


and stratum distendum were formed cannot be deter-

mined without the aid of electron microscopy and isnot within the scope of this communication. As in thecorresponding native epithelia, a signi®cantly larger

number of living cell layers were observed in bothtypes of reconstructed epithelia obtained from themouth than in the epidermis, but the e�ect was slightlyless pronounced than in the native epithelia (Table 1).

The number of living cell layers correlated with theproliferation status of the cultures; Ki67 was more fre-quently expressed in reconstructed epithelia derived

from the mouth than from the epidermis (Fig. 4; Table1).

3.2. Tissue-speci®c expression of di�erentiation markers

3.2.1. EpidermisThe expression of proliferation and di�erentiation

markers was examined in epidermis and epithelia from

the mouth. In the epidermis (as shown in Fig. 2A),

positive immunohistochemical staining for the corni®edenvelope precursors loricrin, involucrin and SPRR2was observed in the stratum granulosum. Keratin 10

was present in all suprabasal cells and SPRR3 wasabsent. As in native epidermis, in reconstructed epider-mis (Fig. 4A) loricrin and SPRR2 were expressed inthe stratum granulosum and keratin 10 in all supraba-

sal layers. In contrast to native epidermis, involucrinwas expressed in all suprabasal layers and SPRR3 wasintermittently and weakly expressed in a very few cells

of the stratum spinosum.

3.2.2. Hard palateIn the hard palate (another example of orthokerati-

nized epithelium) (Fig. 2B), loricrin was weakly

expressed in the stratum granulosum whereas involu-crin and SPRR2 expression extended into the spino-sum. In striking contrast to epidermis, SPRR3 was

Fig. 2 (continued)


strongly expressed in the stratum granulosum andupper spinosum and keratin 10 was expressed only

intermittently in these cell layers. Similarly to thenative epithelium, in reconstructed hard palate (Fig.4B) loricrin was expressed in the stratum granulosum;

involucrin was expressed in all suprabasal layers;SPRR2 and SPRR3 were expressed in the upper spino-sum and the granulosum, and keratin 10 was expressed

intermittently in these two layers.

3.2.3. Buccal mucosa

In buccal mucosa, both in vivo (Fig. 2C) and invitro (Fig. 4C), loricrin and keratin 10 were absentwhereas involucrin, SPRR2 and SPRR3 were expressedin the upper suprabasal layers. The thick upper layer

of nuclei-containing cells formed in cultured buccalmucosa did not stain positively with any of the anti-bodies used.

4. Discussion

Epithelia from the epidermis, hard palate and buccalmucosa represent three distinct types of strati®ed squa-

mous epithelia which have di�erent histological fea-tures and a distinct expression of di�erentiationmarkers (Table 1). Epidermis and hard palate are com-

posed of a stratum basale, spinosum, granulosum andcorneum. Buccal mucosa has the same environment ashard palate (submerged in saliva), but its architecture

is very di�erent. It is composed of a stratum basale, astratum ®lamentosum and a stratum distendum. Thestratum corneum is absent and nuclei are retained in

the terminally di�erentiating cells. The rate of prolifer-ation in both types of oral epithelia is greater than inepidermis, as is illustrated by the larger number of liv-ing cell layers and the higher number of Ki67 positive-

staining cells. The observation that the corni®ed envel-ope precursors are expressed di�erently in these tissues(Table 1) supports the conclusions made by other

researchers (Hohl et al., 1993) that the composition ofthe corni®ed envelope is variable and is dependent onthe body site. It is possible that the composition of the

envelope and the interlinking intermediate ®laments(Steinert and Marekov, 1995) is related to the rigidityand/or permeability of the epithelia, the relative orderof rigidity being epidermis > hard palate > buccal

mucosa and the relative order of permeability beingbuccal mucosa > hard palate > epidermis (Squier etal., 1986). For example, we found that loricrin and

keratin 10 are strongly expressed in the epidermis,weakly expressed in the hard palate and not expressedin buccal mucosa, whereas SPRR3 is strongly

expressed in buccal mucosa and hard palate but isabsent in the epidermis.It is of interest that many of the characteristics that T

able

1

Summary

ofcharacteristics

ofnativeandreconstructed

epidermis,hard

palate

andbuccalmucosa

a

Epidermis

Hard

palate

Buccalmucosa

Native

Reconstructed

Native

Reconstructed

Native

Reconstructed

Tissuearchitecture

SB,SS,SG,SC

SB,SS,SG,SC

SB,SS,SG,SC

SB,SS,SG,SC

SB,SF,SD

SB,SF,SD

Number

oflivingcelllayers

5±8

6±8

20±30

10±20

20±30

10±20

Ki67

SB+

+;SS+

SB+

+SB+

++

;SS(1)+

++

SB+

+;SS(1)+

++

SB+++;SS(1)+

+++

SB+++;SS(1)+

++

Loricrin

SG

strong

SG

strong

SG

weak

SG

weak

Absent

Absent

Involucrin

SG

SS,SG

SS,SG

SS,SG

Suprabasal(u)

Suprabasal

SPRR2

SG

SG

SS(u),SG

SS(u),SG

Suprabasal(u)

Suprabasal(u)

SPRR3

Absent

Interm

ittent/weak

SS(u),SG

SS(u),SG

Suprabasal(u)

Suprabasal(u)

Keratin10

SS,SG

SS,SG

SS(u),SG

SS(u),SG

Absent

Absent

aTissuearchitecture

wasdetermined

withtheaid

oflightmicroscopy.In

nativeepithelia

thenumber

oflivingcelllayerswasestimatedfrom

therangebetweenthetopandbot-

tom

oftherete

ridges;in

reconstructed

epithelia

thenumber

oflivingcelllayerswasestimatedfrom

di�erentregionswithin

theculture.Im

munoreactivityusinganti-K

i67scored

onascale

ofincreasingfrequency

ofnuclearstainingis

shownas+

to+

++

+.Abbreviations:

SB,stratum

basale;SC,stratum

corneum;SD,stratum

distendum;SF,stratum

®lementosum;SG,stratum

granulosum;SS,stratum

spinosum;l,lower

celllayers;u,upper

celllayers.


we observed in buccal epithelia are also characteristicof psoriatic epidermis (Bowden et al., 1983; Hohl etal., 1995; Ishida-Yamamoto et al., 1996). For example,

as in buccal mucosa, lesional psoriatic tissue does notlose its nuclei during di�erentiation and the tissue hassigni®cantly more living cell layers than in healthy epi-

dermis. Involucrin and SPRR2 expression extends dee-ply into the stratum spinosum and granulosum,loricrin is not expressed and K10 expression is

decreased. However, unlike buccal mucosa whereSPRR3 is strongly expressed, SPRR3 is not expressed

in psoriatic epidermis. It is therefore possible that inpsoriasis and healthy buccal mucosa a number of com-mon regulatory pathways are shared which are distinct

from those in healthy epidermis.Most of the morphological and immunohistochem-

ical characteristics observed in the epidermis, hard

palate and buccal mucosa were also observed in recon-structed epithelia derived from these tissues (Table 1).As identical culture systems were used to generate the

reconstructed epithelia the di�erences observed can beattributed to di�erences in intrinsic properties of theepithelial cells rather than to environmental factors. It

is highly unlikely that ®broblasts present in the smalltissue specimen (2 mm2) migrated out of the explant

into the collagen gel (2 cm2) and thus contributed tothe speci®c properties observed in the reconstructedepithelia, and furthermore the number of ®broblasts

present in the biopsy is very small when compared tothe number incorporated within the collagen gel(2 � 105 cells). These reasonings are further supported

by the data reported by Saiag et al. (1985), who, usinga similar culture system, were able to disprove that®broblasts are able to migrate out of the explant. They

showed that reconstructed epidermis from psoriaticnon-involved skin was not hyperproliferative whengenerated on a dermal equivalent populated with nor-

mal human ®broblasts whereas hyperproliferation wasobserved when a normal skin biopsy was implanted on

to a gel populated with ®broblasts derived from psor-iatic non-involved skin.Involucrin is the only corni®ed envelope precursor

which showed a major di�erence between its in vivoand in vitro expression and even then this was onlyobserved in the epidermis and not in the two other

types of epithelia. In native epidermis, involucrin isexpressed in the stratum granulosum whereas in vitroit was expressed in all suprabasal layers. At present it

is not clear whether the enhanced expression of involu-crin can be ascribed to conditions used to reconstruct

the epidermis. In both types of reconstructed oralepithelia, involucrin was also expressed in most supra-basal layers but this does correspond to involucrin ex-

pression in vivo in these tissues.It is clear from our observations that the rate of pro-

liferation in oral epithelia is higher than in the epider-

mis and that this is re¯ected in the correspondingreconstructed epithelia (Table 1). However, slightdi�erences between the number of living cell layers in

native and reconstructed tissues were observed forboth types of oral epithelia. The di�erences can beascribed to the presence of deep rete ridges in vivo,

which are absent in vitro. The number of living celllayers at the top of the rete ridges corresponds to thenumber of living cell layers observed in vitro when the

epithelia are reconstructed on ¯at, ®broblast-popu-lated, collagen gels.

Whether the di�erences in the di�erentiation patternof di�erent epithelia are due to intrinsic properties ofkeratinocytes or to in¯uences from subepithelial con-

nective tissue is an important question that has boththeoretical and clinical implications. There is muchvariation in the data reported so far, but in general it

can be concluded that both intrinsic and extrinsic fac-tors are involved in the formation of a speci®c epi-thelium (Bohnert et al., 1986; Brysk et al., 1995;

Doran et al., 1980; Chung et al., 1997; Karring et al.,1975; Kautsky et al., 1995; Lillie et al., 1988;

Fig. 3. Morphology of reconstructed tissue. Reconstructed epithelia derived from human epidermis (A), hard palate (B) and buccal

mucosa (C) retain the morphological characteristics of their native tissue.


Fig. 4. Reconstructed epithelia retain the speci®c expression of proliferation and di�erentiation markers found in their corresponding

native tissue. Sections derived from reconstructed epidermis (A), reconstructed hard palate (B) and reconstructed buccal mucosa (C)

stained immunohistochemically using antibodies directed against human Ki67, loricrin, involucrin, SPRR2, SPRR3 and keratin 10.

Mackenzie and Fusenig, 1983; Mackenzie and Hill,1984; Mackenzie et al., 1993; Ouhayoun et al., 1988).We clearly show that not all of the di�erences

observed between epithelia are regulated by extrinsicfactors (such as neighbouring cell types Ð oral or epi-dermal ®broblasts, contact with saliva, air exposure,

temperature, relative humidity or contact with aspeci®c dermal matrix), but that at least the expressionof loricrin, involucrin, SPRR2, SPRR3, keratin 10 andKi67 is regulated by intrinsic properties of the kerati-

nocytes. The expression of these proteins is most prob-ably partially responsible for the di�erent phenotypesthat we observe in reconstructed epithelia which are

also characteristic of the di�erent epithelia.

Acknowledgements

We would like to thank Mary Verhoeven and

Johanna Kempenaar for their skilful technical assist-ance. This study was supported by a grant from theDutch Ministry of Health and Education PAD 92-16.

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Intrinsic regulation of differentiation markers in human epidermis, hard palate and buccal mucosa

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