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Theses and Dissertations in Biomedical Sciences College of Sciences
Spring 2006
Modulation of TGFβ-Induced PAI -1 Expression by Changes in Modulation of TGF -Induced PAI -1 Expression by Changes in
Actin Polymerization in Human Mesangial Cells Actin Polymerization in Human Mesangial Cells
Keyur Patel Old Dominion University
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MODULATION OF TGFp-INDUCED PAI-1 EXPRESSION BY CHANGES IN
ACTIN POLYMERIZATION IN HUMAN MESANGIAL CELLS
Keyur Pravinchandra Patel M.B.B.S., January 1999, Gujarat University
A Dissertation Submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University
in Partial Fulfillment of the Requirement for the Degree of
DOCTOR OF PHILOSOPHY
BIOMEDICAL SCIENCES
EASTERN VIRGINIA MEDICAL SCHOOL AND
OLD DOMINION UNIVERSITY May 2006
by
Approved by:
William F. Glass II (Director)
Pamela Harding (MemE^T)"
Tim Bos (Member)
Julie Kerry (Member)
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ABSTRACT
MODULATION OF TGFp-INDUCED PAI-1 EXPRESSION BY CHANGES IN ACTIN POLYMERIZATION IN HUMAN MESANGIAL CELLS
Keyur Pravinchandra Patel Old Dominion University, 2006 Director: Dr. William F. Glass II
Chronic renal diseases show increased deposition of extracellular matrix
(ECM) in the glomerulus (glomerulosclerosis). Glomerulosclerosis is associated
with activation of normally quiescent glomerular mesangial cells into
myofibroblast-like cells. The overall objective of this study is to delineate cellular
mechanism/s of myofibroblast-differentiation in disease states. In cultured
mesangial cells certain characteristics of myofibroblast differentiation (a-smooth
muscle actin (a-SMA) and hypertrophy) are associated with an increase in
polymeric actin microfilaments (stress fibers). It is likely that other genes are also
regulated in an actin cytoskeleton-dependent manner during myofibroblast
differentiation. In these studies, we therefore examined the hypothesis that
changes in the actin cytoskeleton regulate myofibroblast differentiation of
mesangial cells.
In vivo, myofibroblasts play a major role in ECM accumulation and tissue
scarring. TGFp increases ECM deposition by increasing plasminogen activator
inhibitor type-1 (PAI-1) expression. PAI-1 inhibits ECM degradation by tissue-
(tPA) and urokinase-type (uPA) plasminogen activators. Additionally, the Rho
family of GTPases is required for TGFp-induced PAI-1 expression. Since,
regulation of actin cytoskeletal organizaton is a major function of Rho, the
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hypothesis that Rho-mediated changes in actin cytoskeleton modulate PAI-1
expression in glomerular cells was proposed. The effects of modulating the actin
cytoskeleton on TGFp-induced PAI-1 expression were examined in cultured
human glomerular mesangial cells. Inhibitors of Rho signaling decreased basal
and TGFP-induced PAI-1 mRNA and protein expression. These effects were
mimicked by direct inhibition of actin polymerization by Cytochalasin B (CytB)
suggesting that the effects of Rho on PAI-1 expression are mediated through the
actin cytoskeleton. CytB also inhibited the activity of a PAI-1 promoter.
Conversely, stabilization of actin microfilaments with jasplakinolide (JAS) had the
opposite effect on TGFp-induced PAI-1 expression. These results indicate a bi
directional regulation of PAI-1 by the actin cytoskeleton depending on the state of
actin polymerization. In contrast to inhibition of PAI-1, actin-depolymerizing
agents increased tPA mRNA expression.
In summary, the changes in the actin cytoskeleton mediate the effects of
Rho-GTPases in modulation of PAI-1 and tPA expression in response to TGFp.
Thereby, they may control ECM degradation. Overall, the results suggest that
changes in organization of the actin cytoskeleton regulate myofibroblast
differentiation and ECM accumulation by coordinately modulating expression of
multiple genes.
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V
This thesis is dedicated to my wife, Rupal, and, to my parents, Hansa and Pravinchandra Patel. Your unconditional love, silent sacrifices,
continued support and prayers have made my dreams come true.
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ACKNOWLEDGMENTS
First and foremost, I express my sincere gratitude to Dr. William F. Glass
for accepting me as his graduate student and supporting me at every step on the
way. His support, patience and guidance over the years have helped me achieve
things I never thought were possible. His undying enthusiasm for the basic
science research, the energy to successfully handle multiple complex
responsibilities and the ability to maintain a wonderful balance between his
personal and professional life, are and will remain my inspirations and guiding
principles in life. He has been a great mentor and role model. Thank you, Dr.
Glass, for everything you have done for me.
I want to express my sincere gratitude to Dr. Pamela Harding for teaching
me how to take baby steps in basic science research and more importantly how
to get up when down. She helped me learn to cope with the frustrating times in
research and in life. I could always turn to her for discussing topics ranging from
a friendly chat to the most complex issues in life and science. She showed me
the importance of spirituality in becoming a good human being. Thank you, Dr.
Harding, for all your kindness.
I also want to express my sincere gratitude to Dr. Julie Kerry for an
extensive review of my thesis. I feel fortunate to have a mentor with such passion
for science and teaching. I am truly indebted for her help. Her commitment to the
biomedical sciences graduate program is exemplary. As the track coordinator,
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she has been a constant source of motivation and personalized guidance. Thank
you, Dr. Kerry, for the detailed review of my work.
I also want to thank Dr. Tim Bos for helping me understand the principles
and complexities of molecular biology. His meticulous approach to experimental
designs, attention to details and constructive criticisms during molecular biology
classes, journal club discussions and review of my work have helped me become
a better student and researcher. Thank you, Dr. Bos, for your guidance.
I want to thank all four members of my guidance committee for ensuring
that my work was scientifically sound and professionally presented. I feel
privileged to be your student.
I want to thank Dr. Russell Prewitt for serving as an observer during the
thesis defense, Dr. Nicholas D’Amato for constantly being a source of inspiration
and Dr. George Wright for a rewarding rotation on proteomics. I want to thank my
fellow lab technicians, Lisa Haney and Alexandra Stevens, for their help with the
experimental work and their companionship in the lab. I want to thank the
departmental secretaries, Celia Denton and Vera Potts, for always brightening
my days with their friendly smiles. I want to thank the staff of the molecular
pathology laboratory for allowing me to use the Lightcycler instrument amid a
busy work schedule.
I want to thank Toni Dorn, the graduate program coordinator with
unmatched organization skills, for her continuous support on issues ranging from
registration to immigration. I also want to acknowledge the support of my fellow
graduate students and others whom I may have unknowingly left out.
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Special gratitude is owed to all my friends and family for standing by me
through thick and thin. There are too many names to mention and the list of
things you have done for me is endless. I can only say that without your
unconditional love, undying support, silent sacrifices and constant motivation, I
would not have achieved anything in my life. I owe you with everything I have
today.
Last, but not least, I want to humbly thank God for blessing me with
wonderful friends, family and colleagues. Thank you, God.
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TABLE OF CONTENTS
Page
LIST OF TABLES....................................................................................................... xii
LIST OF FIGURES...................................................................................................xiii
LIST OF ABBREVIATIONS.....................................................................................xvi
CHAPTER
1. INTRODUCTION.........................................................................................1MESANGIAL CELLS AND GLOMERULOSCLEROSIS................... 1CENTRAL HYPOTHESIS.................................................................... 9ACTIN CYTOSKELETON AND GENE REGULATION .................. 10THE ACTIN CYTOSKELETON.........................................................11PLASMINOGEN ACTIVATOR SYSTEM AND PLASMINOGEN ACTIVATOR INHIBITOR-1 INCHRONIC RENAL DISEASE............................................................ 15TGFp IN RENAL DISEASES.............................................................22RHO AND PAI-1 EXPRESSION....................................................... 23HYPOTHESIS AND AIMS.................................................................24
2. MATERIALS AND METHODS.................................................................26CELL CULTURE................................................................................26DNA ARRAY ANALYSIS................................................................... 26WESTERN BLOT ANALYSIS............................................................31NORTHERN BLOT ANALYSIS.........................................................34IMMUNOCYTOCHEMISTRY............................................................37QUANTITATIVE REAL-TIME POLYMERASECHAIN REACTION (PCR). ...............................................................38CLONING OF PAI-1 PROMOTER.................................................... 44TRANSIENT TRANSFECTION........................................................ 47LUCIFERASE REPORTER ASSAY.................................................47DEVELOPMENT OF CHRONIC GLOMERULONEPHRITISIN EXPERIMENTAL ANIMALS........................................................ 48STATISTICAL ANALYSIS................................................................. 51
3. PROFILING THE EFFECTS OF ACTIN CYTOSKELETON ON EXPRESSION OF MULTIPLE GENES................................................... 52
INTRODUCTION................................................................................52EXPERIMENTAL MODEL................................................................. 53RESULTS............................................................................................54
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CHAPTER Page
4. EFFECT OF TGF(3 ON PAI-1 EXPRESSION........................................ 61INTRODUCTION................................................................................61EXPERIMENTAL MODEL.................................................................62RESULTS............................................................................................63
5. EFFECT OF CHANGES IN ACTIN POLYMERIZATIONON TGFp-INDUCED PAI-1 EXPRESSION.............................................75
INTRODUCTION................................................................................75EXPERIMENTAL MODEL.................................................................76RESULTS............................................................................................77
6. EFFECT OF ACTIN CYTOSKELETON ON PAI-1PROMOTER..............................................................................................91
INTRODUCTION................................................................................91EXPERIMENTAL MODEL................................................................. 94RESULTS............................................................................................94
7. EFFECTS OF TGFp AND ACTIN CYTOSKELETON ON PLASMINOGEN ACTIVATORS...............................................................98
INTRODUCTION................................................................................98EXPERIMENTAL MODEL.................................................................99RESULTS............................................................................................99
8. DISCUSSION...........................................................................................112MESANGIAL MYOFIBROBLAST ACTIVATIONAS A DISEASE MODEL.................................................................. 112CULTURED MESANGIAL CELLS AS THE EXPERIMENTAL MODEL OF MYOFIBROBLASTDIFFERENTIATION.........................................................................113ACTIN CYTOSKELETON AS A REGULATOR OFGENE EXPRESSION.......................................................................114TGFp-INDUCTION OF PAI-1 EXPRESSION................................ 117INHIBITION OF ACTIN CYTOSKELETON ONTGFp-INDUCED PAI-1 EXPRESSION...........................................119STABILIZATION OF ACTIN CYTOSKELETON ONTGFp-INDUCED PAI-1 EXPRESSION...........................................122THE EFFECT OF ACTIN CYTOSKELETON ONPAI-1 PROMOTER ACTIVITY.........................................................124THE EFFECT OF ACTIN CYTOSKELETON ON PLASMINOGEN ACTIVATORS..................................................... 127
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CHAPTER Page
9. CONCLUSIONS AND FUTURE DIRECTIONS...................................131CONCLUSIONS............................................................................... 131ROLE OF MAPK IN TGF|3-INDUCED PAI-1EXPRESSION.................................................................................. 132EFFECT OF RHO-KINASE INHIBITION ON SCLEROSIS IN CHRONIC MESANGIOSCLEROTICGLOMERULONEPHRITIS.............................................................. 139
REFERENCES........................................................................................................147
APPENDIX.............................................. 166PERMISSION OF THE AMERICAN PHYSIOLOGICALSOCIETY .....................................................................................................166
VITA..........................................................................................................................168
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LIST OF TABLES
Table Page
1. Actin Binding Proteins....................................................................................... 14
2. Primer Sequences for Real-Time PCR Analysis............................................ 42
3. Summary of Changes in Gene Expression on DNAArray Analysis................................................................................................... 56
4. Calculation of PAI-1 cDNA Concentration by Real-Time PCR......................89
5. Effect of Rho-kinase Inhibitor on ExperimentalGlomerulonephritis..........................................................................................144
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xiii
LIST OF FIGURES
Figure Page
1. Normal Glomerulus............................................................................................2
2. Glomerular Morphology in Normal and PathologicStates................................................................................................................... 4
3. Cascade of Events Leading to Chronic Renal Diseases................................7
4. Regulation of Actin Cytoskeleton Organization byRho GTPase......................................................................................................12
5. Regulation of Extracellular Matrix Turnover bythe Plasminogen Activator System................................................................. 16
6. DNA Array Analysis of Effect of Actin Depolymerizationon Gene Expression.........................................................................................55
7. Effect of Actin Depolymerization on PAI-1 mRNAExpression in Human Mesangial Cells............................................................59
8. PAI-1 Standard Curve for Quantitative Real-TimePCR Analysis.................................................................................................... 64
9. UBC Standard Curve for Quantitative Real-TimePCR Analysis.................................................................................................... 66
10. Gel Electrophoresis of Standard Curve PCR Products................................68
11. Representative Images from Quantitative Real-Time PCRof PAI-1 and UBC From Experimental Samples........................................... 69
12. Dose-response Analysis of TGFp Inductionof PAI-1 Expression..........................................................................................72
13. Time-course Analysis of TGFp Induction ofPAI-1 Expression..............................................................................................74
14. Effect of Actin Depolymerization on TGFp-inducedPAI-1 mRNA Expression.................................................................................. 78
15. Effect of TGFp and Actin Depolymerization onPAI-1 Protein Expression................................................................................. 80
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xiv
Figure Page
16. Effect of Actin Depolymerization on TGFp-inducedPAI-1 Protein Expression................................................................................. 82
17. Effect of Actin Depolymerization on TGFp-inducedPAI-1 Expression by Immunocytochemistry................................................... 83
18. Effect of Changes in Actin Polymerization onTGFp-induced PAI-1 mRNA Expression........................................................ 85
19. Effect of JAS and Toxin B Mediated Changes in ActinPolymerization on TGFp-induced PAI-1 Protein Expression........................ 86
20. Effect of Changes in Actin Polymerization onTGFp-induced PAI-1 Protein Expression........................................................88
21. Schematic Representation of PAI-1 Promoter..............................................92
22. Time-course Analysis of TGFp on PAI-1Promoter Activity...............................................................................................95
23. The Effects of TGFp and Actin Depolymerizationon Human PAI-1 Promoter Activity.................................................................97
24. tPA Standard Curve for Real-Time PCR Analysis..................................... 101
25. uPA Standard Curve for Real-Time RT-PCR Analysis...............................102
26. Gel Electrophoresis of tPA and uPA StandardCurve PCR Products.......................................................................................103
27. Representative Images From Real-Time PCR oftPA and UBC From Experimental Samples................................................. 104
28. Representative Images From Real-Time PCR ofuPA and UBC From Experimental Samples................................................ 105
29. Time-course Analysis of TGFp-treatment ontPA and uPA mRNA Expression................................................................... 107
30. The Effects of TGFp and Actin Depolymerizationon tPA mRNA Expression.............................................................................. 109
31. The Effects of TGFp and Actin Depolymerizationon uPA mRNA Expression............................................................................. 110
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Figure Page
32. Effect of MAPK Inhibition on PAI-1 mRNA Expression.............................. 136
33. Effect of MAPK Inhibition on PAI-1 Protein Expression............................. 137
34. Effect of Rho-kinase Inhibition on Sclerosis in a RatModel of Chronic Glomerulonephritis............................................................143
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LIST OF ABBREVIATIONS
a-SMA Alpha-Smooth Muscle ActinADF Actin Depolymerizing FactorAP-1 Activator Protein-1ATS Anti-Thymocyte SerumCytB Cytochalasin BECM Extracellular MatrixEGF Epidermal Growth FactorERK Extracellular Signal Regulated KinaseESRD End-Stage Renal DiseaseFBS Fetal Bovine SerumFGF Fibroblast Growth FactorGAP GTPase Activating ProteinGDI GDP Dissociation InhibitorGDP Guanidine DiPhosphateGEF Guanidine Nucleotide Exchange FactorGRE Glucocorticoid Response ElementsGTP Guanidine TriPhosphateHMC Human Mesangial CellHRE Hypoxia Response ElementsJAS JASplakinolideJNK c-Jun N-terminal KinaseLAP Latency Associated PeptideLatB Latrunculin BL-NAME N-Nitro-L-Arginine Methyl Ester (L-NAME)MAPK Mitogen Associated Protein KinaseMEKK1 Mitogen Associated Protein Kinase Kinase 1MMPs Matrix MetalloProteinasesPAI-1 Plasminogen Activator Inhibitor type-1PCR Polymerase Chain ReactionPKA Protein Kinase APKC Protein Kinase CROCK Rho-KinaseRT Reverse TranscriptionSAPK Stress-Activated Protein KinaseSEM Standard Error of MeanSERPIN SERine Protease INhibitorSRF Serum Response FactorTBS Tris-Buffered SalineTGFp Transforming Growth Factor BetaTPA Tissue-type Plasminogen ActivatorUPA Urokinase-type Plasminogen ActivatorUPAR uPA ReceptorUUO Unilateral Ureteral ObstructionVSMC Vascular Smooth Muscle Cells
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CHAPTER 1
INTRODUCTION
Over 25 million people in the USA suffer from different forms of chronic
renal diseases, of which approximately 419,000 have end stage renal disease
(ESRD) with an annual increase of 4.6% [1]. Dialysis and organ transplant are
the two mainstays of treatment of ESRD, both of which provide a palliative
treatment at best and are associated with many limitations [2], The current
preventive measures are only effective if instituted early in the disease process,
which is clinically difficult to achieve since early disease is generally
asymptomatic [3]. An annual expenditure of $ 25 billion for treatment of ESRD
and lack of definitive treatment warrant further medical research on mechanisms
leading to end state renal diseases [1].
The information in this chapter is organized into two parts: The role of
activated mesangial cells in pathogenesis of chronic renal diseases and the
observations with cultured mesangial cells leading to the central hypothesis are
described first (sections 1.1 and 1.2). The second part contains information on
actin cytoskeleton and the protein of interest of this study (sections 1.3 to 1.7).
Mesangial cells and glomerulosclerosis
The glomerulus
The renal glomerulus, the primary site of filtration of waste products from
The model journal for this dissertation is Kidney International.
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Fig. 1. Normal glomerulus. The renal glomerulus is a highly vascular structure formed by invagination of the afferent arteriole into a double-layered Bowman’s capsule and subsequent branching into capillaries (e) lined by endothelial cells (d). A parietal layer of simple squamous epithelium (a) and a visceral layer of podocytes (b) separated by Bowman’s space (c) form the Bowman’s capsule. The efferent arteriole drains the glomerulus. The point at which the two arterioles penetrate Bowman's capsule is called the vascular pole and the point at which ultrafiltrate leaves the glomerulus is called the urinary pole. The space in between the capillary loops is called the mesangium (meso= in between, angium= vessels) (f), which consists of the mesangial cells (g) and extracellular matrix laid by them.
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the bloodstream, is a highly vascular structure consisting of a network of capillary
loops held together by mesangial matrix (Fig. 1) [4], At the vascular pole, the
afferent arteriole invaginates into the double-layered Bowman’s capsule
consisting of a parietal layer of simple squamous epithelium and a visceral layer
of podocytes separated by the Bowman’s space (urinary space). The afferent
arteriole branches into a tuft of capillaries with multiple loops, collectively called
the glomerulus. The glomerulus is drained by the efferent arteriole, which leaves
near the point of insertion of afferent arteriole. The space in between the capillary
loops is called the mesangium (meso= in between, angium= vessels), which
consists of the mesangial cells and extracellular matrix laid by them.
As the blood flows through the capillary loops, some of its constituents
including the water, waste products and electrolytes are selectively filtered into
the Bowman’s space (Fig. 1) [4], This ultrafiltrate is collected, at the urinary pole,
by the proximal convoluted tubules to be further transferred to a downstream
system of tubules and ducts. After a complex series of absorption, re-absorption
and secretion, the ultrafiltrate is finally converted into the urine.
Glomerulosclerosis
Glomerulosclerosis is a major feature of many renal diseases that
progress to end stage renal disease (ESRD) [5, 6]. Pathologically,
glomerulosclerosis is characterized by increased deposition of extracellular
matrix (ECM) and gradual replacement of functioning renal tissue with non
functioning fibrous tissue (Fig. 2). Different underlying mechanisms lead to a
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B. Chronic Glomerulosclerosis
ECM deposition and_ mesangial expansion
C. Global Sclerosis
Replacement of normal glomeruli by ECM —
Fig. 2. Glomerular morphology in normal and pathologic states. (A)Glomerulus showing relatively low amounts of extracellular matrix in the mesangium (arrow), (B) chronic glomerulosclerosis showing increased amount of extracellular matrix deposition and resultant expansion of the mesangium (arrow), (C) end-stage renal disease showing complete replacement of the glomeruli by extracellular matrix (arrow) in a process called global sclerosis.
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common pathological feature in the form of scarring of renal tissue [7], Increased
formation of extracellular matrix leading to fibrosis begins in the mesangium, the
intercapillary portion of the glomerulus composed of mesenchymal mesangial
cells [8, 9]. Expansion of the ECM occurs as a result of deposition of type IV and
type I collagen, and fibronectin. In chronic renal diseases, fibrosis results from
increased matrix production, decreased matrix breakdown or both. Since filtration
and removal of waste products from the blood are the major functions of
glomerulus, patients with non-functioning glomeruli develop toxicity due to
accumulation of waste products [10],
Renal fibrogenesis
Renal fibrogenesis typically occurs in three distinct phases, with the first
two being similar to physiological wound healing, namely the phase of induction,
the phase of inflammatory matrix synthesis and the phase of post-inflammatory
matrix synthesis [7], The first phase is associated with an influx of mononuclear
cells upon an insult leading to an inflammatory response. The inflammatory cells
liberate cytokines and growth factors, which in turn activate fibroblasts [7], In
particular, transforming growth factor p (TGFp), a pro-fibrotic growth factor, plays
a major role in activating normally quiescent mesangial cells [11], The second
phase of inflammatory matrix synthesis is characterized by increased synthesis
of ECM and de novo expression of proteins, and reduced degradation of ECM.
The structural changes are still potentially reversible throughout this phase [7],
The third phase of post-inflammatory matrix synthesis is marked by autonomous
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fibroblast proliferation, synthesis of ECM independent of the presence of primary
inflammatory stimulus and perpetuation of the scarring process [7],
The mesangial myofibroblast
During the three phases described above, glomerular mesangial cells
contribute to excess matrix deposition in multiple ways. Besides providing
structural support to the mesangium, mesangial cells both secrete and respond
to growth factors and inflammatory mediators, thereby regulating the glomerular
microenvironment [12]. TGFp activates otherwise quiescent mesangial cells
which now start proliferating and expressing proteins that contribute to ECM
deposition (Fig. 3). These activated mesangial cells also express a-smooth
muscle actin (a-SMA) de novo, which is normally seen in smooth muscle cells,
[13-20]. Consequently, these activated mesangial cells with smooth muscle cell
like properties are referred to as myofibroblasts. a-SMA expression and
myofibroblast-like differentiation are also seen elsewhere in tissues such as
heart, lung, liver and skin [21-24],
Along with a-SMA, activated myofibroblasts start expressing profibrotic
plasminogen activator inhibitor-1 (PAI-1) de novo, which in turn inhibits ECM
degradation and favors ECM deposition [19, 25], Thus, TGFp-mediated
myofibroblast-like differentiation is associated with increased PAI-1 expression
and excess ECM deposition that eventually leads to glomerulosclerosis and end-
stage renal disease (Fig. 3).
The overall objective of this study is to characterize the mechanisms of
mesangial myofibroblast-like differentiation in order to obtain useful insights into
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(end result)
(disease phenotype)
(effector molecules)
(effector cells)
(initiating insult)
M esangial Cells
Glomerular Sclerosis
Chronic Renal D isease
Plasminogen Activator System
- More than 25 million patients- Annual health care cost 25 billion
- Major feature of chronic and end stage renal diseases- Excess deposition of extracellular matrix- Glomerulus replaced by non-functional fibrous tissue
- Profibrotic growth factor, associated with renal fibrosis- Increases matrix production directly- Decreases matrix degradation by increasing PAI-1.
- Regulates the rate of matrix turnover- Components:
- Activating: tPA and uPA; anti-fibrotic- Inhibitory: PAI-1; profibrotic
- Mesangial = meso + angium = between blood vessels- Structural support of glomerulus and capillary loops- Site of production & action of growth factors & cytokines- Express PAI-1 de novo in disease states
Fig. 3. Cascade o f events leading to chronic renal diseases. TGFp is a profibrotic growth factor that increases production of PAI-1 by glomerular mesangial cells. Increased production of PAI-1 leads to decreased degradation and increased deposition of extracellular matrix in mesangium resulting in glomerulosclerosis. Replacement of normal glomeruli by non-functional fibrous tissue in glomerulosclerosis leads to clinical presentation in the form of chronic and end-stage renal diseases, both currently irreversible disease processes.
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renal fibrogenesis. The specific aim of this study was to examine regulation of
TGFp-induced PAI-1 expression in human mesangial cells.
Cultured mesangial cells as a model of myofibroblast phenotype
Cultured mesangial cells provide a useful in vitro model of myofibroblast
phenotype. The phases of fibrogenesis can be mimicked in vitro by altering the
culture conditions. Cell culture in the presence of serum provides cytokines and
growth factors as seen in the phase I to activate mesangial cells (refer to section
1.1.3). Cell culture in the absence of serum mimics phase III, which is marked by
the absence of cytokines, development of the myofibroblast-like phenotype (a-
SMA expression) and increased ECM production (refer to section 1,1,3). Thus,
initial culturing in the presence of serum, followed by serum deprivation, mimics
the growth factor and cytokine milieu of myofibroblast activation and
differentiation. Primary cultures of human and rat mesangial cells were used in
this study to understand mechanisms of myofibroblast differentiation.
Regulation of g-SMA expression in cultured mesangial cells
As a part of an overall focus on understanding the myofibroblast-like
phenotype, our laboratory previously examined the regulation of a-SMA,
expressed de novo upon myofibroblast differentiation, in cultured mesangial
cells. Stephenson et al showed that serum-deprivation caused mesangial cells to
spread, form abundant stress fibers, express more a-SMA and undergo
hypertrophy [26]. Likewise, proliferating dermal fibroblasts are initially devoid of
microfilaments [24], but their appearance and subsequent disappearance
paralleled the expression of a-SMA. The observations of Stephenson et al and
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the in vivo data regarding dermal fibroblasts, suggested that clearance of growth
factors increases actin polymerization thereby controlling myofibroblast
differentiation. Since, expression of a-SMA paralleled changes in actin
cytoskeleton organization, it was hypothesized that changes in actin
polymerization regulate a-SMA expression and hypertrophy in mesangial cells.
The Rho family of small GTPases regulate actin polymerization through the
downstream effector, Rho-kinase (ROCK), in a variety of cell types [21]. Inhibition
of actin polymerization by agents that inhibit Rho-kinase (Y-2632, HA-1077), as
well as by the agents that directly bind to actin and affect its degree of
polymerization (CytB, LatB), inhibited a-SMA expression and promoter activity
[28]. On the other hand, stabilization of actin polymerization by JAS and
phalloidin increased a-SMA expression and promoter activity. Similarly,
mesangial cell hypertrophy was blocked by actin depolymerization. These results
confirmed the hypothesis that changes in actin polymerization regulate a-SMA
expression and hypertrophy in mesangial cells [28].
Central hypothesis
Like a-SMA expression and hypertrophy, de novo expression of PAI-1 is
also seen with myofibroblast differentiation [29]. It is possible that a-SMA, PAI-1
and multiple other genes are regulated by a common underlying mechanism that
regulates overall myofibroblast differentiation. Therefore, a central hypothesis
that changes in actin polymerization regulate myofibroblast-like phenotype by
simultaneously regulating expression of multiple genes was proposed.
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The following sections contain information on the actin cytoskeleton,
regulation of its organization, its role in gene expression, introduction to
plasminogen activator system, regulation of PAI-1 expression and the specific
hypothesis for this study.
Actin cytoskeleton and gene regulation
Traditionally, actin cytoskeletal organization is considered important for
controlling processes such as cell shape and cell motility [30-32], At the same
time there is increasing evidence suggesting that the actin cytoskeleton and cell
shape are important regulators of gene expression and cell function. Early
observations of Folkman and Moscona, showing an association between
inhibition of cell spreading and decreased DNA synthesis, proposed that cell
shape regulates cell function [33]. Over the past 20 years, studies by Ingber have
supported the idea that mechanical transduction through cytoskeletal elements
affect cell functions [34-38], Recent studies have shown that networks of actin
polymers, also known as stress fibers, provide a highly specific and organized
network of fibers for processes such as signal transduction and gene regulation.
Localization of signaling molecules at specific cytoskeletal locations allows
integration of signaling pathways and effective transmission of a signal [39, 40].
For example, MEKK1 that activates JNK mitogen associated protein kinase is
associated with the actin cytoskeleton [41]. During keratinocyte differentiation,
cytoskeletal association facilitates coupling of ErbB1 and ERK [42], Also,
expression of genes such as a-SMA seems to be regulated by the state of actin
polymerization [28]. Localization of mRNA and protein translation machinery at
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11
highly specific intracellular pockets of actin stress fibers have also been identified
[43], Thus, current evidence points towards regulation of gene expression by
actin cytoskeleton.
The actin cytoskeleton
The actin cytoskeleton, also known as the microfilament system, is an
important structural component of all eukaryotic cells. Actin, a 42-kDa globular
polypeptide, is the building block of the microfilamentous cytoskeletal system.
Monomeric actin (G-actin) molecules polymerize via non-covalent interactions
into filamentous actin also known as F-actin or stress fibers [44], Actin filaments
cross-link with each other and with various cellular and membranous structures
to form a highly organized network. The actin filaments traverse the cytoplasm
and provide structural support to the cells. They transmit tension and resist
deformation of the cells. Other cellular functions regulated by the actin
cytoskeleton include but are not limited to cell shape, cell motility, cell division,
cell-cell and cell-substrate interaction, transmembrane signaling, endocytosis,
secretion and muscle contraction [45, 46],
The actin filaments are very dynamic structures undergoing constant
growth in length at one end (barbed end, plus end) and attrition at the opposite
end (pointed end, minus end). The rates of growth and attrition determine the
amounts of monomeric and polymeric actin in the cell. Actin polymerization is
regulated at multiple levels in response to extracellular stimuli or intracellular
needs; and involves various cell-signaling pathways, actin binding and
associated proteins (Fig. 4) [46],
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12
Rho-kinase
(-)<* .
LIM-kinasei
Cofillinstabilization
I -RhoA
(-) Iloxin 1J mDia
Profilm
F-actm
(.'ytochald^in IJ I u trum uiin b
(-) 1olymerization
G-actin
IJ ^?? Other factors
ranscription factors4D>
Regulation of Gene Expression
a-SMA p a h tPA
Fig. 4. Regulation of actin cytoskeleton organization by Rho GTPase. RhoGTPase is a major regulator of actin polymerization. Rho-kinase (ROCK) and mDia are known downstream effectors of Rho that increase polymeric F-actin and decrease monomeric G-actin content by regulating the actions of actin binding proteins such as cofillin and profilin. The hypothesis examined in this thesis proposed that Rho-mediated changes in actin polymerization regulated expression of specific genes through involvement of some yet unknown factors. Agents that increase (Jasplakinolide) or block (Toxin B, HA-1077, Y-2632, Cytochalasin B and Latrunculin B) actin polymerization were used to test the hypothesis by modifying actin polymerization at different levels.
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13
Actin binding proteins
The actin cytoskeleton undergoes different stages of organization such as
nucleation, polymerization, cross-linking, folding and movement. As the name
implicates, actin-binding proteins bind to actin monomers or polymers during
different phases of assembly and regulate the rate and the extent of actin
polymerization. The activities of these proteins are in turn regulated by signaling
molecules [46], Major actin-binding proteins are briefly reviewed in Table 1 [47-
49],
RhoA and actin cytoskeleton
Rho is a member of Rho family of small GTPases (Rho, Rac and Cdc42)
that plays a major role in organizing actin cytoskeleton in the form of stress fibers
(Fig. 4) [27, 50, 51]. The Rho GTPases exist in an inactive GDP-bound form
complexed with a GDP-dissociation inhibitor (Rho GDI) and an active GTP-
bound form. In response to stimuli, guanine nucleotide exchange factors (GEFs)
dissociate GDI from Rho and allow Rho to bind to GTP. Activated Rho-GTP in
turn activates downstream effecters. GTPase activating proteins (GAP) inactivate
Rho by catalyzing GTP hydrolysis [52], Rho-kinase or ROCK, is the main
downstream effector of Rho that inactivates proteins that depolymerize actin
such as cofilin and destrin and thereby facilitates actin polymerization. Other
effectors of Rho include PIP-5 kinase and mDia, a 140 kDa protein that increases
actin polymerization through the actin-binding protein profilin [53-55],
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Table 1. Actin Binding Proteins
14
Actin Binding Protein/s Effect on Actin Cytoskeleton
Profilin In resting state, sequesters monomeric actinthereby inhibits actin polymerization, In
stimulated state, promotes assembly of actinfilaments
P-thymosin, twinfilin, DNAse I Binds to monomeric actin and sequesters it,
thereby inhibits actin polymerizationCofilin Binds and severs actin filaments, Also
known as actin depolymerizing factor (ADF)Gelsolin, villin, fragmin, adseverin, Bind and sever actin filamentsscinderin
Fascin, villin, fimbrin, a-actinin Cross-link actin filaments, Also known asactin-bundling proteins
Srv2/cyclase-associated protein Stimulate nucleotide exchange on actin(CAP) family proteins monomers and relieve the inhibitory effects
of ADF/cofilin on this exchange
Wiskott-Aldrich Syndrome Protein Increases nucleation of actin monomers(WASP)Arp 2/3 Binds to actin dimers and increases
nucleation of stress fibers
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15
Plasminogen activator system and plasminogen activator inibitor-1 in
chronic renal disease
The plasminogen activator system, which controls activation of
plaminogen into plasmin, is the key regulator of matrix degradation (Fig. 5).
Activated plasmin, in turn, not only degrades some matrix proteins itself, but also
amplifies the fibrolytic response by activating another set of powerful matrix
degrading enzymes called matrix metalloproteinases (MMPs) [56]. PAI-1, tissue-
type plasminogen activator (tPA) and urokinase-type plasminogen activator
(uPA) are components of the plasminogen activator system (Fig. 5). PAI-1
inhibits matrix breakdown by inhibiting tPA and uPA. Although tPA and uPA have
a limited capacity to directly degrade ECM proteins, they trigger activation of a
cascade of proteases that lead to ECM degradation (Fig. 5) [56]. The balance
between the anti-fibrotic effects of plasminogen activators and the pro-fibrotic
effects of PAI-1 determines the rate of matrix turnover and the amount of matrix
deposition in the tissue [57, 58]. Transforming growth factor beta (TGFp) is a
major profibrotic growth factor responsible for increased PAI-1 expression and
resulting glomerulosclerosis seen in chronic renal diseases [59]. The
plasminogen activator system also regulates fibrinolysis in the intravascular
space [60].
tPA
Tissue-type plasminogen activator (tPA), Mr of 70,000, is secreted in the form of
a single or double chain enzyme from endothelial and mesangial cells [61].
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16
Plasminogen Activators
^Plasminogen' I (Inactive) J
uPAv______
Matrix Metalloproteinases (MMPs)
IExtracellular Matrix Degradation
Fig. 5. Regulation of extracellular matrix turnover by the plasminogen activator system. Plasminogen activator system consists of two activators of plasminogen namely tissue-type (tPA) and urokinase-type (uPA) plasminogen activators and their inhibitor called plasminogen activator inhibitor type I (PAI-1). Both tPA and uPA convert inactive plasminogen into active plasmin. Active plasmin, in turn, stimulates extracellular matrix (ECM) degradation mainly by activating matrix metalloproteinases (MMPs) and to a lesser degree by directly degrading ECM. PAI-1 acts as a gatekeeper of matrix degradation cascade by inhibiting tPA and uPA mediated plasminogen activation.
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17
The modular structure of tPA include five domains homologous to those of
fibronectin, epidermal growth factor (EGF), plasminogen and trypsin-like
protease. The extensive homology of domains of tPA to that of components of
ECM, proenzymes and growth factors facilitates the interaction between the
components and effective activation of plasminogen. tPA alone activates
plasminogen at a very low rate. Binding of tPA to proteins of extracellular matrix,
such as thrombospondin and collagen IV, increases the rate of plasminogen
activation. Vascular smooth muscle cells (VSMCs), which are phenotypically
similar to activated mesangial cells, produce the majority of tPA in vivo and
express a tPA binding receptor which not only increases tPA activity but also
decreases the inhibition of tPA by PAI-1 [62],
Administration of recombinant tPA provides a protective effect by
decreasing glomerular matrix accumulation in an anti-Thyl model of glomerular
matrix expansion [63], On the other hand, over-activation of tPA can produce
harmful effects due to increased MMP activation and tubular basement
membrane degradation [64]. Therefore it appears that tPA expression and
activity have to be carefully regulated for normal functioning of renal tissue.
uPA
Urokinase-type plasminogen activator (uPA), Mr of 55,000, having trypsin
like protease activity, was first isolated from urine. Like tPA, uPA is also
composed of domains that facilitate effective activation of plasminogen. Domains
of uPA are homologous to those of EGF, plasminogen and trypsin-like protease.
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Many cell types express a cystein-rich 65 kDa receptor for uPA (u-PAR)
[62], uPA binds to N-terminal domain of u-PAR with high affinity. Binding of uPA
to uPAR initiates intracellular signal transduction and triggers local proteolysis by
decreasing the Km for plasminogen activation by up to 200-300 fold, both of
which play a critical role in tissue development. Activated plasmin in turn
amplifies the fibrinolytic potential by activating zymogen pro-uPA. Thus an initial
activation of small amounts of plasminogen can generate large amounts of
plasmin through a positive feedback loop [62], Mice expressing an inducible uPA
transgene or following administration of an adenoviral vector containing a uPA
gene had reduced fibrosis [65].
Studies have shown that tPa and uPA are complimentary activators of
plasminogen with a high degree of functional overlap. Conventionally, tPA
regulates plasminogen activation in intravascular compartment, whereas uPA
does so in tissues. [66], Also tPA plays a major role in fibrinolysis, whereas uPA
has been associated with cell migration [67],
PAI-1
PAI-1 (Serpin E1) is a member of the SERPIN (SERine-Protease-lnhibitor)
family of protease inhibitors [68]. It is a single-chain, 379 amino acid, 52-kDa
glycoprotein (~13% carbohydrate) that exists in active, inactive and latent forms.
The active form spontaneously converts to a latent form, which can be partially
reactivated by denaturing agents [56], The reactive center of the inhibitor is
contained within the exposed “strained loop region” at the carboxy terminal of the
molecule. This serves as a pseudosubstrate for the target serine proteases.
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PAI-1 is not normally expressed in kidneys but is rapidly induced in a
variety of pathological states. Newly synthesized PAI-1 is released into the
extracellular space where it is converted into its inactive latent form in a few
minutes. Binding of PAI-1 to matrix proteins such as vitronectin increases its half-
life >10 times, thus enabling matrix the to resist proteolysis by invading cells [69],
[25], Mesangial cells along with glomerular endothelial and epithelial cells have
been identified as a source of PAI-1 expression under different conditions. [25,
70, 71]. The major function of PAI-1 is to regulate extracellular matrix turnover by
inhibiting the activation of proteases that break down the matrix. PAI-1 does so
by forming a 1:1 stoichiometric reversible complex with tPA and uPA, thereby
inhibiting them [56]. PAI-1-plasminogen activator complexes can be endocytosed
and subsequently degraded by lysosomes, thereby providing a clearance
mechanism for fibrolytic activity [69].
The inhibition of plasminogen activators by PAI-1 is the rate-limiting step
in matrix degradation. Not surprisingly, increased PAI-1 expression has been
associated with increased matrix deposition and tissue fibrosis. PAI-1 is nearly
undetectable in normal kidneys by immunohistochemistry and in situ
hybridization. However, increased PAI-1 expression is seen in many human and
experimental glomerular diseases including acute (thrombotic microangiopathy,
renal vasculitis, proliferative glomerulonephritis and membranous nephritis) and
chronic human diseases (diabetic nephropathy, hypertensive nephropathy, focal
segmental glomerulonephritis and lupus nephritis) and animal models in different
renal cell types including mesangial cells [25]. Studies with knock-out mice
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20
clearly established the role of PAI-1 in fibrosis. PAI-1 knock out mice were
resistant to renal injury caused by unilateral ureteral obstruction [71] and
pulmonary fibrosis caused by bleomycin (an antineoplastic drug producing
pulmonary fibrosis as a side effect) [72]. Similarly, PAI-1 knockout mice were
protected against N-nitro-L-arginine methyl ester (L-NAME)-induced perivascular
fibrosis [73]. Conversely, over expression of PAI-1 worsened bleomycin-induced
fibrosis [72]. By virtue of its action on matrix turnover, PAI-1 also plays an
important role in neointima formation, angiogenesis, atherosclerosis, tumor
growth and metastasis, and wound healing. PAI-1 may play a role in cell
adhesion and migration as a result of its high affinity binding to vitronectin.
PAI-1 acts as a gatekeeper guarding against fibrinolysis by inhibiting
unnecessary activation of the plasminogen cascade. Hence it is one of the most
highly regulated components of the plasminogen activation system. The human
PAI-1 gene is located on the long arm of chromosome 7. It is comprised of nine
exons and eight introns, and is ~12.2 kb long. Alternative polyadenylation of PAI-
1 mRNA results in two distinct transcripts, one 2.8 kb long and the other 3.2 kb
long. The PAI-1 gene and its flanking region contain 12 Alu repeat elements and
5 long poly (Pur) repeat elements. Also, the promoter contains a 4G/5G
polymorphism at -675. It is suggested that the presence of 4Gs in the promoter
is associated with increased production of PAI-1 [74],
PAI-1 expression is induced by a variety of factors including growth
factors, coagulation factors, metabolic factors and hormones such as
transforming growth factor-(B (TGF(3), epidermal growth factor (EGF), fibroblast
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21
growth factor (FGF), fibrin fragments, thrombin, glucose, insulin, Angiotensin,
hypoxia, glucocorticoids and radiation [25]. Regulation is primarily at the
transcription level, although cyclic nucleotides may regulate PAI-1 message
stability [25, 75]. The PAI-1 promoter has two AP-1 like sites, two SP-1 sites,
three SMAD sites, one NF-kB site, glucocorticoid response elements (GRE) and
hypoxia response elements (HRE) allowing the regulation of promoter by multiple
pathways [76-79], Indeed, inflammatory mediators such as TGF(3, triglycerides,
fatty acids and phorbol esters increase PAI-1 expression. PAI-1 expression is
regulated by signal inputs from many intracellular signaling pathways including
MAPK, Smad, PKA, PKC, nuclear receptors and Rho signaling [76, 80-84],
PAI-1, in turn, inhibits uPA-plasminogen activation, which normally
activates latent TGFp, a major inducer of PAI-1 expression in mesenchymal cells.
Thus TGFp turns off its own production through induction of PAI-1. The
autoregulatory feedback loop between TGFp and PAI-1 plays an important role in
wound healing and in limiting ECM deposition [69]. A detailed understanding of
signal transduction involved in TGFp induced PAI-1 expression is required to
identify potential targets for the intervention of TGFp mediated PAI-1 expression
and fibrosis.
In a previous study we have shown that changes in actin cytoskeleton
regulate a-SMA gene expression and hypertrophy in mesangial cells [28]. Like a-
SMA expression and hypertrophy, de novo expression of PAI-1 is also seen with
myofibroblast differentiation [85, 86], It is possible that regulation of both the
genes share common or similar underlying regulatory mechanisms, suggesting
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22
regulation of PAI-1 by actin cytoskeleton. Also, recently Schnaper et al have
shown that the inhibition of actin polymerization blocks TGFP-mediated collagen
expression [87]. It is logical to think that changes in actin cytoskeleton may also
similarly modulate expression of PAI-1, another TGFp-dependent gene and a
regulator of ECM. Thus two independent lines of evidence suggest regulation of
PAI-1 expression by changes in actin polymerization.
TGFp in Renal Diseases
Transforming Growth Factor-p (TGFp) regulates extracellular matrix
production by increasing the production of matrix components such as collagen,
fibronectin, laminin and heparan sulfate proteoglycans, and simultaneously
decreasing matrix degradation by increasing production of plasminogen activator
inhibitor-1 (PAI-1) [88]. The net result is an increase in extracellular matrix
production and glomerulosclerosis [89, 90]. TGFp regulates a range of cellular
processes including the cell cycle, differentiation, wound healing, extracellular
matrix production and the immune response [91-94], TGFp circulates in an
inactive form bound with latency-associated peptide (LAP) and is activated
locally when LAP binds to integrin avp6 and TGFp is cleaved.
Not surprisingly, TGFp is present in human glomeruli in several
glomerular diseases including diabetic nephropathy [95] and is associated with
increased mesangial matrix production [96]. During pathogenesis of these renal
diseases, TGFp signaling causes tubular atrophy, podocyte depletion, loss of
capillary endothelial cells, mesangial cell activation and epithelial-to-
mesenchymal transdifferentiation. The combination of these multiple pathogenic
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23
events leads to loss of functioning renal tissue [91]. The role of TGFp in renal
fibrosis has also been proven in a variety of animal models [97, 98]. Integrin av|36
knockout mice, which are unable to release LAP inhibition of TGFp, were
protected against pulmonary fibrosis [99], Administration of anti-TGFp antibody
protected against scarring in an animal model of chronic renal disease confirming
the role of TGFp in fibrosis [100].
Rho and PAI-1 expression
Rho proteins regulate gene expression by increasing activity of
transcription factors such as c-jun, ATF2a, ELK and STAT3 through intracellular
signaling pathways such as MAPK and src [101]. Recently, the protective effects
of Rho-kinase inhibitors against tissue fibrosis have established the role of Rho in
the pathogenesis of tissue fibrosis. The Rho-kinase (ROCK) inhibitor Y-27632
markedly decreased bleomycin-induced pulmonary fibrosis and collagen
accumulation in mice [102], Similarly, Y-27632 protects against hepatic fibrosis
[103]. The inhibition of Rho-kinase also protects against tubulointerstitial fibrosis
in mouse kidneys with unilateral ureteral obstruction [104]. The basis for the
protective effects of Rho-inhibition is not fully understood. Inhibition of expression
of profibrotic molecules such as PAI-1 appears to be a potential mechanism.
Inhibition of Rho by C3 exoenzyme decreases PAI-1 production in rat proximal
tubules [105] and aortic endothelial cells [106]. Co-transfection with dominant
negative RhoA decreases PAI-1 promoter activity, whereas constitutively-active
RhoA increases PAI-1 in chicken atrial cells [107], ROCK inhibition blocks PAI-1
expression in response to Angiotensin II. [108], Also, ROCK inhibition blocks
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24
PAI-1 promoter activity [81]. Thus, both inhibition of Rho activation and inhibition
of signaling downstream of Rho lead to inhibition of PAI-1 expression.
Collectively, these facts suggest that inhibition of Rho protects against fibrosis
through inhibition of PAI-1.
Although the role of Rho in the regulation of PAI-1 expression is becoming
increasingly clear, the mechanism for this effect has not been examined. Since
actin polymerization is one of the major functions of Rho-ROCK signaling (Fig.
3), it is plausible that the effect of Rho-inhibition is mediated by depolymerization
of actin. Taken together, these observations suggest that Rho-mediated
changes in actin cytoskeletal structure modulate PAI-1 expression. Given the
diversity of signaling pathways that interact with Rho, it is possible that Rho
mediates and integrates the effect of these pathways on PAI-1 expression by
modulating actin cytoskeleton organization.
Hypothesis and aims
The Central hypothesis is that changes in actin polymerization regulate
the myofibroblast-like phenotype of kidney mesangial cells by simultaneously
regulating expression of multiple genes.
The overall objective is to characterize the mechanisms of mesangial
myofibroblast-like differentiation.
The working hypothesis is that Rho-mediated changes in actin
cytoskeletal structure modulate PAI-1 expression in glomerular mesangial cells.
My specific aims are to examine the role of Rho in the regulation of TGF[3-
induced PAI-1 expression in human mesangial cells as follows:
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25
Specific Aim 1:
Specific Aim 2:
Specific Aim 3:
Specific Aim 4:
Specific Aim 5:
To profile the effects of actin cytoskeleton on the expression
of multiple genes in human mesangial cells.
To examine the effect of TGFp on PAI-1 expression
To examine the role of Rho and actin cytoskeleton in TGFp-
induced PAI-1 expression.
To examine the role of Rho and actin cytoskeleton in TGFp-
induced PAI-1 promoter.
To examine the effect of TGFp and actin cytoskeleton on
plasminogen activators.
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26
CHAPTER 2
MATERIALS AND METHODS
Cell culture
Human mesangial cells (HMCs) were cultured in RPMI 1640 (Mediatech
Inc., Herndon, VA) with 16.7% fetal bovine serum (Invitrogen, Carlsbad, CA).
from renal glomeruli isolated by the sieving method. HMCs between passages 5
and 9 (P5 -P9) were used for experiments. After plating in serum-containing
media, cells were serum-starved at 60-80% confluence. After 24 hours of serum
starvation, cells were pre-treated with agents that depolymerize (Cytochalasin B:
1.0 nM, Latrunculin B: 0.1 pM, Toxin B: 10 pM, Y-27632: 10 pM, HA-1077: 20
pM) and/or stabilize actin cytoskeleton (Jasplakinolide: 50 nM) for one hour prior
to addition of 10 ng/ml TGFp for the indicated time periods. Cytochalasin B was
purchased from Sigma Chemical Co., St. Louis, MO. The Rho-kinase inhibitor, Y-
27632, was purchased from Welfide Corporation, Osaka, Japan. Another Rho-
kinase inhibitor, HA-1077, was purchased from AG Scientific Inc., San Diego,
CA. Clostridium Difficile Toxin B (Toxin B), Latrunculin B (LatB) and
Jasplakinolide (Jas) were purchased from Calbiochem, San Diego, CA. Human
recombinant TGFp was purchased from R&D Systems, Minneapolis, MN.
DNA Array Analysis
The RNA isolation, DNAse treatment, Poly A+ enrichment, cDNA probe
synthesis, hybridization and analysis of scanned arrays were performed as per
manufacturer’s protocol [109],
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27
RNA Isolation
Human mesangial cells were plated on five 10 cm dishes per condition. At
the end of the desired treatment, cells were trypsinized and spun down at 680xg
for 5 minutes. The pellet was resuspended in 1 ml denaturing buffer. A 0.5 ml
aliquot was transferred to sterile 1.5 ml tube and incubated on ice for 5-10 min.
After vortexing briefly, the homogenate was centrifuged at 13,370xg for 15 min at
4°C to remove cellular debris. The supernatant was transferred to a new 2 ml
tube and 1 ml of Tris-EDTA saturated phenol was added to it. The tube was
vortexed for 1 min. After a 5 min incubation on ice, 0.3 ml chloroform was added.
The sample was vortexed vigorously for 1-2 min and then incubated on ice for 5
min. The homogenate was centrifuged at 13,370xg for 10 min at 4°C. The upper
aqueous phase containing RNA was transferred to a new 2 ml tube. A second
round of phenol chloroform extraction using 0.8 ml saturated phenol and 0.3 ml
chloroform was performed. Next, 1 ml isopropanol was added with incubation on
ice for 10 min. The samples were centrifuged at 13,370xg for 15 min at 4°C. The
pellet was washed with 0.5 ml of 80% ethanol. The samples were centrifuged at
13,370xg for 15 min at 4°C. The supernatant was discarded and the pellet was
air-dried. The pellet was re-suspended in 25 pi RNase-free H20.
DNase Treatment of Total RNA
The DNAse treatment reaction was set up in a final volume of 200 pi
containing 100 pg total RNA, DNase (1 unit/pl) and 10 x DNase buffer (400 mM
Tris-HCI (pH 7.5), 100 mM NaCI, 60 mM MgCh). After a 30 min incubation at
37°C, the reaction was stopped by adding 20 pi termination mix (0.1 M EDTA (pH
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28
8.0), 1 mg/ml glycogen). The reaction was split into two 1.5 ml tubes. To each
tube, 100 pi of saturated phenol and 60 pi chloroform were added followed by
centrifugation at 16,170xg for 10 min at 4°C to separate phases. Top aqueous
layer was carefully removed and placed in a 2.0 ml tube. To each tube, 1/10
volume of 2 M (10 pi) NaOAc and 2.5 volumes (0.3 ml) of 95% ethanol were
added. The mixtures were vortexed thoroughly and incubated on ice for 10 min.
The tubes were centrifuged in a microfuge tube at 16,170xg for 15 min at 4°C.
The pellets were washed with 100 pi of 80% ethanol followed by centrifugation at
16,170xg for 5 min at 4°C. The supernatant was removed and the pellet was air-
dried. The pellets were dissolved in 50 pi RNase-free H20.
Poly A+ RNA Enrichment and Probe Synthesis
Streptavidin Magnetic Bead Preparation
Magnetic beads were re-suspended by inverting and gently tapping the
tube. Aliquots containing 15 pi of beads per probe synthesis reaction were
transferred into 0.5 ml tubes. Beads were separated on magnetic particle
separator. The supernatant was pipetted off and discarded. Beads were washed
with 150 pi of 1x binding buffer [109] and separated again on a magnetic particle
separator. The supernatant was pipetted off and discarded. The cycle was
repeated three times. The beads were resuspended in 15 pi 1x binding buffer per
reaction.
Poly A+ RNA Enrichment
The PCR thermal cycler was preheated to 70°C. Aliquots of 10-50 pg total
RNA were transferred into 0.5 ml tubes. Deionized H20 was added to make up a
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29
final volume of 45 jj.1. To each tube, 1 pi Biotinylated Oligo (dT) was added
followed by incubation at 70°C for 2 min in the preheated thermal cycler. The
reaction was cooled at room temperature for 10 min. To each tube, 45 pi 2x
binding buffer was added. The washed beads were resuspended by pipetting up
and down. Fifteen pi of beads were added to each RNA sample and mixed on a
vortexer or shaker for 30 min at room temperature. Beads were separated using
the magnetic separator. The supernatant was pipetted off and discarded. Beads
were resuspended in 50 ul 1x wash buffer. Beads were again separated and
washed once. Beads were resuspended in 6 pi dH20.
cDNA Probe Synthesis
A master mix containing 5X reaction buffer (250 mM Tris-HCI (pH 8.3),
375 mM KCI, 15 mM MgCh), 10x dNTP mix (5 mM each dCTP, dGTP, dTTP), [a-
32P] dATP (3000 Ci/mmol, 10 pCi/pl, 5 pl/reaction) and 10 mM DTT was
prepared. One pi cDNA synthesis primer mix [109] was added to the
resuspended beads. The beads and primer mix were incubated in the preheated
thermal cycler at 65°C for 2 min. Meanwhile, 2 pi MMLV reverse transcriptase
per reaction was added to master mix. To each reaction tube, 13.5 pi master mix
was added followed by thorough mixing. Tubes were incubated at 50°C for 25
min. The reactions were stopped by adding 2 pi 10x termination mix (0.1 M
EDTA [pH 8.0], 1 mg/ml glycogen).
Column Chromatography
The probe synthesis reaction mixture was diluted to a final volume of 200
pi with Buffer NT2 [109]. The sample was added to a NucleoSpin Extraction Spin
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30
Column and centrifuged at 16,170xg for 1 min. The NucleoSpin column was
inserted into a fresh 2-ml collection tube and 400 pi Buffer NT3 [109] was added
to the column. After centrifugation at 16,170xg for 1 min, the collection tube and
the flow-through were discarded. The wash with Buffer NT3 was repeated twice.
The NucleoSpin column to was transferred to a clean 1.5-ml microcentrifuge
tube. One hundred pi of Buffer NE [109] was added to the column for 2 min
followed by centrifugation at 16,170xg for 1 min to elute purified probe. The
radioactivity of the probe was checked by scintillation counting.
Hybridization Procedure
The hybridization solution (BD ExpressHyb + sheared salmon testes DNA
heated at 95°C for 5 min) was prewarmed at 68°C. The membrane was
prehybridized for 30 min with continuous agitation at 68°C. To prepare the probe
for hybridization, 5 pi Cot-1 DNA [109] was added to the entire pool of labeled
probe followed by incubation in a boiling (95-100°C) water bath for 2 min. Next,
the probe was incubated on ice for 2 min. The probe was added to the
membrane and hybridized overnight with continuous agitation at 68°C.
The next day, the array membrane was washed with continuous agitation
at 68°C with Wash Solution 1 (2X SSC, 1% SDS; 3 X30 min washes) and Wash
Solution 2 (0.1X SSC, 0.5% SDS; 1X 30 min wash). The final 5-min wash was
performed in 200 ml of 2X SSC with agitation at room temperature. The blot was
wrapped with plastic and exposed to phosphorimager plates at room
temperature.
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31
Analysis
The phosphor imager plates were scanned after a 3 day exposure to
radio-active blots. Atlas Image software (Clontech, Palo Alto, CA) was used to
analyze the scanned blots. According to manufacturer’s instructions, the signals
from cDNA spots were normalized to a panel of three house-keeping genes
(GAPDH, cytoplasmic [3-actin, 60S ribosomal protein L13A) on the same blot.
The software takes into account the changes in the level of multiple house
keeping genes and corrects the test gene signals accordingly. This provides a
more accurate normalization of test genes as compared to using a single house
keeping gene for normalization. Three housekeeping genes that were relatively
unaffected by the treatment were selected to increase the accuracy of
normalization. The ratios of normalized gene-expression values for CytB treated
and untreated control were prepared. Arbitrarily, either more than a 2-fold
increase or more than 50% inhibition in gene-expression were considered
significant. The genes that failed to show a signal in either of the blots or showed
a near-background signal were excluded from the analysis.
Western blot analysis
Western blot analysis was performed using alkaline phosphatase or
enhanced chemiluminescence detection as described below.
Electrophoresis
Since PAI-1 is secreted upon synthesis, conditioned media in which the
cells were cultured was used to detect changes in PAI-1 protein. Equal amounts
of conditioned media were analyzed. After a 1:10 dilution of conditioned media in
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1X sample buffer (0.12M Tris 1.51 g, 2% SDS, 10% glycerol, pH 6.8), 20 pi of
diluted samples per lane were electrophoresed on reducing polyacrylamide gels
at 4°C. Proteins were separated first on 4% stack gel at 50 V for 30 min and
then on 11 % resolving gel at 200 V for 90 min. The gels were run in tank buffer
(Tris: 3.04 g/l, Glycine 14.4 g/l, SDS 1 g/l) at4°C. An HRP-labeled molecular
weight marker, MagicMark (Invitrogen, Carlsbad, CA) was run on each gel along
with the samples. The samples were transferred onto PVDF overnight at 15 V in
cooled (4°C) Western blot buffer (Tris 3 g/l, Glycine 14.5 g/l, methanol 20%).
Immunodetection
Alkaline-Phosphatase Detection
After overnight transfer, membranes were washed five times with dH20 to
remove excess methanol. Membranes were blocked overnight with 5% milk in
high salt-TBS (Tris 20 mM, NaCI 500 mM). After two 10-min washes with TBS-
Tween (Tris 20 mM, NaCI 500 mM, Tween 20 0.05%, pH 7.5), membranes were
incubated one hour each at room temperature, first with goat anti-PAI-1 antibody
(American diagnostica, Stamford, CT) diluted 1:500 in TBS/Tween and then with
a biotinylated secondary anti-goat antibody (Pierce, Rockford, IL) diluted 1:500 in
TBS-Tween. Between and after incubations with antibodies, membranes were
washed four times for 15 min each with TBS-Tween. After washing, PAI-1
antigen was detected using the Vectastain ABC system (Vector laboratories,
Burlingame, CA). Blots were incubated for one hour with Vectastain ABC reagent
for alkalaline phosphatase detection. Blots were washed twice for 15 min each
wash with high salt TBS-Tween and once for 15 min with high salt TBS. After
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briefly washing membranes with BCIP-NBT buffer (Tris 0.1 M, MgCI2 0.5 mM, pH
9.5), BCIP-NBT detection reagent (Bromo Chloro Indolyl Phosphate-Nitrozolium
Blue in BCIP-NBT buffer) was added to the membrane. After a sufficient amount
of signal was obtained, blots were washed with dH20 and air-dried.
Enhanced Chemiluminescence Detection
After overnight transfer, membranes were washed five times with dH20 to
remove excess methanol. Membranes were blocked for one hour with NAP-Sure
Blocker (Geno Technology, St. Louis, MO). Membranes were incubated one hour
each at room temperature, first with Goat Anti-PAI-1 antibody (American
diagnostica, Stamford, CT) diluted 1:1000 in TBS/Tween and then with horse
raddish peroxidase (HRP)-labeled secondary anti-goat antibody (Jackson
Immunoresearch, West Grove, PA) diluted 1:20,000 in TBS/Tween. Anti-tPA
antibody (PAM-3 clone, American diagnostics, Stamford, CT) was used at 1:200
dilution and anti-uPa antibody (American diagnostics, Stamford, CT) was used at
1:1000 dilution. HRP-labeled secondary antibodies were used at 1:20,000
dilution. Between blocking and incubations with antibodies, membranes were
washed once for 15 min and twice for 5 min with TBS/tween ( Tris 20 mM, NaCI
500 mM, Tween 20 0.05%, pH 7.5) At the end of the incubation with the
secondary antibody, membranes were washed once for 15 min and four times for
5 min with TBS/Tween. Substrate buffer from Enhanced Chemiluminescence
Western blotting analysis system (Amersham Biosciences, Piscataway, NJ) was
added to the blots for one minute to detect HRP-signal on the membrane. X-ray
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34
films were exposed to the blot for varying amounts of time to detect optimum
signal from the blot.
Analysis
X-ray films were scanned on a flatbed scanner with a transparency
adapter and analyzed using NIH Image software, available on the internet at
http://rsb.info.nih.gov/nih-image/Default.html (National Institute of Health,
Bethesda, MD).
Northern Blot Analysis
Northern blot analysis for PAI-1 mRNA was performed using partial a PAI-
1 cDNA as probe (Clontech, Palo Alto, CA).
Electrophoresis
Ten pg total mRNA was incubated with 5.4 pi 6M glyoxal, 16 pi DMSO,
3.0 pi 0.1 M Sodium Phosphate at 50°C in heat block for 1 hr. Samples were
cooled to 0°C on ice. After adding 4 pi loading dye, samples were run on a 1.2 %
agarose gel with ethidium bromide in running buffer (1.9 ml 1M NaH2P04, 6.1 ml
1M Na2HP04,990 ml DEPC H20) at 50 V for 30 min to allow all the samples to
migrate out of the well and into the gel. Next, the gel was run at 75 V for
approximately 3 hours with buffer circulation. A digital photograph of the gel was
taken using Eagle Eye II (Stratagene, LA Jolla, CA) under UV light to document
18S/28S loading in each lane. RNA was transferred onto positively-charged
nylon membrane (Roche, Indianapolis, IN) overnight in 1X TAE (Tris 4.84 g/l,
Glacial Acetic Acid 1.14 g/l, EDTA 0.744) at 25 V and 4°C.
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UV Cross linking
After overnight transfer, the membranes were air dried for 10 min. RNA
was cross-linked to membranes using UV cross-linker 1800 (Stratagene, LA
Jolla, CA) at 120 m Joules. Hybridization was then performed or the membrane
was stored at -20°C for probing at a later time.
Hybridization
The blots were incubated at 42°C for 1 hr to overnight in pre-hybridization
solution (5%Formamide, 5X SSPE, 5XDenhardt’s, 0.1 mg/ml Salmon Testes
DNA, 0.5% SDS) in a Techne Hybridizer HB-2D hybridization oven (Burlington,
NJ). After sufficient pre-hybridization, 25-50 ng/ 2 pi PAI-1 cDNA probe (0.5 pi in
case of 18S fragment) (Clontech, Mountain View, CA) was brought up to 15 pi
using DEPC H20. Probe was denatured in boiling H20 for 15 min. While probe
was denaturing, the following reagents from Random Primer DNA Labeling
System ( Invitrogen, Carlsbad, CA) were combined in a final volume of 49 pi on
ice: 2 pi dATP, 2 pi dGTP, 2 pi dTTP, random primer buffer solution (0.67 M
HEPES, 0.17 M Tris-HCI, 17 mM MgCI2, 33 mM 2-mercaptoethanol, 1.33 mg/ml
BSA, 18 OD260 units/ml oligodeoxyribonucleotide primers, pH 6.8), 32P-dCTP
(approximately 50 pCi or 20pCi for the 18S probe). The denatured probe was
immediately transferred to ice and the probe added to the preassembled labeling
mix. To the probe mix, 1 pi Klenow fragment (3U/pl) was added and mixed
gently. Probe synthesis was allowed at 37°C for 30 min and then stopped with 5
pi stop buffer (0.5 M EDTA, pH 8.0). Radio-labeled probe was denatured by
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boiling at 100°C for at least 10 min. While the probe was being labeled, the pre
hybridization solution was replaced by 10 ml hybridization solution per blot (50%
Formamide, 5X SSPE (NaCI 43.8 g/l, Na phosphate (mono) 6.9, EDTA 1.9 g/l,
pH 7.4), 5X Denhardt’s (Ficoll 1 g/l, polyvinylpyrrolidone 1 g/l, BSA 1 g/l), 0.11
mg/ml Salmon Testes DNA, 0.5% SDS, 4% Dextran Sulfate). Radio-labeled
probe was carefully added to the tube and the probe was hybridized with the blot
in hybridization oven overnight at 42°C.
Washing
After overnight hybridization, blots were washed twice for 10 min each with 100
ml 5X SSPE (NaCI 43.8 g/l, Na phosphate (mono) 6.9, EDTA 1.9 g/l, pH 7.4) and
0.5 % SDS to remove non-specific binding. Blots were washed for 5 min at 65°C
in 0.5% SSPE and 1% SDS. If the blots were still radioactive at the edges, an
additional wash with 0.1X SSPE and 1% SDS was performed for 5 min at 65°C.
The blots were wrapped in saran wrap and exposed to a phosphor-imaging
cassette (Molecular Dynamics-Amersham, Piscataway, NJ). For PAI-1 mRNA
detection, the phosphorimager plates were scanned after 2-3 days.
The membranes were stripped before probing for 18S. The blots were
washed in 50-100 ml nearly boiling DEPC H20 for 15 min 2-3 times.
18S Hybridization
The membranes were probed with radio-labeled 18S fragment using the
Northern blot protocol described above. The blots were exposed to phosphor
imager plates for 30 min to few hours.
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Analysis
The blots were scanned using phosphor imager scanner. The densitometric
analysis for PAI-1 bands was performed using Image Quant Software (Molecular
Dynamics-Amersham, Piscataway, NJ).
Immunocytochemistry
Adherent cells on coverslips were immunofluorescently stained using a
two-day protocol. For PAI-1 staining, cells were plated on serum-coated dishes in
serum-free media.
At the end of desired period of treatment, cells were washed 3 times with
2 ml Hank’s balanced salt solution. Cells were fixed with 2 ml 3.7%
paraformaldehyde in PBS ( monobasic sodium phosphate 0.5 g/l, dibasic sodium
phosphate 2.3 g/l, NaCI 8.5 g/l) at room temperature for 10 min.
Paraformaldehyde was washed off with 3x 2 ml washes with TBS. Cells were
permeabilized with 2 ml 0.5% Triton X-100 in TBS (Tris 25 mM, NaCI 0.9%, pH
7.5) for 5 min at room temperature, after which they were washed 3x with 2 ml
TBS. Cells were incubated with 5% blocking milk (centrifuged at 13,370 G for 1
hour) in TBS for at least one hour at room temperature. Coverslips were
incubated with primary goat anti-PAI-1 antibody diluted 1:100 in milk-TBS
overnight at 4°C in a humidified chamber.
After overnight incubation, coverslips were washed 1x with milk and 2x
with TBS for 10 min each. Coverslips were incubated with secondary antibody
Biotinylated anti-Goat diluted 1:100 in milk-TBS for 2 hr at 37°C in a humidified
box. Excess antibody was washed off once with milk and twice with TBS for 10
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38
min each. Coverslips were incubated with mixture of FITC-avidin and Texas red
phalloidin diluted 1:200 in TBS at 37°C for 1 hr. Cells were washed 3x with TBS
briefly and then once with 1 iM TBS-Biotin (10 ml TB S /1 00jj.I 1mM biotin).
Finally after washing once with dH20, excess water was drained off. Coverslips
were air dried and mounted on glass slide with 25 (j,l gelvatol.
Images were captured using a Spot digital camera attached to a
fluorescent microscope.
Quantitative Real-time Polymerase Chain Reaction (PCR)
Principle of Quantitative real-time PCR
In real-time PCR, amplification of DNA is monitored during each cycle
allowing accurate quantitative estimation of nucleic acid concentration. The cycle
number at which the signal starts to increase exponentially over the background
is called the crossover point. Comparison of crossover points of samples with
those of known concentrations of standard cDNAs gives the concentration of a
particular template in the sample. Relative quantification with external standards
as a method for comparing gene expression was used in this study. In this
method of quantification, a reference, typically a house-keeping, gene is used to
normalize the concentration of the target gene. First, the concentrations of target
and reference genes are obtained by using an external standard curve of known
amounts of nucleic acid. The results are expressed as target/reference ratio. The
use of an endogenous control, corrects for the factors affecting PCR efficiency.
Following is the protocol used in this study.
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39
RNA Isolation and Quantification
Total cellular RNA was isolated using TRIzol method as described earlier
and quantified using a UV spectrophotometer.
DNAse treatment
The protocol for real time PCR analysis was modified from a previous
protocol [110]. Briefly, 1 pg total RNA sample was DNAse treated and
subsequently reverse transcribed using specific anti-sense primers for PAI-1 and
Ubiquitin (UBC, reference gene) in the same reaction (Table 2). In a typical
reaction, a total of 4 pg mRNA was treated with 4 pi of 1 U/pl DNAse I
(Invitrogen, Carlsbad, CA) in the presence of 2 pl of 10 x reaction buffer in a total
reaction volume of 20 pl. After a 15-minute incubation at room temperature,
DNAse treatment was stopped by incubating the mixture at 65° C for 10 min with
2 pl of 25 mM EDTA. DNAse-treated total RNA was stored at -80° C until further
use in the reverse transcription (RT) reaction.
RT reaction
DNAse treated mRNA was reverse-transcribed using a thermocycler (MJ
Research, Reno, NV). The test gene and housekeeping gene were reverse-
transcribed simultaneously using their specific anti-sense primers in the same
reaction. Performing RT for both genes, the gene of interest and the reference
gene, in the same reaction minimizes the error due to the efficiency of the RT
reaction and hence provides a good way of normalizing for a reference gene in
the RT reaction. 1 pg of DNAse treated RNA (1 pg/5 pl) was reverse transcribed
in a final reaction volume of 25 pl. The reaction mix also contained RT buffer
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(Promega, Madison, Wl), 60 U RNAse inhibitor, 3 pM anti-sense primer for
sample gene, 3 pM anti-sense primer for Ubiquitin, 10 pM DTT and 500 pM
dNTPs. Reverse transcription reaction was performed at 37°C for one hour in a
thermocycler followed by heat inactivation of the enzyme at 70°C for 10 min. RT
products were immediately used for real-time PCR analysis on the Roche
lightcycler PCR instrument.
Preparation of cDNA standards for real time PCR
The standards for the quantification were prepared as described
previously [110], by amplifying 10-fold serial dilutions of known concentrations of
templates for each gene. Briefly, cDNA was prepared for each gene from 1 pg
human mesangial cell total RNA using specific anti-sense primers and amplified
using a PCR reaction for 35 cycles. PCR products from 5 identical reactions for
the same gene were pooled in a single tube. An aliquot of PCR products was run
on 8% acrylamide to confirm the product size. PCR products were purified using
“High pure PCR products purification kit” (Roche Diagnostics, Indianapolis, IN).
The concentration of purified PCR products was measured using picoGREEN
dsDNA quantification kit (Molecular probes, Eugene OR) [111]. Briefly, a 2 pg/ml
stock solution of lambda dsDNA standard was prepared in TE (10 mM Tris-HCI,
2 mM EDTA, pH 7.5). The stock solution was diluted in duplicate vials with TE to
provide 0, 1.25, 2.5, 6.25, 12.5, 18.75 and 25 pg/pl concentration of standard.
Duplicate vials containing 1 jal or 2 pl of test sample in a final volume of 1000 pl
TE were prepared. To each vial 1 ml of PicoGreen dsDNA quantitation reagent
[111] was added for 5 min. The vials were incubated in dark. After incubation,
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fluorescence from the standards and the samples were measured using a
fluorometer using an excitation wavelength of 480 nm and emission wavelength
of 520 nm. The values for standard concentrations were plotted on a graph. The
concentration values for samples (pig/pil) were derived using the linear graph
equation.
After fluorometric measurement, the concentration of purified product was
adjusted to 5000 pg/pl. Ten-fold serial dilutions of PCR products were prepared
to cover a range from 0.001 pg to 1 ng for the standard curve. These serial
dilutions were amplified on lightcycler using specific parameters for each gene.
Melting curve analysis was performed at the end of the PCR run to ensure the
purity of the product and to eliminate the possibility of primer-dimers. Roche
Lightcycler software was used to prepare a standard curve. The identities of
PAI-1 and ubiquitin cDNAs were confirmed by sequencing (Midland Sequencing,
Midland, TX)
Real-time PCR
After diluting RT products 1:10 in dH20, 2 pl of diluted sample was
amplified using the LightCycler (Roche, Indianapolis, IN). Each gene of interest
and the reference gene were analyzed in separate glass capillaries. The
reactions were set up in the capillaries in a final volume of 20 pl, which contained
2 pl of standard or diluted sample, MgCL (4 mM for PAI-1 or uPA, 1 mM for tPA),
1 pM each of both the primers (table 2) and 10 pl of 2x SYBR green PCR mix
(Qiagen, Valencia, CA). The hot start method was used to initiate PCR reaction
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Table 2. Primer sequences for real-time PCR analysis
Gene Sense Primer (5’-3’) Anti-Sense Primer (5’-3’)
PAI-1 TGCTGGTGAATGCCCTCTACT CGGTCATTCCCAGGTTCTCTA
UBC ATTTGGGTCGCGGTTCTTG TGCCTT GACATT CT CGAT GGT
TPA CCCCAGGCAAACTTGCAC GCCTTT GCAGT GT CTT CT GAA
uPA CACGCAAGGGGAGATGAA ACAGCATTTTGGT GGT GACTT
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by heating the samples at 95° C for 15 min prior to PCR cycling. The following
cycling parameters were used for the amplification: denaturation at 95° C for 15
s, annealing at 60° C (PAI-1, uPA) or at 56° C (tPA) for 5 s, and extension at 72°
C for 18 s. Samples were amplified for 35 (PAI-1, tPA) or 40 cycles (uPA). Gene
amplification was monitored in real-time with SYBR green dye. The crossing
points of sample genes were compared against the crossing points of known
standards to determine the absolute concentration of a gene in a particular
sample. Values for the gene of interest were normalized to Ubiquitin amplified
from the same sample. At the end of PCR cycling, the melting curve of the
products was analyzed to ensure the specificity of the reaction. PCR products
from representative experiments were run on the gel and visualized by ethidium
bromide staining to further ensure the specificity of the reaction.
Table 2 shows the primers used for real-time PCR analysis of PAI-1, tPA,
uPA and Ubiquitin. The primers for PAI-1, uPA and Ubiquitin were purchased
from a commercial vendor (Midland Reagents, Midland, TX) using previously
published primer sequences [110]. The protocol for tPA detection was developed
in our lab using the primers designed and synthesized by TIB Molbiol LLC
(Adelphia, NJ).
Melting curve analysis
Melting curve analysis was performed at the end of each run to ensure
specific amplification of a single product in three steps:
a. Rapid heating to 95°C to denature all the DNA present in the mixture
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b. Cooling to below annealing temperature to allow formation of double
stranded DNA.
c. Gradual heating to 95°C to melt double stranded DNA
Melting curve analysis showed a single peak suggesting a pure population
of PAI-1, tPA, uPA and UBC cDNA in PCR products. The PCR products were run
on a gel to confirm the size of the product and to detect the purity of the reaction.
Subsequently, products from representative experiments were run on a gel to
ensure amplification of single template of expected size.
Cloning of PAI-1 promoter
PCR cloning of the promoter
The PAI-1 promoter containing major regulatory elements as listed in chapter 6
was cloned from human mesangial cells using specific primers. Human genomic
DNA was isolated from primary culture human mesangial cells using a DNA
isolation kit (Clontech, Palo Alto, CA). Primers for a 1106 bp promoter (- 973 to
+133) were purchased from a commercial vendor (Midland Reagents, Minland,
TX) as published by Grenette et al [112].
Forward primer: 5’-CGATCGGTACCTAAAAGCACACCCTGCAAAC-3’
Reverse primer: 5’-CGATCAGATCTCAGAGGTGCCTTGCGATTG-3’
A total of three reactions were set up with 2 pg genomic DNA, 4 pg DNA or no
DNA. The reactions were set up in a 100 pl reaction mix containing 150 pmol of
sense and anti-sense primers, 1.5 mM MgC^and 1 pl platinum taq polymerase.
PCR reaction was performed for 30 cycles using the following parameters:
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denaturation at 94°C for 45 seconds, primer annealing at 58°C for 45 seconds
and primer extension at 72°C for 45 seconds. An additional 5-min incubation at
72°C was performed to complete the reaction. PCR products from all three
reactions were run on a 1% agarose gel in TAE buffer (Tris 4.84 g/l, Glacial
Acetic Acid 1.14 g/l, EDTA 0.74 g/l) to ensure the product size. Bands were seen
at the expected sizes of 1100 bp. No bands were seen in the negative control,
which contained no DNA.
Ligation
Next, pGL3 basic vector (restriction sites at 5 and 36) and PCR products
(restriction sites in cloning primers) were cut with Kpnl and Bgl II restriction
enzymes. Digested fragments were cleaned with geneclean spin kit (Qbiogene,
Carlsbad, CA). The approximate concentration of digested fragments was
determined by comparing the densities of bands with the density of ladder.
Sticky ends of PAI-1 promoter were ligated into pGL3 basic vector at an insert:
vector ratio of 1:3 using either 2 pl or 4 pl DNA ligase (1 U/pl) in a final volume of
15 pl at room temperature overnight. Top 10 competent E. coli were transformed
with pGL3 vectors to select the plasmid containing the PAI-1 promoter. The
transformed Top 10 competent cells were grown on ampicillin plates overnight.
Representative colonies of bacteria were inoculated into 10 ml of LB media
(Bacto-tryptone 10 g, Bacto-yeast extract 5 g, NaCI 5 g, dH20 1 L, pH 7.0)
containing 125 pg/ml ampicillin and incubated overnight at 37°C with vigorous
shaking. The tubes were incubated on ice for 30 minutes. Bacteria were
harvested by centrifugation at 1540 G for 15 min at 4° C. The pellet was washed
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in 1 ml ice-cold STE buffer (100 mM NaCI, 10 mM Tris, 1 mM EDTA, pH 8.0)
followed by centrifugation at 281 Oxg for 15 min at 4°C. The pellet was
resuspended in 300 pl of Buffer P1 (50 mM Tris, 10 mM EDTA, pH 8.0). Next,
300 pl of Buffer P2 (200 mM NaOH, 1 % SDS) was added with gentle mixing.
Finally, 300 pl Buffer P3 (2.55 M Potassium Acetate adjusted to pH to 4.8 with
acetic acid) was added with gentle mixing. The tubes were centrifuged at
15,000xg for 15 min at 4° C. The supernatant was transferred to a fresh, sterile
microfuge tube. Plasmid DNA was precipitated by incubation with 800 pl of 100%
isopropanol at room temperature for 30 minutes. The tubes were centrifuged at
16,000 G for 10 min at 20° C. The pellet was washed twice with 70% ethanol
followed by centrifugation at 16,000xg for 10 min at 20°C. The pellet was dried
under vacuum and resuspended in 100 pl of sterile dH20 containing 1 pl of
RNAse A (1 pg/pl). The plasmid concentration was measured by measuring
260/280 absorbance with a UV spectrophotometer.
A restriction enzyme digest of the isolated plasmid with Kpnl and Bglll was
set up to ensure that the isolated plasmid contained the desired PAI-1 promoter.
The digested products were run on the gel. The preparation that showed a band
corresponding to expected size of PAI-1 promoter (1106 bp) was presumed to be
the successful cloning of PAI-1 promoter. Sequencing of the insert in this plasmid
confirmed successful cloning of 1100 bp human PAI-1 promoter in pGL3 basic
vector.
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Transient Transfection
Human and rat mesangial cells were co-transfected with 1 jjg pGL3 vector
containing PAI-1 promoter and 1 pg renilla luciferase vector per well (control for
transfection efficiency). Control cells were co-transfected with empty pGL3 basic
vector and renilla luciferase vectors. Fugene 6 (Roche, Indianapolis, IN) was
used to transfect cells in 6-well pates with PAI-1 promoter. Cells were transfected
at 60-80% confluence with 1 pg PAI-1 promoter-containing plasmid and 1 pg
renilla luciferase plasmid. The cells were serum-starved at the time of
transfection. First, 4 pl Fugene 6 per pg DNA was incubated with 100 pl serum-
free media/well for 15 minutes. Next, 2 pg DNA/well was added to the tube. The
resulting transfection mixture was incubated at room temperature for 30 min.
Meanwhile, the cells were washed and the media was changed to 1 ml fresh
serum-free media. To each well 100 pl Fugene-DNA complexes were added
drop wise. After three hours of transfection, CytB was added to appropriate wells.
After 24 hours, cells were treated with 10 ng/ml TGF(3 for 6 hours.
Luciferase Reporter Assay
Upon completion of the experiments, transfected cells were washed 3
times with ice-cold PBS and 150 pl 1X passive lysis buffer (Promega, Madison,
Wl) was added to each well. Plates were wrapped in Parafilm and frozen at
-80°C for at least 1 hour. Cells were scraped after thawing the plates at room
temperature for 3 min. Lysates were centrifuged at 13,370xg for 1 min at 4°C to
remove cellular debris. After equilibrating to room temperature, 20 pl of
supernatant was added quickly to 100 pl of reporter assay reagent containing
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firefly luciferase substrate. Luciferase activity was measured by using TD 20/20
luminometer (Turner Diagnostics, Sunnyvale, CA) set for dual luciferase
detection mode. The sensitivity of the detection was maintained at
manufacturer’s default level (50.9%) for all the assays. The samples were read
after a delay of 2 seconds to allow luciferase activity to begin. The samples were
read for a total of 15 seconds. Stop n Glo buffer (Promega, Madison, Wl) was
added to stop the firefly luciferase activity and to provide substrate for the renilla
luciferase activity. The activity of renilla luciferase was measured on the
luminometer using the same parameters as firefly luciferase on luminometer. In
rat cells, the values for firefly luciferase activity for each sample were normalized
to the values for renilla luciferase for the same sample. In human cells, the
values for renilla luciferase were not detected above the baseline and hence a
different method of normalization was necessary. For human cells, protein
concentration of each sample was determined by BCA protein assay kit (Pierce,
Rockford, IL). Luciferase activity was normalized for the protein concentration of
each sample to get the activity in counts/mg protein for each sample. For each
treatment, cells were transfected at least in duplicates.
Development of chronic glomerulonephritis in experimental animals
The following protocol was approved by the institutional animal care and
use committee of Eastern Virginia Medical School under the title “Prevention of
glomerulosclerosis by rho-kinase inhibition" (Approval # 02-010).
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49
Production of injury
Repeated Injections of anti-thymocyte serum (ATS) were used to induce
chromic glomerulonephritis in experimental rats. In three experimental groups,
six male Sprague-Dawley rats were injected with 25 mg/kg sheep anti-thymocyte
serum (30 mg/ml) through tail vein injections. Four weekly injections of ATS were
given to the animals. The ATS injections were immediately followed by injection
of 1.2 ml normal sheep serum as a source of complement. The doses of ATS
were adjusted each week according for the weight gain. A control group (Group
1) of six animals was injected with equal volumes of normal saline.
Treatment
Out of the three groups that received ATS-complement, one group did not
receive any treatment and hence served as the injury group (Group 2). The
remaining two of the three ATS-complement groups received 30 mg/kg/day HA-
1077 in drinking water. The concentration of the drug in the drinking water was
determined according to the average weight of the animal and average daily
water consumption. Within the two treatment groups, one group (Group 3) was
treated from one day before the first injection to see if the treatment prevents the
onset of the injury, whereas the other group (Group 4) was treated one week
after the injury to see if the progression of the disease was altered. The animals
were fed normal rat diet and were allowed to drink ad lib. After four weekly
injections, animals were treated and observed for 6 more weeks (total of 4+6=10
weeks) to allow progression of the chronic disease. At the end of the tenth week,
animals were killed and samples were collected for analyses.
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50
Study groups
1: Normal saline injection alone
2: ATS-complement injection
3: ATS-complement+ HA-1077 treatment started 1 day before the first injection
4: ATS-complement+HA-1077 treatment started 1 week after the first injection
Sample collection
Urine
After 24-hour acclimatization in metabolic collection chambers, urine
samples representing next 24-hour urine collection were collected one day
before the first injection and at the end of 2, 4, 6, 8, 10 weeks. The first two
collections showed contamination by food particles and hence, the food was
removed from the chambers for the later set of collections.
Blood pressures
The tail-cuff method was used to measure blood pressure at the end of
the study.
Serum
At the end of the study animals were anesthetized with intraperitoneal
injection of sodium pentobarbital (Nembutal). Blood was collected from
abdominal aorta for serum preparation.
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51
Tissue samples
Kidneys were harvested in ice-cold Hanks’s balanced salt solution.
Animals were killed by removal of the heart. One kidney from each animal was
pooled with others from the same group for isolation of glomeruli through
differential sieving method. Most of the glomeruli (90%) were suspended in Trizol
for isolation of RNA and the remaining glomeruli were suspended in
electrophoresis sample buffer for SDS-PAGE and Western Blot analysis. The
second kidney was bisected and one half was cut into thin slices, some of which
were fixed in paraformaldehyde (4% in PBS) overnight, and then snap frozen for
immunohistochemistry. The remaining slices were fixed in 10% buffered formalin
for paraffin section preparation. The other half was cut into multiple thin slices
and snap frozen in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA)
for immunohistochemistry or laser capture microdissection (LCM).
Histology and Immunohistochemistry
The sections from formalin-fixed tissue were stained with mason trichrome
staining to visualize ECM deposition. The sections from snap frozen tissue were
stained with a-SMA antibody as described in immunocytochemistry section.
Statistical Analysis
The statistical significance of the results was determined using Student’s
paired t-test. A p-value of <0.05 was considered statistically significant.
Experimental variation is reported as standard error of the mean in the bar
graphs (except where indicated differently).
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52
CHAPTER 3
PROFILING THE EFFECTS OF ACTIN CYTOSKELETON ON EXPRESSION
OF MULTIPLE GENES
Introduction
As described in chapter 1, studies in this lab showed that changes
in actin polymerization regulate a-SMA expression and hypertrophy in cultured
mesangial cells suggesting that the actin cytoskeleton may regulate
myofibroblast differentiation [28]. The role of the cytoskeleton as an important
regulator of gene expression has also been supported by many other studies.
Studies by Folkman and Moscona on cell-spreading [33], and Ingber on
mechanical transduction and cell functions [34, 113, 114] provided early
evidence for the role of actin cytoskeleton in the gene regulation. More recently
specialized actin cytoskeleton locations have been identified for integration of
signaling such as JNK signaling [39-42], The importance of localization of mRNA
in effective protein translation has also become evident [43], The collective
evidence suggests that the actin cytoskeleton is a highly specialized network of
fibers that precisely regulate multiple cellular responses. But the precise
regulatory pathways and mechanisms have not been characterized. Also the list
of genes that are affected by actin polymerization is far from complete.
The regulatory effect of actin cytoskeleton on a-SMA expression in
myofibroblast differentiation and the evidence from the literature suggested that
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53
other genes could also be regulated in a similar manner. Also, the inhibition of
hypertrophy suggested that multiple genes could be simultaneously regulated by
the actin cytoskeleton. It was therefore hypothesized that other genes associated
with the myofibroblast phenotype are also regulated by changes in actin
polymerization. The aim of the next experiment was to identify the effect of the
actin cytoskeleton on a panel of genes related to myofibroblast differentiation.
Experimental model
Recently, DNA arrays consisting of multiple genes spotted on a glass
coverslip or a nylon membrane have become available to facilitate rapid and high
throughput analysis of large populations of genes. Commercially available arrays
contain functionally related genes on the same array allowing a highly relevant,
focused, and time- and cost-effective analysis of gene expression.
Since myofibroblasts are largely responsible for extracellular matrix
production in fibrotic diseases, one such array (cell interaction array) containing
genes related to cell-interaction and extracellular matrix was used to study the
role of actin cytoskeleton in myofibroblast differentiation. The Cell Interaction
array (Clontech, Palo Alto, CA) contains a total of 265 cDNAs immobilized on a
nylon membrane. The genes on this Cell-lnteraction array include extracellular
matrix proteins; cell-cell adhesion receptors; matrix adhesion receptors; growth
factors, chemokines, cytokines and their receptors; neuropeptides; cell surface
antigens; extracellular transporters and carrier proteins; metalloproteinases;
serine proteases; protease inhibitors, oncogenes and tumor suppressor genes;
intracellular transducers, effectors and modulators; G proteins; cell-cycle related
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54
proteins and house-keeping genes covering a broad range of relevant gene
population. A detailed list of these genes can be found on the internet at
http://www.clontech.com/clontech/atlas/genelists/7746-1_HuCelllnt.pdf. The gene
expression profile of cells with depolymerized actin cytoskeleton (CytB treated
cells) was compared with that of cells containing intact actin cytoskeleton
(untreated serum-starved cells) as described in Chapter 2.
Results
Actin depolvmerization affects expression of multiple genes
Between control and CytB-treated cells a total of 74 genes were detected
on a 265-gene cell-interaction array, out of which 65 genes were detected in both
the conditions and 9 genes were detected under only control conditions (Fig. 6).
Using an arbitrarily defined criteria of either more than a 2-fold increase or more
than 50% inhibition as a significant change, 10 genes were decreased and 4
genes were increased upon CytB treatment. All 9 genes expressed only under
control conditions were excluded from the final analysis since their expression
was very close to the base-line. Table 3 shows the ratio of signal intensities in
CytB treated cells and untreated control cells in serum-free media. The genes
that decreased after CytB treatment, mainly represented extracellular matrix
components or other molecules that favor increased matrix deposition. In
contrast, 3 of the 4 genes that increased after CytB treatment, decrease matrix
deposition either by direct fibrolysis (tPA, MMP11) or by inhibiting pro-fibrotic
growth factors (decorin). These changes suggest that the overall effect of actin
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55
I. Serum-free control
lHouse
keepingGenes"
Negative; control
Figure Key
A. UBCB. KCEP1 C H P R T iD. G A PD HE. T U B A !F. H LA CG. AC TBH. RPL13AI. RPS9
Fig. 6. DNA array analysis of effect of actin depolymerization on gene expression. Serum-starved human mesangial cells were treated with 1.0 pM CytB for 24 hours in serum-free media. 32P-labeled gene-specific probes prepared from cellular poly A mRNA were hybridized with the membrane overnight. After washing, membranes were exposed to phosphorimager plates for 3 days. Scanned blots were analyzed by Atlaslmage software for differences in gene expression (n=1). The top blot represents gene expression profile of cells in serum-free media, whereas the bottom panel represents gene expression profile of cells treated with 1.0 pM CytB. The panel on left shows house-keeping genes and negative control spots. The spots corresponding to PAI-1 (oval) and tPA (rectangle) genes are highlighted for comparison on both the blots.
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Table 3. Summary of changes in gene expression on DNA array analysis
Gene CytB:S- ratio
A. Genes showing decreased expression after Cytochalasin B treatment
Endothelial plasminogen activator inhibitor-1 precursor (PAI-1) 0.093
Insulin-like growth factor binding protein 5 precursor (IGFBP5) 0.210
Fibronectin receptor alpha subunit (FNRA) 0.310
Insulin-like growth factor binding protein 3 precursor (IGFBP3) 0.314
Tumor metastatic process associated protein (NM23) 0.326
Fibronectin precursor (FN) 0.339
Hypoxanthine-guanine phophoribosyltransferase (HPRT) 0.370
Collagen 3 alpha 1 subunit precursor (COL3A1) 0.382
Collagen 1 alpha 2 subunit precursor (COL1A2) 0.393
Collagen 4 alpha 2 subunit precursor (COL4A2) 0.397
B. Genes showing increased expression after Cytochalasin B treatment
Tissue-type plasminogen activator precursor (tPA) 2.840
Bone proteoglycan II precursor (PGS2), decorin (DCN) 3.250
Junction plakoglobin (JUP); desmoplakin 3 (DP3) 3.875
Matrix metalloproteinase 11 (MMP11), stromelysin 3 9.000
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57
depolymerization favors increased matrix degradation and decreased matrix
accumulation. The net effect may be a decrease in extracellular matrix.
Confirmation of DNA array results using Northern and Western Blotting
The results obtained by the initial screening experiments required both
confirmation and more accurate quantification of the changes using conventional
methods such as Northern blot analysis. The changes in protein expression were
also examined to confirm similar effects on mRNA and protein expression. Out of
14 genes identified by the array experiment, PAI-1 and tPA, the members of
plasminogen activator system, are particularly relevant to the overall objective of
understanding mechanisms of myofibroblast-like differentiation and regulation of
ECM accumulation. PAI-1 is a natural inhibitor of the plasminogen activation by
tPA. CytB not only decreases PAI-1 expression, but also increases tPA
expression. These changes may result in increased and unopposed actions of
tPA leading to more activation of plasminogen into plasmin. Activated plasmin in
turn degrades ECM. Consequently, the balance between tPA and PAI-1
determines the rate of ECM turnover. A relative increase in tPA leads to
increased degradation of ECM. In contrast, a relative increase in PAI-1 leads to
inhibition of ECM degradation and increased ECM deposition as seen in renal
fibrosis. Such an increase in PAI-1 expression and resulting ECM deposition are
associated with myofibroblast-like activation of mesangial cells. Thus, changes in
tPA and PAI-1 expression on gene array are directly pertinent to myofibroblast
differentiation and fibrosis.
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Therefore, the changes in PAI-1 and tPA expression seen on DNA array
were confirmed first using conventional methods such as Northern blot and
Western blot analyses. mRNAs from cells treated with actin depolymerizing
agents CytB (direct inhibitor of actin polymerization) and Y-27632 (inhibitor of
signal-mediated actin polymerization) were analyzed. The Northern blot analysis
showed two PAI-1 transcripts at 2.8 kb and 3.2 kb positions resulting from two
polyadenylation sites in the PAI-1 gene [115]. The results showed that in
presence of serum (S+ conditions), inhibition of actin polymerization with CytB
decreased PAI-1 mRNA expression in a time and dose dependent manner (Fig.
7). The effect of CytB was apparent at 24 hours and continued at 48 and 72
hours (Fig. 7A). The dose response shows that the minimum dose of CytB
required to produce a visible effect on PAI-1 mRNA was 0.4 pM at 24 hours.
CytB caused >50% inhibition of PAI-1 mRNA expression at 1 pM concentration
(Fig. 7B). We used 1 pM (0.5 pg/ml) CytB for the rest of the study. In serum-
starved cells, inhibition of Rho-signaling by 10 pM Y-27632 also inhibited PAI-1
mRNA expression in a time-dependent manner (Fig. 7C). The effect was evident
as early as 4 hours and continued at 8, 24 and 48 hours. Similar inhibitory effects
of actin depolymerization on PAI-1 protein expression were confirmed in the
laboratory by other investigators. An increase in tPA expression by actin
depolymerization as shown in the array experiment was also confirmed at mRNA
and protein levels by Northern blot and Western blot analyses respectively in the
lab.
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59
A
PAi-
B
PAI-
C
PAI-
Fig. 7. Effect of actin depolymerization on PAI-1 mRNA expression in human mesangial cells. (A) Time-course of effect of 1.0 pM CytB (8, 24, 48 and 72 hours) on PAI-1 mRNA expression in serum-fed cells by Northern blot analysis (n=1). CytB treatment blocks PAI-1 expression at 24, 48 and 72 hours, (B) Dose-response analysis of effect of CytB (0.1, 0.2, 0.4, 1 and 2 pM) on PAI-1 mRNA expression in serum-fed cells by Northern blot analysis (n=1). CytB concentrations of 0.4, 1 and 2 pM block PAI-1 mRNA expression. (C) Time- course of effect of 10 pM Y-27632 (8, 24, 48 and 72 hours) on PAI-1 mRNA expression in serum-starved cells by Northern blot analysis (n=1). Y-27632, an inhibitor of Rho-kinase that blocks Rho-mediated actin polymerization, blocks PAI-1 mRNA expression at 24, 48 and 72 hours.
Time (hi 0_ __8_ Cyt B
24 48 72
3.2 kb
2.8 kb
Cyt B (jxM) 0 0.1 0.2 0.4 1 2
1 C3.2 kb
2.8 kb - mm llil
Time (h) 0_ 8Y-27632 - - +
3.2 kb
2.8 kb
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60
In summary, the DNA array analysis showed that actin depolymerization
coordinately regulated cell-interaction and ECM-related genes in human
mesangial cells. The inhibition of PAI-1 and increase in tPA expression were
confirmed by Northern blot and Western blot analyses. To further investigate the
mechanisms of actin cytoskeleton mediated regulation of PAI-1 and tPA
expression in pathophysiological context, TGF|3 treatment was used as a model
system as described in the next chapter.
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61
CHAPTER 4
EFFECT OF TGFp ON PAI-1 EXPRESSION
Introduction
Results of the array experiment and subsequent confirmation with Northern
and Western blot analysis showed that actin depolymerization changed PAI-1
and tPA in opposite directions. PAI-1 was studied first and in more detail than
tPA because PAI-1 expression has been correlated with myofibroblast
differentiation [19, 25], Moreover, PAI-1 acts as the gatekeeper of the fibrolytic
cascade regulating the activity of plasminogen activators including tPA.
Therefore, functionally changes in PAI-1 expression are more directly related to
tissue fibrosis.
Since transforming growth factor (3 (TGFP) is the major regulator of PAI-1
expression in disease, the ability of actin depolymerization to antagonize its
effect on PAI-1 expression was examined. Also, TGFp was chosen for further
studies particularly since it plays a central role in regulating myofibroblast
differentiation and tissue fibrogenesis [116-120]. In the kidney, expression of
TGFp by mesangial cells increases extracellular matrix (ECM) production and
inhibits matrix degradation [88, 89, 91, 121]. The net result is an increase in
ECM deposition and glomerulosclerosis [90, 122]. Consequently, inhibition of
TGFp signaling was shown to protect against renal fibrosis [88, 123]. It was
therefore important to understand the pathways and mechanisms involved in
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62
TGFP-induced fibrosis. Experimentally, serum-free culture conditions were
selected to allow the study of TGFp without other serum-factors.
The overall experimental approach was to induce PAI-1 expression by TGFp
and to determine the effects of changes in actin polymerization on PAI-1
expression in basal as well as TGFp-induced states. Since the effects of growth
factors can vary according to cell-type and cell-culture conditions; the effective
dose, effective time period and the magnitude of effect of TGFP on PAI-1
expression in human mesangial cells were determined first.
Experimental model
Primary cultures of human mesangial cells between passages 5 and 9 (P5-
P9) were used in the study. Initially, the cells were cultured in RPMI media
containing 16.7% fetal bovine serum (FBS). Cells were serum-starved at 60-80%
confluence. After 24 hours of serum starvation, TGFp was added to the cells.
The effects on PAI-1 expression were examined at the indicated time-points.
Serum-free media, being free of other growth factors, allows the study of effects
of TGFp alone. Also, serum starvation increases the stress fiber content of the
cells which is consistent with myofibroblast-like phenotype [26]. The effects of
inhibition of actin polymerization can be easily compared to untreated control
cells that have abundant stress fibers. A culture in serum-free media helps in
studying the regulation of basal expression of PAI-1, where as treatment with
TGFp allows the study of induced PAI-1 expression.
To determine the optimum dose of TGFp and the optimum duration of
treatment, human mesangial cells were treated with increasing doses of TGFp
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63
for 0, 1, 2, 4, 8, 24 and 48 hours. At each time point, supernatants (conditioned
media) were stored for detection of PAI-1 protein and the cells were lysed in
TRIzol for RNA isolation. PAI-1 protein was detected from conditioned media
using Western blot analysis with enhanced chemiluminescence detection,
whereas PAI-1 mRNA was detected using quantitative real-time RT-PCR.
Results
Quantification of PAI-1 mRNA using real-time PCR analysis
PAI-1 mRNA from cultured human mesangial cells was successfully
detected by a reverse transcription reaction in a thermal cycler (MJ Research,
Reno, NV) followed by a quantitative real-time PCR analysis on Roche
Lightcycler (Roche Diagnostics, Indianapolis, IN). The Lightcycler instrument
measures fluorescence emitted by SYBR Green I, a double-stranded DNA
binding dye, at the end of each cycle during the log-linear phase of the PCR and
generates an amplification curve by plotting changes in fluorescence intensities
against cycle number (Fig. 8A, Fig. 9A). The cycle at which the fluorescence
signal rises above the background signal is called the crossing point, which is
directly proportional to the concentration of the target DNA. The crossing point for
an unknown sample can be compared to crossing points of known standards for
quantification of sample concentration. The values for PAI-1 concentration were
derived in picograms, which were further normalized to similarly obtained values
for a reference gene, ubiquitin (UBC), in each sample. Ubiquitin was selected as
the reference or house-keeping gene because it is expressed constitutively in the
cells and its level did not change upon the experimental treatments (refer to table
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64
Standard CurveAmplification Curve
60-r = -1.00
Slope = -3.77cDNA template0.001 pg -------------
0.01 pg -------------0.1 pg -------------
1 pg ------------ :10 pg — y15-
15 20 25 30 35Cycle Number Log Concentration
Melting PeakMelting Curve
Temperature (°C)Temperature (°C)
Fig. 8. PAI-1 standard curve for quantitative real-time PCR analysis. (A)Amplification of PAI-1 plotted as fluorescence vs. cycle number. Each sample is represented in a uniquely colored line on the real-time PCR software, allowing tracing of amplification of individual samples. (B) PAI-1 standard curve plotted as cycle number vs. log concentration showing a linear standard curve (r= -1). (C) Real-time monitoring of melting curve analysis of PCR products plotted as fluorescence vs. cycle number showing only one PCR product corresponding to PAI-1. (D) Melting curve analysis data plotted as changes in fluorescence signal overtime (dF/dT) vs. temperature. Presence of only one melting peak in this reaction corresponding to melting temperatures of PAI-1 (-85.5 C) indicates specificity of the amplification reaction and absence of primer-dimers.
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65
4). Also it is expressed at relatively lower levels, which is a desired property for
real-time PCR analysis allowing detection of smaller changes in the expression
unlike genes that are too abundant in the cells such as 18S.
Standard curves for PAI-1 and UBC were developed by performing real
time PCR on known concentrations of purified cDNA (Fig. 8, Fig. 9). For every
experiment, one purified standard cDNA (0.01 pg for PAI-1 and 100 pg for UBC)
with similar crossing point as a representative experimental sample was also run
with the samples. The sample concentrations were calculated by the software in
a two-step process. First, the software compared the crossing point of the known
standard in each experiment with the previously saved standard curve to account
for the PCR efficiency. Next, the concentrations of the unknown samples were
derived by comparing the sample crossing point to the standard crossing point.
Following completion of the PCR, a melting curve analysis of PCR
products was performed by the instrument to confirm the purity of the reaction.
Melting curve analysis measures changes in fluorescence intensities as the
temperature is gradually increased. Separation of double stranded DNA with
increasing temperature caused an initial gradual fall in the fluorescence signal
followed by a precipitous fall (Fig. 8C, Fig. 9C). When these changes in
fluorescence intensity with time were plotted against the temperature, a melting
peak corresponding to the maximum changes in signal intensity (dF/dT) at
melting temperature (Tm) was seen (Fig. 8D, Fig. 9D). The number of melting
peaks in each reaction indicates the number of target sequences being amplified.
A unique peak at the melting temperature of the target sequence indicated
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66
Fluo
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Amplification Curve A
cDNA template0 1 pg ,
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“V
Ip g ----------------------------------►10 pg ------------------------- /■ ’ • - *■
50 pg ---------------------- -----►100 pg -----------------~ 7~ + ’
0 4 8 12 16 20 24 28 32 36
Cycle Number
0 -0.4 0.2 0.8 1.4 2.0 2.6
Log Concentration
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- ” ■■ ■ — " | V —U I I I I I I
66 70 74 78 82 86 Temperature (°C)
1 1 1 1 1 68 72 76 80 84
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Fig. 9. UBC standard curve for quantitative real-time PCR analysis. (A)Amplification of UBC plotted as fluorescence vs. cycle number. Each sample is represented in a uniquely colored line on the real-time PCR software, allowing tracing of amplification of individual samples. (B) UBC standard curve plotted as cycle number vs. log concentration showing a linear standard curve (r= -1). (C) Real-time monitoring of melting curve analysis of PCR products plotted as fluorescence vs. cycle number showing only one PCR product corresponding to UBC. (D) Melting curve analysis data plotted as changes in fluorescence signal over time (dF/dT) vs. temperature. Presence of only one melting peak in this reaction corresponding to melting temperatures of UBC (~82°C) indicates specificity of the amplification reaction and absence of primer-dimers.
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67
absence of non-specific amplification and primer-dimers in the reaction. As an
added quality control measure to ensure the size of the product and to rule out
non-specific amplification, the PCR products from standard curve samples and
representative experimental samples were run on 8% agarose gel (Fig. 10, Fig.
11D). Gel electrophoresis was used as a quality control tool only to visualize the
number and size of products in each reaction. It was not used for quantification of
mRNA.
Figures 8 and 9 show standard curves for real-time PCR analysis of PAI-1
and UBC respectively. Figure 10 shows gel electrophoresis of PCR products
from PAI-1 and UBC PCR standard curves.
TGFB dose-response on PAI-1 mRNA expression
The effect of TGFp on PAI-1 mRNA were studied using real-time
quantitative PCR analysis with lightcycler PCR instrument. Figure 11 shows a
representative real-time monitoring of PAI-1 and UBC amplification at the end of
each cycle in panel A. Panels B and C represent melting curve analysis of PAI-1
and UBC at the end of PCR cycling showing a single peak each for both the
genes. Panel D show gel electrophoresis of PCR products showing a single
product for each gene correlating with the expected band size.
To determine the effective dose of TGFp, human mesangial cells were
serum-starved at 60-80% confluence. After a 24-hour serum-starvation, 1, 2, 5,
7.5 or 10 ng/ml TGFp was added to serum-free media for 8 hours. Total cellular
RNA was isolated using TRIzol method. The changes in PAI-1 mRNA expression
were detected using quantitative real-time RT-PCR analysis. In two separate
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B UBC
UBC (133 bp)4
concentration ('pg)
Fig. 10. Gel-electrophoresis of standard curve PCR products. Aliquots of PCR products (2 pi out of 20 pi total) were run on 8% acrylamide gel followed by ethidium bromide staining. (A) PAI-1 and (B) UBC PCR products show a single band at expected base-pair location confirming specific amplification of respective templates.
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69
Amplification Curve M elting Curve80-70-60-50-40-30-20-
10-
6 0 -5 0 -4 0 - Samples and
standards3 0 -UBC PAI-120 -
10-Crossing points
o-
Temperature (°C)Cycle Number
M elting Peak
u b c A i
Gel Electrophoresis20-
PAI-1
P A I-1 I B(Temperature (°C)
Fig. 11. Representative images from quantitative real-time PCR of PAI-1 and UBC from experimental samples. (A) Amplification of PAI-1 and UBC plotted as fluorescence vs. cycle number. Each line is represented in different color on the real-time PCR software, allowing tracing of amplification of individual samples. The crossing points for both PAI-1 and UBC fall within the bars marking approximately cycles 15 and 22. (B) Real-time monitoring of melting curve analysis of PCR products plotted as fluorescence vs. cycle number showing two PCR products corresponding to PAI-1 and UBC. (C) Melting curve analysis data plotted as changes in fluorescence signal over time (dF/dT) vs. temperature. Presence of only two melting peaks in this reaction corresponding to melting temperatures of UBC (~82°C) and PAI-1 (~85.5°C) indicate specificity of the amplification reaction and absence of primer-dimers. (D) Gel electrophoresis of PCR products from representative experimental samples show a single PCR product of expected base-pair size in each reaction.
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experiments, treatment with 1, 2, 5, 7.5 and 10 ng/ml TGFp increased average
PAI-1 mRNA expression to 381.9%, 323.1%, 443.3%, 485.1% and 287.9% of
untreated control respectively (Fig. 12A).
TGFB dose-response on PAI-1 protein expression
To determine the effective dose of TGFp on PAI-1 protein expression,
human mesangial cells were serum-starved at 60-80% confluence in 2-wells per
condition in 6-well plates. After 24-hour serum-starvation, 1, 2, 5, 7.5 or 10 ng/ml
TGFP was added to serum-free media for 24 hours. Conditioned media was
analyzed for changes in PAI-1 protein using Western blot analysis. In a single
experiment, treatment with 1, 2, 5, 7.5 and 10 ng/ml TGFp changed average PAI-
1 protein expression to 125.9%, 100.9%, 79.3%, 133.6% and 283.6% of
untreated control respectively (Fig. 12B). The maximum increase in PAI-1 protein
was seen with 10 ng/ml TGFp. Although TGFp showed an effect at lower
concentrations, it was inconsistent. Fan et al. previously showed that renal
tubular epithelial-myofibroblast transdifferentiation in vitro required TGFp at
concentrations between 10 to 50 ng/ml [119]. Others have also shown 10 ng/ml
TGFp as an effective dose [124, 125], The effectiveness of higher concentration
of TGFp correlates with high serum TGFp levels seen in patients with chronic
and end-stage renal disease [126].
Since 10 ng/ml TGFp showed consistent effects on both mRNA and
protein expression, that dose was used in the remaining experiments.
Previously, Motojima et al. also reported 10 ng/ml TGFp to be the most effective
dose for the induction of PAI-1 in rat mesangial cells [82],
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71
TGFB time-course of PAI-1 mRNA and protein expression
To determine the optimum length of time of TGF|3-treatment for maximum
PAI-1 mRNA and protein expression, human mesangial cells were serum-starved
at 60-80% confluence and after 24-hours of serum starvation, 10 ng/ml TGF(3
was added to serum-free media for 1,2,4, 8, 24 and 48 hours. At each time
point, conditioned media were saved for protein analysis and cells were used for
RNA analysis. In three separate experiments, treatment with 10 ng/ml TGFp for
1, 2, 4, 8, 24 and 48 hours changed PAI-1 mRNA expression to 180.4±22.8%,
186.0±30.7%, 191.0±23.5%, 100.1 ±13.5% and 90.6±8.7% of respective time-
matched controls (Fig. 13A). At the same time, the basal expression of PAI-1
mRNA at 2 and 4 hours decreased to 90.9±10.9% and 59.7±9.4% (p<0.05) of 0-
time point control. At 8, 24 and 48 hours, basal PAI-1 mRNA expression changed
to 105.0± 7.5%, 134.7±14.7% and 146.2% of 0-time point control.
In the same set of experiments, the effect on PAI-1 protein expression was
examined using Western blot analysis at 8, 24 and 48 hours in conditioned
media. PAI-1 expression could not be detected at 8 hours. At 24 hours TGFp
increased PAI-1 protein to 424 ± 104.6% of time-matched control (Figre 13B). At
48 hours, the increase in PAI-1 protein was 181.9% and 131.7% in two separate
experiments (average 158%). Thus, 10 ng/ml TGFp increased PAI-1 mRNA
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PAI-1 Protein B
TGFp (ng/ml) 0 7.5 10
Fig. 12. Dose-reponse analysis of TGFp induction of PAI-1 expression.Serum-starved human mesangial cells were treated with 0, 1, 2, 5, 7.5 and 10 ng/ml TGFp for 8 (mRNA) or 48 (protein) hours (A) PAI-1 mRNA expression measured by quantitative real-time PCR analysis shows an increase with TGFp treatment. Each bar represents the average increase at corresponding concentration normalized to untreated control (n=2). The ends of error bars show value for each experiment. (B) PAI-1 protein expression was measured in conditioned media using Western blot analysis with chemiluminiscence detection. Treatment with 10 ng/ml TGFp increased PAI-1 protein expression to 283.6% of untreated control (n=1).
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73
expression maximally and consistently at 8 hours, whereas it increased PAI-1
protein expression maximally at 24 hours. The maximum increase in PAI-1
protein expression at 24 hours is consistent with previous studies by Matsumoto
et al [127].
In summary, the results of time-course and dose-response analysis of
TGFP on PAI-1 expression showed 10 ng/ml TGFp as the effective dose and, 8-
hour and 24-hour as effective treatment duration for mRNA and protein studies
respectively. The remaining studies were performed using these time points.
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74
PAI-1 mRNA
TGFp- - + - + - + - + - +
Time (h) 0 2 4 8 24 48
600 n'o0)JS 500 - ejt3 'o 400 " S ui t i 3 0 0 -MS S 200 -VsO 100 -0s
PAI-1 Protein *
B
TGFp — + +
Time (h) Matchedcontrol
24 48
Fig. 13. Time-course analysis of TGFp induction of PAI-1 expression.Serum-starved mesangial cells were treated with or without 10 ng/ml TGFp for indicated lengths of time. (A) Changes in PAI-1 mRNA were detected using quantitative real-time PCR analysis (n=3 except 48 hours: n=2). (B) Changes in PAI-1 protein expression were measured in conditioned media using Western blot analysis with chemiluminescence detection at 24 (n=3) and 48 (n=2) hours. The vertical bars and the error bars represent the mean PAI-1 values and SEM respectively. The value of either 0-hour (A) or time-matched (B) untreated control is set at 100%. *:p<0.05 compared to time-matched control, f : p<0.05 compared to 0-hr time-point.
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75
CHAPTER 5
EFFECT OF CHANGES IN ACTIN POLYMERIZATION ON TGFp-INDUCED
PAI-1 EXPRESSION
Introduction
The results of earlier chapter confirmed induction of PAI-1 by TGFp in
human mesangial cells. The role of actin cytoskeleton in this induction was
examined next. Previous studies in this lab showed that changes in actin
cytoskeleton regulate a-SMA gene expression, a marker of myofibroblast
differentiation, and hypertrophy in mesangial cells [28]. Like a-SMA expression,
de novo expression of PAI-1 is also seen with myofibroblast differentiation. It is
possible that the expression of PAI-1 is also regulated in a cytoskeleton-
dependent manner similar to that of a-SMA.
The Rho family of small GTPases controls organization of the actin
cytoskeleton [27], Rho activates Rho-kinase, a serine-threonine kinase that acts
through downstream kinases to stabilize actin polymers e.g. stress fibers and
adhesion. The small GTPases, Rho and Rac are both involved in the regulation
of PAI-1 expression. Inhibition of Rho by the C3 exoenzyme decreased PAI-1
production in rat proximal tubules [105] and aortic endothelial cells [106]. In
chicken atrial cells, co-transfection with dominant-negative RhoA decreased PAI-
1 promoter activity, whereas dominant-active RhoA increased promoter activity
[107], In smooth muscle cells, Y-27632, a Rho-kinase inhibitor, attenuated
expression of PAI-1 in response to Angiotensin II (Angll) [81]. These suggest a
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76
role of Rho GTPases as intracellular mediators of diverse stimuli leading to PAI-1
expression. Rho has also been shown to regulate TGFp-mediated gene
expression. In human mesangial cells, inhibition of Rho and its downstream
effector Rho-kinase blocked TGFp-mediated expression of a1 collagen, a
component of ECM [87]. It is possible that Rho may play a similar role in
regulating TGFp-mediated expression of PAI-1.
Since regulation of actin polymerization is a major function of the Rho
family, the working hypothesis that Rho-mediated changes in actin
polymerization modulate TGFp-induced PAI-1 expression was examined. If Rho-
signaling worked through actin cytoskeleton for regulation of PAI-1 expression,
inhibition of actin cytoskeleton would produce similar results as the inhibition of
Rho.
Experimental model
Pharmacologic agents that stabilize or destabilize the polymeric actin
cytoskeleton by acting at different levels (Fig. 4) were used to test the hypothesis
that Rho GTPase regulates TGFp-induced PAI-1 expression through changes in
actin polymerization. The treatment conditions included (a) inhibition of signal-
mediated actin polymerization by inhibiting Rho GTPase (Clostridium difficile
Toxin B, 10 pM) or its downstream effector Rho-kinase (HA-1077 (20 pM), Y-
27632 (10 pM)) [87, 128, 129]; (b) inhibition of actin polymerization by agents
directly binding to actin polymers (Cytochalasin B, 1 mM) or monomers
(Latrunculin B, 0.1 pM) [130, 131]; and (c) increase in polymeric actin content by
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77
stabilizing the actin polymers (Jasplakinolide, 50 nM) [132], The final outcome
was either an increase or a decrease in the actin polymers in the cells.
Results
Actin depolvmerization inhibits TGFB-induced PAI-1 mRNA expression
First, the effect of actin depolymerizing agents on TGFp-induced PAI-1
mRNA expression was examined (Fig. 14). Either Toxin B (Fig. 14A), a direct
inhibitor of Rho, or Y-27632 (Fig. 14B) and HA-1077 (Fig. 14B), both inhibitors of
a downstream effector of Rho, Rho kinase (ROCK), were used to inhibit Rho-
mediated actin polymerization. ToxinB, Y-27632 and HA-1077 decreased basal
PAI-1 mRNA expression to 19.5±5.6%, 52.8±5.4%, 59.3±17.9% of untreated
control respectively, and decreased TGFp-induced PAI-1 expression to
30.1±7.6%, 51.1 ±6% and 55.8±15.6% of TGFp-treated control respectively (Fig.
14A-C). Direct inhibition of the actin polymerization by CytB and LatB decreased
basal PAI-1 expression to 29.9 ±3.3% and 45.3±16% of untreated control
respectively, and decreased TGFp-induced PAI-1 expression to 35.2±6.5% and
49.7±16.5 of TGFp-treated control respectively (Fig. 14D, E). Northern blot
analysis (Fig. 14G) confirmed these results. This observation, that the effects of
Rho-inhibition are mimicked by direct actin depolymerization, suggests that the
effects of Rho on PAI-1 expression are mediated by the changes in actin
cytoskeleton.
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78
PAI-1 mRNA
Serum-free media TGFP350 -
* A 300 - sfcsfcsfc B 300
"3 300 - "E 250 ■ r "3 250Im-ws 250 - ImMMs 200 - + a 200© 200 '
4-ou 150 ■ t t t
ou 150
o '150 ‘
T N®O' 100 - —f-| N°100
1UU - T50 - * * *
i—*—i
1 50 - n 50
— + + 0 — — + +Toxin B
300
"E 250 -ImMMa 200 -ou 150 -N?O' 100 ■
50 -
0
iD 300
T tti
^250-§ 2 0 0OU 150
— — + CytB
100
50 -
0
Y 27632
* * E
t
— — + LatB
400 1_ 350 O
300
S 230 oU 200
o ' 150 100
50 -
0
* *
Hhc
*
trh
p h
— — + +H A -1077
* *
+
F f t f
- f -
— — + JAS
p a h -
18S
* * -TGFP — + — + — + — +
S- CylB LalB Y-27632
Fig. 14. Effect of actin polymerization on TGFp-induced PAI-1 mRNA expression. Serum-starved mesangial cells were treated with (A) 10 pM Toxin B, (B) 10 pM Y-27632, (C) 20 pM HA-1077, (D) 1 mM CytB, (E) 0.1 pM LatB or (F) 50 nM JAS for 1-hour prior to 8-hour treatment with 10 ng/ml TGFp (n=3). The changes in PAI-1 mRNA were measured using real-time PCR analysis (A-F) and Northern blot analysis (G). The vertical bar and the error bars represent the mean PAI-1 mRNA values and SEM for the experimental condition. The value for untreated control is set at 100%. The value for untreated control is set at 100%. *:p<0.05, **:p<0.01 and ***:p<0.005 compared to S- control, t : P<0.05 and t t t : p<0.005 compared to TGFP treated control. (G) Representative Northern blot showing inhibition of basal and TGFp-induced PAI-1 expression with actin depolymerization (n=2).
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79
Actin depolvmerization inhibits TGFB-induced PAI-1 protein expression
To ensure that the changes in PAI-1 mRNA expression were reflected at
the level of protein expression, the effect of the actin-depolymerization on PAI-1
protein expression was examined using Western blotting.
The effect on cellular PAI-1 protein was examined first (Fig. 15A). Cell-
lysates scraped in 1x sample buffer were used to detect changes in PAI-1
protein. Serum-starved mesangial cells were pre-treated with CytB for one hour
before 24 or 48 hour treatments with TGFp. TGFp had no apparent effect on
PAI-1 expression at 24 or 48 hours. This is possibly due to saturation of PAI-1
binding sites (vitronectin) on the substratum during prior culture [133], CytB
blocked PAI-1 expression in both basal and TGFp-treated conditions. At 24
hours, CytB blocked basal and TGFp-treated PAI-1 expression to 14.4% and
38.8% of their respective controls. At 48 hours, CytB blocked basal and TGFP-
treated PAI-1 expression to 4.2% and 46.1% of their respective controls.
Active PAI-1 is rapidly secreted from the cells upon synthesis [25].
Therefore, the conditioned media was examined for changes in PAI-1 protein
expression in response to TGFp. In the same experiment, changes in PAI-1
levels were clearly apparent in conditioned media (Fig. 15B). At 24 hours, TGFp
increased PAI-1 expression to 273% of control. CytB blocked basal and TGFp-
induced PAI-1 expression to 7.9% and 20.7% of their respective controls. At 48
hours, TGFp increased PAI-1 expression to 217% of control. CytB blocked basal
and TGFp-induced PAI-1 expression to undetectable levels.
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80
Cell-Lysates
PAI-1
Conditioned Media
PAI-1
m m m m>» *o o
Fig. 15. Effect of TGFp and actin depolymerization on PAI-1 protein expression. Serum starved mesangial cells were pre-treated with 1 mM CytB prior to 24-hour treatment with 10 ng/ml TGFp. Cell-lysates (A) and Conditioned media (B) were analyzed for PAI-1 detection using Western blot analysis (n=1). An increase in PAI-1 upon TGFp treatment is seen in conditioned media (B) but not in cell lysates (A). CytB blocks PAI-1 expression, under basal and TGFp- treated conditions, in both cell lysates and conditioned media.
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81
Next, the effect of a panel of actin cytoskeleton inhibitors on TGFp-
induced PAI-1 expression was examined using conditioned media (Fig. 16). The
changes in PAI-1 protein expression were consistent with the changes seen in
mRNA. Densitometric analysis of the Western blots showed that TGFp maximally
stimulates PAI-1 expression to 304.3±31.4% of control at 24 hours. Toxin B, Y-
27632, FIA-1077, CytB and LatB inhibited basal PAI-1 expression to 31.7±4.4%,
42.2±7.8%, 31±3.7%, 37.3±5.6% and 44.4±7.8% of control respectively, and
inhibited TGFP-induced PAI-1 expression to 26.8±7%, 38.6±4.9%, 33.6±6.9%,
21.1 ±4.1 % and 31±8.3% of TGFp-treated control respectively. A representative
Western blot is shown in Fig. 16A. These results suggest that agents with actin
depolymerizing activity blocks TGFp-induced PAI-1 expression regardless of
their mechanism of action.
Immunocvtochemical analysis of effect of actin depolvmerization on
TGFB-induced PAI-1 expression
In culture, secreted PAI-1 binds to vitronectin on the pericellular
substratum and shows characteristic intracellular and extracellular distribution
[134], Indirect immunofluorescence microscopy was used to visualize the effects
of Y-27632 and CytB on PAI-1 deposition on the substratum (Fig. 17). These
cells were plated and stained at relatively low density. Thus the substratum is not
saturated with PAI-1 and it is possible to visualize the effect of TGFp on PAI-1
protein expression.
Cells were counterstained with Texas Red-phalloidin to allow concurrent
examination of filamentous actin (F-actin) content. PAI-1 staining was seen
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82
TGFP - - + + - - + + - - + + - - + +
Serum - CytB LatB Y-27632
B
w8
5Ok*r-t
<Ph
400 -
350 -
300 -
250 -
200 150 100 50
0
TGFp + TGFp
- * - 1
* * * *
n - f -* *
ETL
t t i t
C/5 CQ
U1 s■J
i>
t*-t>o▼H
35
s►»u
CQt §■J £
I><
£l>o
Jl35
Fig. 16. Effect of Actin depolymerization on TGFP-induced PAI-1 protein expression. Serum-starved mesangial cells were treated with 1 mM CytB, 0.1 pM LatB, 10 pM Y-27632 or 20 pM HA-1077 for 1-hour prior to 24-hour treatment with 10 ng/ml TGFp. The effect of actin depolymerization on PAI-1 protein expression was measured in conditioned media using Western blot analysis (n=3). (A) A representative Western blot showing increase in PAI-1 expression with TGFp treatment in serum-free media. (B) Inhibition of actin polymerization by agents using different mechanisms blocks basal and TGFp-induced PAI-1 expression. The vertical bars and the error bard represent mean values for PAI-1 protein and SEM respectively. The value for untreated control is set at 100%.*: p<0.05 compared to S- control, **: p <0.005 compared to S- control, t : P <0.05
compared to TGFp treated control.
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83
F-Actin PAI-1
S-
TG Fp
TG Fp+
CytB
TG Fp+
Y-27632
MM^ Mi■1
Fig. 17. Effect of Actin depolymerization on TGFP-induced PAI-1 expression by immunocytochemistry. Serum-starved mesangial cells were treated with 1 mM CytB (E, F) or 10 pM Y-27632 (G, H) before 24-hour treatment with 10 ng/ml TGFp (C-H). Cells were stained with Texas-Red phalloidin to visualize stress fibers (arrow) (A, C, E, G) and PAI-1 antibody (arrowhead) (B, D, F, H).Phalloidin staining shows abundant stress fibers (red staining) in S- control (A) and TGFp treated (C) cells. TGFp treated cells show more intense staining for PAI-1 (green staining) (D) compared to S- control (B). Loss of stress fibers with cytoskeleton inhibitors (E, G) correlates with loss of staining for PAI-1 (G, H)(n=1)-
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84
intracellularly and on the substratum. TGFp increased staining for PAI-1 after 24-
hour treatment. Y-27632 and CytB reduced stress fiber content and staining for
PAI-1 in both basal and TGF(3-stimulated conditions (Fig. 17). Thus PAI-1
expression changes in parallel with changes in actin polymerization.
Stabilization of actin cvtoskeleton increases TGFB-induced PAI-1 expression
The results showed that inhibition of actin polymerization inhibited basal
as well as TGFp-induced PAI-1 expression. If the actin cytoskeleton controls PAI-
1 expression, then an increase in actin fibers may increase PAI-1 expression. To
test the possibility that stabilization of actin polymers can increase PAI-1
expression, serum-starved cells were pre-treated for 1 hour with Jasplakinolide
(JAS), an agent that increases the F-actin content of cells by directly binding to
actin polymers and increasing nucleation. Cells were treated with 10 ng/ml TGFp
for indicated time periods, with and without JAS. In contrast to the actin
depolymerizing agents, JAS increased basal and TGFp-mediated PAI-1 mRNA
expression to 154.3±14% and 142.7±10.3% of respective controls as determined
by quantitative real-time RT PCR analysis (Fig. 14F). To ensure that the effects
of JAS were mediated by increased F-actin content, cells were co-treated with
JAS and actin depolymerizing agents in the presence or absence of TGFp (Fig.
18). Co-treatment with CytB, Y-27632 or Toxin B depolymerized actin
cytoskeleton and antagonized the JAS-mediated increase in PAI-1 mRNA to
24.6±4.6%, 52.6±8.9% and 39.1±8.7% of their respective JAS-treated controls.
CytB, Y-27632 or Toxin B also blocked JAS-mediated increase in TGFp-induced
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85
— TGFp
<a*
400
3 0 0 “
io o -
JAS - + +
JASY-27632
*
_ * rh- k t t ti 1 i
+ TGFp Att
- + +
3 0 0
g - 200 -
I<Oh
IO O
IO O
Toxin B '
B
Fig. 18. Effect of changes in actin polymerization on TGFp-induced PAI-1 mRNA expression. Serum-starved mesangial cells were treated with 50 nM JAS and, 1 mM CytB (A), 10 pM Y-27632 (B) or 10 pM Toxin B (C) for one hour prior to 8-hour treatment with 10 ng/ml TGFp (n=3). PAI-1 mRNA expression was measured using quantitative real-time PCR analysis. The vertical bars and the error bars represent mean PAI-1 mRNA values and SEM respectively. The value for untreated control is set at 100%. *: p<0.05 compared to S-, **: p<0.01 compared to S-, ***: p<0.005 compared to S-, P< 0.05 compared to TGFp, t t f : p<0.005 compared to TGFp, $: p<0.05 compared to JAS, #: p<0.05 compared to TGFp+JAS, ###: p<0.005 compared to TGFp+JAS.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
86
APAI-1
JAS TGF0 Toxin B
B
— +
Co"400 "
a 350 *« rHI
300 -O5-1
Ph 250 -200 -150 -
Pi too -50 -
TG Fp
*
o-JAS
Toxin B
F *—
*n
«*|»
m
— + + + — +
+ + + +
+ TG Fp
i t
#
□— + + + — +
Fig. 19. Effect of JAS and Toxin B mediated changes in actin polymerization on TGFp-induced PAI-1 protein expression. Serum-starved mesangial cells were treated with 50 nM JAS and 10 pM Toxin B for one hour prior to 24-hour treatment with 10 ng/ml TGFp. PAI-1 protein was measures in conditioned media using Western blot analysis. (A) Representative Western blot analysis of conditioned media for PAI-1 detection using Western blot analysis (n=3). (B) The vertical bars and the error bars represent mean PAI-1 protein values and SEM respectively. The value for untreated control is set at 100%. *: p<0.05 compared to S-, f . p< 0.05 compared to TGFp, $: p<0.05 compared to JAS, #: p<0.05 compared to TGFp+JAS.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
87
increase in TGFp-induced PAI-1 mRNA expression; decreasing PAI-1 mRNA to
17.9±1.1 %, 51.3±14.8% and 19.1±10.5% of TGFp+JAS-treated controls.
To confirm the effect of JAS on PAI-1 protein expression under the same
conditions, cells were treated with combinations of TGFp, JAS and actin
depolymerizing agents for 24 hours. Western blot analysis of conditioned media
showed JAS increased basal and TGFp-induced PAI-1 protein to 148.5±5 % and
148.1 ±7.1 % of respective control at 24 hours. Inhibition of actin polymerization
by Toxin B (Fig. 19), CytB (Fig. 20) and Y-27632 (Fig. 20) inhibited JAS-
mediated increase in PAI-1 protein to 28.7±4.9%, 19.3±6.2% and 46.4±11.3% of
their respective JAS-treated controls (Figs. 19, 20). At the same time, Toxin B,
CytB and Y-27632 blocked JAS-mediated increase in TGFp-induced PAI-1
protein expression to 18.9±3.4%, 13.1 ±2.9% and 29.1 ±7.6% of their respective
TGFP+JAS-treated controls (Figs. 19, 20).
Table 4 shows representative calculation of the changes in PAI-1 cDNA
concentration by real-time PCR analysis. The software automatically calculates
the concentration based on the crossing point of each sample. The values for
PAI-1 expression were normalized to the values for UBC, which remained
relatively unaffected by the experimental conditions.
In summary, the bi-directional changes in actin polymerization were
followed in the same direction by the changes in TGFp-mediated PAI-1
expression. An increase in stress fiber content of the cells caused an increase in
PAI-1 expression, whereas inhibition of actin polymerization caused inhibition of
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JASTGFp
B
/-“Nyn—HO£oo
c* y —«<Do
&
Oh
— + + + — +
“ s=
-T G F p500
400
300
200 *
— 4- + + — +
CytB
— — + +
— + — +
Y-27632
+ TGFpit
*
CO CO m PJ
S* rSo i>^ c ti>h
“ _± ± ±CO CO f f i $
VOO r -c iI
Fig. 20. Effect of changes in actin polymerization on TGFp-induced PAI-1 protein expression. Serum-starved mesangial cells were treated with 50 nM JAS and, 1 mM CytB or 10 pM Y-27632 for one hour prior to 24-hour treatment with 10 ng/ml TGFp. PAI-1 protein expression was measured in conditioned media using Western blot analysis. (A) Representative Western blot analysis of conditioned media for PAI-1 detection using Western blot analysis (n=3). (B) The vertical bars and the error bars represent mean PAI-1 protein values and SEM respectively. The value for untreated control is set at 100%. *: p<0.05 compared to S-, t : P< 0.05 compared to TGFp, p<0.05 compared to JAS, #: p<0.05 compared to TGFp+JAS.
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89
Table 4. Calculation of PAI-1 cDNA concentration by real-time PCR
CONDITIONCr.Pt.
PAI-1Calc.Con.(pg)
Cr.Pt.
UBCCalc.Con.(pg)
PAI-1 / UBC(x1 O'5)
%CONTROL
Serum - control 23.2 0.0118 20.2 234.2 5038 100.0TGFp 20.9 0.0427 19.9 289.8 14734 292.4JAS 22.6 0.0167 20.2 237.7 7025 139.4TGFp + JAS 20.6 0.0501 19.9 288.5 17365 344.7CytB 25.1 0.0041 19.8 306.4 1344 26.7TGFp + CytB 23.8 0.0084 20.0 278.0 3025 60.0JAS + CytB 26.3 0.0022 20.1 263.9 814 16.2TGF + JAS + CytB 23.7 0.0087 19.8 301.7 2903 57.6Toxin B 25.2 0.0039 20.1 254.2 1534 30.5TGFp + Toxin B 22.5 0.0177 20.0 280.4 6312 125.3JAS + Toxin B 24.9 0.0047 20.3 220.0 2113 42.0TGFp + JAS + Toxin B 22.7 0.0156 20.2 247.0 6315 125.4PAI-1 standard 23.5 0.0100 - - - -
UBC standard - - 21.5 100.0 - -
Abbreviations are: Cr. Pt, crossing point; Calc. Con, calculated concentration.
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90
PAI-1 expression. The inhibition of actin polymerization also prevented actin
stabilization by JAS.
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91
CHAPTER 6
EFFECT OF ACTIN CYTOSKELETON ON THE PAI-1 PROMOTER
Introduction
The results discussed in previous chapter showed that the changes in
actin polymerization regulated PAI-1 mRNA and protein expression. The
mechanism of this regulation was examined next.
The expression of PAI-1 is tightly regulated by a variety of cytokines,
growth factors, hormones and other agonists [78], These agents include TGFp,
Angiotensin II, thrombin, glucocorticoids, tumor necrosis factor-a and endotoxin
[78,135]. The regulation of PAI-1 mRNA expression is primarily at the level of
transcription [136, 137], Functional analysis of the regulatory region of the PAI-1
promoter shows the presence of multiple regulatory elements that respond to
diverse stimuli listed above [79]. Schematic representation of selected consensus
sequences in human PAI-1 promoter is shown in fig. 21 [69, 76, 115]. The human
PAI-1 promoter has at least 3 AP-1 sites in its distal promoter region (-800 to -
636) and one in the proximal promoter region (-81 to -75) [78], These AP-1 sites
are critical for induction of PAI-1 by the agents listed above [78, 138]. The
regulation of PAI-1 promoter by TGFp has been extensively studied [135, 78,
139]. Two TGFP-inducible, cis-acting regulatory elements were first identified in
the 5’ flanking DNA of the human PAI-1 gene; a relatively strong element
between -791 and -328, and a weaker element between -328 and -187
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Reproduced
with perm
ission of the
copyright ow
ner. Further
reproduction prohibited
without
permission.
■118 SplCTF/NFlcB-730 Smad 3/4 k j L -580 Smad 3/4
VLDL-(E ■280 Smad 3/4-79 to -72 and-58 to -50
P M A -& /T R E-214
CCAATBox■443 Spl CTF/NFkB
AP-1 AP-2 -73 Spl
■42 SplNF-1
CCAAT Box TATAAP-1 AP-1 AP-1
GRE GREtoo
Fig. 21. Schematic representation of PAI-1 promoter. -RE, response element; TGFp, transforming growth factor; VLDL, very low density lipoprotein; CTF, CCAAt box binding transcription factor; NF1, nuclear factor 1; PMA, phorbol 12-myristate 13-acetate; AP, activator protein; TRE, tetradecanoyl phorbol acetate response element;GRE, glucocorticoid response element. Used with permission from Binder et al [69].
CDro
93
[139]. Later, three SMAD-binding sites were identified between -794 and - 532
that were sufficient for induction by TGF(3 [140]. PAI-1 promoter also contains
NF-1 like sequences, one p53 binding site between -139 and -160 [141], two
glucocorticoid response elements (GREs) between -100 and +75, and -800 and
-549 [76], CRE, five Hypoxia Responsive Elements (HREs) [142] and two Sp1
binding site-like sequences at -72 to -67 and -45 to -40 [143]. The rat PAI-1
gene shares many structural similarities with the human PAI-1 gene and contains
similar regulatory elements in the promoter [144], For example, the rat PAI-1
promoter contains binding sites for AP-1 and Sp1, glucocorticoids response
elements and CRE. Although the rat and human PAI-1 promoter are similar, the
number of each type of regulatory elements may be different. For example, the
rat PAI-1 promoter contains five GREs as compared to two contained in the
human PAI-1 promoter. Also, the rat PAI-1 promoter contains two regions that
have at least 80% sequence similarities to its human counterpart. The first is
between -91 and TATA box, and the second between -800 and -549.
The serum response factor (SRF), a transcription factor, has been shown
to mediate effects of changes in actin cytoskeleton on expression of some genes
[145]. AP-1-dependent genes are direct targets of SRF. For example, inhibition of
actin polymerization by inhibitors of direct and Rho-mediated polymerization
blocked inducible nitric oxide gene expression in human epithelial cells [145].
Similarly, inhibition of Rho by C3 toxin blocked activation of AP-1 in rat-1 cells
[146]. TGF(3-mediated SMAD 2/3 activation also required Rho signaling in human
breast carcinoma cells [147]. Thus, the evidence from the literature suggests a
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94
role for Rho and actin cytoskeleton in regulation of genes through similar
regulatory elements as contained in PAI-1 promoter. The hypothesis that Rho-
mediated changes in actin polymerization regulate TGFp-induced PAI-1
expression at the promoter level was therefore examined.
Experimental Model
To examine if PAI-1 expression was at the transcriptional level, the human
PAI-1 promoter, spanning -973 to +133 nucleotides, was cloned into a luciferase
vector as described in chapter 2. Human and rat mesangial cells were serum-
starved and transfected with human PAI-1 promoter vector for 24 hours. Cells
were then pre-treated with CytB to inhibit actin polymerization followed by
treatment with TGFp. The effect on promoter activity was determined by
luciferase assay.
Results
TGFS induces PAI-1 promoter activity in rat mesangial cells
The initial time-course analysis of the effect of TGFp on PAI-1 promoter
activity was conducted in rat mesangial cells due to their better transfection
efficiency. Rat mesangial cells (p27) were co-transfected with PAI-1 promoter
vector and renilla luciferase vector in serum-free media. After 24 hours, the cells
were treated in triplicate wells with 10 ng/ml TGFp (dose shown to be effective on
PAI-1 mRNA and protein expression) for varying amounts of time to identify the
time-point for the maximum increase in PAI-1 promoter activity. In a single
experiment, TGFp-treatment for 2, 4, 6, 8 and 24 hours increased PAI-1
promoter activity to 108.4±3.7% 124.2±7.7%, 128.5±3.1%, 109.0±3.9% and
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95
140 - i
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Fig. 22. Time-course analysis of TGFp on PAI-1 promoter activity. Ratmesangial cells were co-transfected with PAI-1 promoter plasmid and renilla luciferase plasmid in serum-free media. After 24 hours, 10 ng/ml TGFp was added to the media for the indicated times. Duplicate wells of control cells in serum-free media and triplicate cells of TGFp-tread cells were used for the experiment. Luciferase activities in samples were measured using dual luciferase assay (n=1). Each bar represent average luciferase activity for PAI-1 promoter normalized to renilla luciferase activity in the same sample. The normalized Luciferase activity of time-matched untreated control is set as 100%.
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96
116.6±4.2% of their respective time-matched controls. The values for renilla
luciferase activities were above background and hence were used to normalize
for transfection efficiency.
The effect of TGFp was maximum at 6-hours with the minimum variation.
In the following set of experiments, cells were treated with TGFP for 6 hours.
Inhibition of actin polymerization blocks TGFS-induced PAI-1 promoter activity
In rat mesangial cells, the effect of TGFp on PAI-1 promoter (128.5 %)
was much smaller than the effects on PAI-1 mRNA (~250%) and protein (~250%)
in human mesangial cells. Therefore the remaining experiments were performed
in human cells. Serum-starved mesangial cells were transfected with PAI-1 and
renilla luciferase plasmid as described above. After 24 hours, cells were pre
treated with 1 pM CytB followed by 10 ng/ml TGFp for six hours. Since, co
transfection with renilla luciferase was not successful, luciferase counts were
normalized to the protein concentration. Previously, Owens et al showed
normalization for protein concentration as an acceptable method for
normalization for transfection efficiency [148, 149], The treatment with TGFp
increased PAI-1 promoter to 293.6±36.5% of control. CytB inhibited basal as well
as TGFp-induced PAI-1 expression to 45.1 ±6.1% and 34.6±12.7% of their
respective controls. The effects of other cytoskeleton inhibitors could not be
determined due to limited transfection efficiency.
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97
o
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300
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150
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Fig. 23. The effects of TGFp and actin depolymerization on human PAI-1 promoter activity. Human mesangial cells were co-transfected at 60-80% confluence with 1 pg human PAI-1 promoter-containing plasmid and 1 pg renilla luciferase plasmid. The cells were serum-starved at the time of transfection.After three hours of transfection, CytB was added to appropriate wells. After 24 hours, cells were treated with 10 ng/ml TGFp for 6 hours. Luciferase activity was normalized for the protein concentration of each sample to get the activity in counts/mg protein for each sample. For each treatment, cells were transfected at least in duplicates (n=3). Each bar represent average luciferase activity for PAI-1 promoter normalized to cellular protein concentration. The normalized Luciferase activity of untreated control is set as 100%. *: p<0.05 compared to S-, t : P< 0.05 compared to TGFp.
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98
CHAPTER 7
EFFECTS OF TGFp AND ACTIN CYTOSKELETON ON PLASMINOGEN
ACTIVATORS
Introduction
The effects of TGFP and actin cytoskeleton on PAI-1 expression were
determined as shown in chapters 4 to 6. Next, the effects of cytoskeleton
disruption on two other components of plasminogen activator system namely
tissue- (tPA) and urokinase- (uPA) type plasminogen activators were examined.
Two activators (tPA, uPA) and one inhibitor (PAI-1) of plasminogen
activation constitute the plasminogen activator system, which regulates ECM
degradation. The mesangial extracellular matrix determines the physical,
mechanical and functional properties of glomerulus in normal and pathological
conditions [57], Plasminogen activators convert inactive plasminogen into active
plasminogen. Plasmin activates matrix metalloproteinases (MMPs), which then
digest ECM. Plasmin itself has some matrix digesting activity. PAI-1 binds to tPA
and uPA, and blocks the activation of plasminogen into plasmin. Thus, tPA and
uPA favor ECM degradation whereas PAI-1 favors ECM deposition. The balance
between plasminogen activators and PAI-1 thus determines the rate and the
amount of ECM deposition.
The DNA array analysis showed a 2.8-fold increase in tPA expression
upon actin depolymerization (Table 3, Chapter 3), suggesting a coordinated
regulation of the plasminogen activator system by the actin cytoskeleton. The
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99
effects on uPA still remained to be examined. Moreover, treatment with TGFp is
shown to have varying effects on the expression of plasminogen activators
depending on the cell-type [61, 124, 150-152], The following set of experiments
was therefore performed to understand the role of TGFP and the actin
cytoskeleton on the regulation of plasminogen activator system as a whole.
Experimental Model
Serum-starved human mesangial cells were treated with 10 ng/ml TGFp
for 4, 8, 24 and 48 hours to determine optimum treatment duration. The changes
in tPA and uPA mRNA expression were examined by real-time quantitative PCR
analysis. In subsequent experiments cells were pre-treated with CytB, LatB or Y-
27632 for one hour to inhibit actin polymerization, followed by 8-hour treatment
with TGFp. These treatment conditions were identical to those used to examine
the effects on PAI-1 expression in earlier chapters. The effect of TGFp and actin
depolymerization on tPA and uPA expression was determined at the mRNA
level. Real-time PCR analysis was used to detect changes in tPA and uPA
mRNA expression. The changes in tPA and uPA protein expression were
examined in conditioned media at 24 hours to match the experimental conditions
used for PAI-1 protein using Western blot analysis.
Results
Quantification of tPA and uPA mRNA expression by real-time PCR analysis
The existing literature show conflicting evidence about production of tPA
and uPA by human mesangial cells and the regulation of plasminogen activator
system by TGFp [61, 89, 152-158]. Therefore, the expression of uPA and tPA
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100
mRNA by serum-starved mesangial cells was confirmed first prior to examining
the effect of TGF(3 on tPA and uPA expression. Using real-time PCR detection,
both uPA and tPA mRNAs were successfully detected in human mesangial cells.
Briefly, a reverse transcription reaction on thermocycler (MJ Research, Reno,
NV) was followed by a quantitative real-time PCR analysis on Roche lightcycler
(Roche Diagnostics, Indianapolis, IN). First, standard curves fortPA (Fig. 24) and
uPA (Fig. 25) cDNA were generated using known concentrations of cDNA
templates as described in chapter 2. The products of standard curve polymerase
chain reactions were analyzed by gel electrophoresis to ensure amplification of a
single, specific product (Fig. 26).
Subsequently, one known concentration of standard (0.001 pg for tPA and
0.0001 pg for uPA) was amplified along with experimental samples. The values
for tPA and uPA concentrations were derived in picograms, which were further
normalized to similarly obtained values for a reference gene, Ubiquitin (UBC), in
each sample. The sample concentrations were calculated by the software by
comparing the crossing point of experimental sample with that of the known
standard normalized to previously saved standard curve as described in chapters
2 and 4. Ubiquitin was used as the reference gene to normalize the
concentrations for tPA and uPA. Figures 27 and 28 show amplification (panel A),
melting curve analysis (panel B), melting peak analysis (panel C) and gel
electrophoresis (panel D) of tPA and uPA respectively from representative
experimental samples.
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101
2 -
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Amplification Curve
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D
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70 74 78 82Temperature (°C)
70 75 80 85Temperature (°C)
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Fig. 24. tPA standard curve for real-time PCR analysis. (A) Amplification of tPA cDNA plotted as fluorescence vs. cycle number. Each sample is represented in a uniquely colored line on the real-time PCR software, allowing tracing of amplification of individual samples. (B) tPA standard curve plotted as cycle number vs. log concentration showing a linear standard curve (r= -1). (C) Realtime monitoring of melting curve analysis of PCR products plotted as fluorescence vs. cycle number showing only one PCR product corresponding to tPA. (D) Melting curve analysis data plotted as changes in fluorescence signal over time (dF/dT) vs. temperature. Presence of only one melting peak in this reaction corresponding to melting temperatures of tPA (~85.5°C) indicates specificity of the amplification reaction and absence of primer-dimers.
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102
Amplification Curve A
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cDNA template0.0001 pg ________
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66 70
Melting PeakI
-Melting temperature (Tm)—*.1
78 82 86Temperature (°C)
D
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68 75 7§ 85 84 8§ 9?Temperature (°C)
Fig. 25. uPA standard curve for real-time RT-PCR analysis. (A) Amplification of uPA plotted as fluorescence vs. cycle number. Each sample is represented in a uniquely colored line on the real-time PCR software, allowing tracing of amplification of individual samples. (B) uPA standard curve plotted as cycle number vs. log concentration showing a linear standard curve (r= -1). (C) Realtime monitoring of melting curve analysis of PCR products plotted as fluorescence vs. cycle number showing only one PCR product corresponding to uPA. (D) Melting curve analysis data plotted as changes in fluorescence signal over time (dF/dT) vs. temperature. Presence of only one melting peak in this reaction corresponding to melting temperatures of PAI-1 (-83.5 C) indicates specificity of the amplification reaction and absence of primer-dimers.
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103
A tPA
tPA (265 bp)
concentration (p i)
B uPA
uPA (3 10 bp)
concentration (pg)
Fig. 26. Gel-electrophoresis of tPA and uPA standard curve PCR products.Aliquots of PCR products (2 pi out of 20 pi total) were run on 8% acrylamide gel followed by ethidium bromide staining. (A) tPA and (B) uPA PCR products show a single band at expected base-pair location confirming specific amplification of respective templates.
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104
M elting CurveA m plification Curve
H 40-
40-Samples and standards
a 30-Oq 20 -
tPAUBCCrossing points ,10- 10-
0 ' .
Temperature (°C)Cycle Number
Gel E lectrophoresisM elting Peak
U B cii |tPA
265 bp133 bp
t P A UBCTemperature (°C1
Fig. 27. Representative images from real-time PCR of tPA and UBC from experimental samples. (A) Amplification of tPA and UBC plotted as fluorescence vs. cycle number. Each line is represented in different color on the real-time PCR software, allowing tracing of amplification of individual samples. The crossing points for both tPA and UBC fall within the bars marking approximately cycles 15 and 22. (B) Real-time monitoring of melting curve analysis of PCR products plotted as fluorescence vs. cycle number showing two PCR products corresponding to tPA and UBC. (C) Melting curve analysis data plotted as changes in fluorescence signal overtime (dF/dT) vs. temperature. Presence of only two melting peaks in this reaction corresponding to melting temperatures of UBC (~82°C) and tPA (~85.5°C) indicate specificity of the amplification reaction and absence of primer-dimers. (D) Gel electrophoresis of PCR products from representative experimental samples show a single PCR product of expected base-pair size in each reaction.
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105
A m plification Curve M elting Curve
UBC40 -Samples and standards
30 -uPA cA 30-
20 -
10 -
20 -
Crossing points10 - 4lPA
Temperature (°C)Cycle Number
M elting Peak
UBCk IuPA
Gel E lectrophoresis
133 bp
- 2 .
uPATemperature (°C)
Fig. 28. Representative images from real-time PCR of uPA and UBC from experimental samples. (A) Amplification of uPA and UBC plotted as fluorescence vs. cycle number. Each line is represented in different color on the real-time PCR software, allowing tracing of amplification of individual samples. The crossing points for uPA and UBC fall within the bars marking approximately cycles 23-28 and 15-19 respectively. (B) Real-time monitoring of melting curve analysis of PCR products plotted as fluorescence vs. cycle number showing two PCR products corresponding to uPA and UBC. (C) Melting curve analysis data plotted as changes in fluorescence signal overtime (dF/dT) vs. temperature. Presence of only two melting peaks in this reaction corresponding to melting temperatures of UBC (~82°C) and uPA (~83.5°C) indicate specificity of the amplification reaction and absence of primer-dimers. (D) Gel electrophoresis of PCR products from representative experimental samples show a single PCR product of expected base-pair size in each reaction.
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106
TGFB has opposite effects on tPA and uPA mRNA expression
In previous experiments, the effective dose of TGFp was determined to be
at 10 ng/ml (chapter 4). In a time- course analysis 10 ng/ml TGFp treatment
increased uPA mRNA levels to 117.8%, 207.2%, 137.9% and 35.4% of
respective time-matched control at 4, 8, 24 and 48 hours (n=1) (Fig. 29).
Whereas, 10 ng/ml TGFp inhibited average tPA mRNA to 89.3%, 69.5%, 70.65
and 28.8% of respective time-matched control at 4, 8, 24 and 48 hours (n=2)
(Fig. 29). Thus, TGFP increased uPA mRNA expression maximally at 8 hours.
On the contrary, TGFp inhibited tPA mRNA expression at most of the time points
examined. Maximum increase in PAI-1 mRNA was seen at 8-hours (chapter 4)
which corresponds to maximum increase in uPA expression and consistent
inhibition of tPA expression at 8 hours. Therefore 8 hours was determined to be
an optimum time-point for TGFp treatment.
Actin depolvmerization regulates tPA and uPA mRNA expression
The rate of plasminogen activation depends on the balance between
plasminogen activators and PAI-1. Therefore the effects of inhibiting actin
polymerization on expression of the plasminogen activators was examined first in
order to understand the potential net effect on plasminogen activation. The
samples from the same experiments in which actin depolymerization had shown
inhibition of TGFp-induced PAI-1 expression were examined for tPA and uPA
mRNA expression using quantitative real-time PCR analysis. In three separate
experiments, TGFp decreased tPA mRNA expression slightly, but significantly, to
80.7 ± 5.5% of untreated control (p<0.05), consistent with the profibrotic effects
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107
T3 120<0 100Q
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uPAB
24h 48hTGFp treatment
Fig. 29. Time-course analysis of TGFp-treatment on tPA and uPA mRNA expression. Human mesangial cells at 60-80% confluence were serum starved for 24 hours. Cells were treated with 10 ng/ml TGFp for 4, 8, 24 and 48 hours. At each time point, the effect of TGFp treatment on tPA (A) and uPA (B) mRNA was measured by real-time PCR analysis. (A) TGFp inhibited tPA mRNA at all the time-points examined (n=2). Each bar represent mean value for tPA expression. The ends of error bars represent the value for each of the two experiments. (B) TGFp treatment maximally increased uPA mRNA at 8 hours (n=1).
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108
of TGFp (Fig. 30). Actin depolymerization by Y-27632, CytB and LatB increased
basal tPA expression to 232 ± 22.3%, 195.4 ± 26.7% and 288.9 ± 29.8% of
untreated controls. Actin depolymerization also effectively reversed the inhibitory
effects of TGFp on tPA expression. Y-27632, CytB and LatB increased tPA
mRNA to 260.9 ± 22.6%, 208.3 ± 33.8% and 244.9 ± 39% of their respective
TGFP-treated controls. This suggests that actin depolymerization coordinately
regulates the plasminogen activator system. Actin depolymerization
simultaneously inhibits PAI-1 expression and increases tPA expression, the net
result of which favors activation of plasminogen activation and degradation of
extracellular matrix. In contrast, TGFp increased uPA expression by
246.7±61.4% of control (p<0.05). Inhibition of actin polymerization by CytB and
Y-27632 changed uPA mRNA expression to 259.1 ± 119% and 115.6 ± 24.5% of
respective controls under serum-free conditions. In the presence of TGFP, CytB
and Y-27632 changed uPA mRNA expression to 95.8 ± 37.7% and 77.7 ± 16.3%
of TGFP-treated control respectively. The effect of CytB and Y-27632 were not
statistically significant (Fig. 31). In summary, TGFp increased uPA mRNA
expression, whereas actin depolymerization did not have a statistically significant
effect on the same.
Effect TGFB and actin depolvmerization on plasminogen activators protein
expression
Since tPA and uPA are secreted upon synthesis, changes in tPA and uPA
were determined by analyzing conditioned media using Western blot analysis.
Neither tPA nor uPA proteins could be detected in conditioned media using
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109
350 n
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Fig. 30. The effects of TGFP and actin depolymerization on tPA mRNA expression. Human mesangial cells at 60-80% confluence were serum starved for 24 hours. Cells were then pre-treated for one hour with 1.0 mM CytB, 0.1 pM LatB or 10 pM Y-27632 to inhibit actin polymerization. TGFP (10 ng/ml) was added to the media for next 8 hours. At the end of 8-hour treatment with TGFp, mRNA was isolated and analyzed by quantitative real-time PCR analysis for tPA mRNA expression (n=3). The vertical bars and the error bars represent mean tPA mRNA value and SEM respectively, The value for untreated control is set as 100% *: p<0.05 compared to S-, t : p<0.05 compared to TGFp, t t t - P<0.005 compared to TGFp.
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110
400 -INO
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Fig. 31. The effects of TGFp and actin depolymerization on uPA mRNA expression. Human mesangial cells at 60-80% confluence were serum starved for 24 hours. Cells were then pre-treated for one hour with 1.0 mM CytB or 10 pM Y-27632 to inhibit actin polymerization. TGFp (10 ng/ml) was added to the media for next 8 hours. At the end of 8-hour treatment with TGFp, mRNA was isolated and analyzed by quantitative real-time PCR analysis for uPA mRNA expression (n = 5). The vertical bars and the error bars represent mean uPA mRNA value and SEM respectively, The value for untreated control is set as 100%. *: p<0.05 compared to S - .
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111
Western blot analysis. Multiple optimization efforts including using smaller
amounts of media for incubation (to reduce dilution of secreted tPA and uPA),
using larger amounts of media for detection, concentrating the media using spin
concentrators and using varying amounts of primary antibodies were
unsuccessful in detecting tPA and uPA proteins in conditioned media. Previously
uPA was successfully detected in this lab in a preparation of substratum attached
extracellular matrix and membrane proteins called adhesion plaques but not in
conditioned media [159, 160].
In summary, the results show an increase in tPA mRNA expression by
actin depolymerization. PAI-1 expression was inhibited under similar
experimental conditions, suggesting a coordinated regulation of these two genes
with opposite functions by actin cytoskeleton.
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112
CHAPTER 8
DISCUSSION
Mesangial myofibroblast activation as a disease model
Diverse cells and tissues react to injury with similar responses, including
myofibroblast differentiation and formation of scar tissue from increased
extracellular matrix accumulation [22-24, 29, 161-166]. In the event of tissue
injury, native fibroblasts or mesenchymal cells with fibroblast-like properties can
transdifferentiate into activated myofibroblasts. De novo expression of genes,
possibly required to cope with demands of injury and tissue repair, is seen upon
myofibroblast differentiation [19, 119, 167], For example, the activation of a-SMA
expression, a property associated with smooth muscle phenotype and hence the
name myofibroblast, is a common feature of myofibroblasts regardless of the
tissue affected [168], In the skin, dermal fibroblasts transdifferentiate into
myofibroblast during the process of wound healing and start expressing a-SMA
de novo [11]. Similarly, in the kidneys, glomerular mesangial cells, mesenchymal
cells that hold the capillary tufts of glomeruli together, do not normally express a-
SMA. But in the event of injury, the activated mesangial cells transdifferentiate
into myofibroblasts and start expressing a-SMA [19]. They also undergo
hypertrophy to meet the functional demands in disease states [168]. Hypertrophic
cells also produce more ECM proteins leading to scar tissue formation. Indeed,
myofibroblast differentiation is associated with excess ECM deposition in
glomeruli in chronic glomerulosclerotic diseases [165]. The association of the
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113
myofibroblast differentiation with ECM deposition and scarring suggests that the
knowledge of the mechanisms that regulate myofibroblast differentiation may
provide new insights into the cellular processes that control tissue scarring. The
overall objective of the current study was to identify the mechanisms regulating
myofibroblast differentiation. In this study, the role of actin cytoskeleton in the
regulation of gene expression and phenotype changes in glomerular mesangial
cells was examined.
Cultured mesangial cells as the experimental model of myofibroblast
differentiation
Previously, Glass et al showed that cultured mesangial cells mimic in vivo
changes during myofibroblast activation[26]. Initial culture in presence of serum
causes mesangial cell proliferation similar to that seen in early stages of
glomerular injury. Whereas, in the absence of serum, the mesangial cells stop
proliferating, spread more, become hypertrophic, increase their stress fibers, and
express more a-SMA [26], The phenotype seen during the serum-deprivation
closely resembles that of myofibroblasts seen in the later stages of glomerular
injury. Thus, the cultured mesangial cells share similarities both in phenotypes
and in the sequence of phenotype changes with in vivo myofibroblasts.
Therefore, cultured mesangial cells serve as a good model system to study the
myofibroblast phenotype in vitro.
Primary culture human mesangial cells between passages 5 and 9 were
therefore used in the current study to understand the regulation of myofibroblast
differentiation. The serum-free media was used as the basal culture condition to
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114
allow the mesangial cells to acquire myofibroblast-like phenotype. The use of
already activated mesangial cells resembles the clinical scenario in chronic
glomerular diseases, where a majority of the patients present with already
established glomerular lesions containing activated mesangial cells. Also, the
role of individual growth factor can be studied when added to the serum-free
media used as the basal culture condition.
Actin cytoskeleton as a regulator of gene expression
The concordant increase in stress fiber formation with a-SMA expression
and hypertrophy in serum-starved mesangial cells lead to the hypothesis that the
actin cytoskeleton regulates a-SMA expression and hypertrophy in mesangial
cells. In a further study, the use of agents that modulate actin cytoskeleton
showed that the changes in actin cytoskeleton regulated both hypertrophy and a-
SMA expression in serum-starved mesangial cells [28], The a-SMA expression
was regulated at the level of transcription and message stability. These findings
were consistent with the regulation of a-SMA mRNA by the actin cytoskeleton in
different cell types [169, 149]. These results showed the evidence for the role of
actin cytoskeleton in gene regulation and myofibroblast-differentiation in
mesangial cells.
Since cellular hypertrophy is associated with an increase in global protein
synthesis, it was hypothesized that the actin cytoskeleton regulates myofibroblast
phenotype by simultaneously regulating the expression of multiple genes. Recent
availability of DNA arrays containing multiple genes with related functions on one
array provides opportunity to study the expression of multiple genes
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115
simultaneously. Since the myofibroblast differentiation is associated with
increased ECM, the effects of actin cytoskeleton on a DNA array containing
genes related to ECM and cell-interaction were examined.
The array experiment way performed only once, but the results were
consistent with the hypothesis that cytoskeletal organization coordinately
regulates genes involved in myofibroblast function and sclerosis. Also the results
were confirmed, in particular, for PAI-1 and tPA. The expression of 74 out of 265
genes was detected on the array (Chapter 3). Cell-type specific and/or cell-
culture conditions could account for the detection of limited number of genes.
Alternatively, optimization of the protocol could help detect some of the genes not
detected in this study. Also, repeating the experiment with the same and other
actin depolymerizing agents could show the consistency of changes in pattern of
gene expression.
A noticeable pattern of gene expression was observed among the
detected genes. Out often down-regulated genes, four were precursors of
extracellular matrix components, namely procollagen 3 a1 subunit, procollagen 1
a2 subunit, collagen 4 a2 subunit and fibronectin. Endothelial plasminogen
activator inhibitor-1 (PAI-1) precursor also decreased. Since the role of PAI-1 is
to favor ECM deposition, a decrease in PAI-1 would also decrease ECM. Thus
actin depolymerization decreases both ECM components as well as profibrotic
regulators of ECM turnover. The collective effect is a decrease in ECM
deposition. Thus, actin depolymerization correlates with decreased PAI-1
expression and ECM deposition. Since, PAI-1 expression is an attribute of
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116
myofibroblast differentiation and the ECM deposition is an outcome of such
differentiation, these findings support the overall hypothesis that changes in actin
polymerization regulate the myofibroblast phenotype of mesangial cells.
At the same time, out of four genes that increased, three favor
degradation of ECM. These include tissue-type plasminogen activator (tPA),
decorin (DCN) and matrix metalloproteinases 11 (MMP 11). tPA converts inactive
plasminogen into active plasmin with fibrolytic properties [56]. MMP11, although
not a major fibrolytic enzyme, digests ECM under certain conditions [170].
Decorin inhibits profibrotic TGFp and thereby prevents deposition of ECM [122,
171]. Together these genes lead to increased removal of ECM.
The cut-off ratios for increase (>2-fold) or decrease (>50%) in gene
expression set during the DNA array analysis were arbitrarily selected, requiring
further validation of the findings. Also, the experiment was performed only once.
Thus, further validation of the changes in gene expression seen on DNA array
was required. Therefore, the changes in the expression of two genes, relevant to
myofibroblast differentiation and ECM regulation, seen on DNA arrays were
confirmed by Northern blot and Western blot analysis. The components of
plasminogen activator system, PAI-1 and tPA, were selected for the confirmation
first since they are the major regulators of fibrinolysis and are directly related to
ECM turnover. Also they both were regulated in opposite directions by actin
cytoskeleton consistent with their opposite functions on ECM. The findings on
PAI-1 and tPA were confirmed at mRNA and protein levels, providing strong
evidence for coordinated gene regulation by the actin cytoskeleton. Indeed,
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inhibition of actin depolymerization by CytB and Y-27632 inhibited PAI-1 mRNA
and protein expression, whereas CytB increased tPA protein expression. Thus
the findings of DNA array experiments were reflected at the protein level. These
experiments were set under different cell-culture conditions, some in the
presence of serum and some in the absence of serum, making the comparison of
results difficult. The aim of the next set of experiments was to understand the
significance of regulation of PAI-1 and tPA in the context of their
pathophysiological regulation using a uniform cell culture condition.
TGFp induction of PAI-1 expression
Since PAI-1 is a major mediator of the ability of TGFp to cause fibrosis
[172] and it acts as a gate-keeper of the plasminogen activation cascade by
inhibiting both tPA and uPA, the regulation of PAI-1 expression was studied first
and more extensively than that of tPA or uPA. The cells were treated with TGFp,
a major regulator of PAI-1, plasminogen activator, ECM turnover and
myofibroblast differentiation; to resemble in vivo regulation of plasminogen
activator system. The effect of actin cytoskeleton on TGFp-mediated expression
of the plasminogen activator system was studied in human mesangial cells. The
use of TGFp as an agonist provides a model that mimics in vivo regulation of
plasminogen activators, and helps to understand myofibroblast differentiation and
ECM regulation.
The dose-response analyses of TGFp on PAI-1 mRNA and protein
expression were performed first (Chapter 4). Although treatment with 1 ,2 ,5 and
7.5 ng/ml TGFP increased average PAI-1 mRNA expression to 381.9%, 323.1%,
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443.3% and 485.1% of control in two different experiments, the effects on PAI-1
protein were inconsistent at these lower doses. At 10 ng/ml, TGFp increased
both PAI-1 mRNA and protein expression to 287.9% and 283.6% of respective
controls. Additionally, this concentration is very commonly used in published
studies by numerous investigators. In a similar dose-response analysis, Motojima
et al also found 10 ng/ml as the most effective dose of TGFP for PAI-1 induction
in rat mesangial cells [82]. Other researchers in this lab have also used TGFp
successfully at 10 ng/ml for studying effect on COX-2 expression (Harding et al,
unpublished observations). Also, this concentration of TGFp is pathological which
makes the experiments more relevant to an actual pathological state [126]. The
failure to show an increase with lower doses of PAI-1 expression was difficult to
interpret since the experiment was performed only once. It may result from
inefficient translation and/or secretion of PAI-1, inactivation of lower doses of
TGFp in the treatment media at 24-hour treatment or lack of activation of co
stimulatory pathways.
The time-course analysis showed that TGFP increased PAI-1 mRNA
maximally to a value of 191.0 ± 23.5% at 8 hours (Chapter 4). PAI-1 mRNA
decreased to 100.1 ± 13.5% and 90.6 ± 8.7% of time-matched controls at 24 and
48 hours respectively suggesting either loss of TGFP activity or inhibition of
TGFp activity at those time points. Although not tested directly at protein level,
the array experiment did show presence of decorin mRNA, a known inhibitor of
TGFp [122]. The analysis for PAI-1 protein showed maximum activation at 24
hours. At earlier time-points PAI-1 could not be detected in the conditioned
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119
media. Since PAI-1 is a secreted protein, there may be a lag period before PAI-1
is synthesized and secreted in sufficient amounts to reach the level of detection
in the culture media. Even using a more sensitive detection method such as
enzyme-linked immunosorbent assay (ELISA), Matsumoto et al found 24 hr
treatment with TGF(3 to maximally induce PAI-1 protein expression [127],
Therefore, the use of more sensitive detection method may still show 24 hours as
optimum duration of treatment. Although PAI-1 is a rapidly secreted protein,
protein synthesis takes longer than mRNA synthesis, partially accounting for the
differences in the time-frame of their detection. Therefore, PAI-1 mRNA reached
a peak at 8 hours and protein reached a peak at 24 hours. At 48 hours, PAI-1
protein was still elevated (146%), whereas mRNA returned to the control levels
(90.6 ± 8.7%). Alternatively, this sustained increase at 48 hours in protein
expression could represent accumulation in the media. Since a consistent and
comparable increase was seen in PAI-1 mRNA at 8 hours and protein at 24
hours, these time points were used for subsequent experiments.
Inhibition of actin cytoskeleton on TGFp-induced PAI-1 expression
In vivo and in vitro evidence suggest that Rho and the actin cytoskeleton
regulate PAI-1 expression and matrix deposition. The small GTPase, Rho, is
known to regulate expression of PAI-1 [81, 108, 173, 174], Also, inhibition of Rho
signaling prevents fibrosis in animal models, an effect consistent with PAI-1
inhibition [102-104], Since one of the major functions of Rho is to regulate actin
polymerization, the effects of Rho on PAI-1 expression could be mediated
through the actin cytoskeleton. Secondly, activated mesangial cells or
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120
myofibroblasts express a-SMA and PAI-1 de novo in various forms of renal
fibrosis. Recently this lab showed that Rho-mediated changes in actin
polymerization regulate a-SMA expression and hypertrophy, both attributes of
myofibroblast differentiation, in mesangial cells [28], Although the injurious
agents and cytokines involved are diverse, it is likely that common cellular
mechanisms underlie the regulation of myofibroblast differentiation and increased
ECM deposition common to sclerotic diseases. PAI-1, another attribute of
myofibroblast differentiation, could also be similarly regulated by the changes in
actin polymerization. Thus two separate lines of evidence suggest the role of
actin cytoskeleton in the regulation of PAI-1 expression. Therefore the hypothesis
that Rho GTPase regulates PAI-1 expression by modulating actin polymerization
was examined.
To test the hypothesis, the effects of either inhibition of Rho-mediated
signaling or direct inhibition of actin polymerization was examined on TGFp-
induced PAI-1 mRNA and protein expression (Chapter 5). The inhibition of Rho
blocked both basal and TGF(3-induced PAI-1 mRNA expression in human
mesangial cells. Similarly, direct inhibition of actin polymerization with CytB and
LatB also inhibited basal and TGF(3-induced PAI-1 mRNA expression. The
results seen by real-time PCR were comparable to those seen by conventional
methods such as Northern blot analysis. These confirmed the utility of real-time
quantitative PCR in detecting changes in mRNA expression. Since the effects of
Rho inhibition on PAI-1 mRNA expression are mimicked by direct inhibition of
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121
actin polymerization, it suggests that the effects of Rho on PAI-1 mRNA
expression are mediated by changes in actin polymerization.
Surprisingly, TGFP did not show any effect on PAI-1 expression in cell-
lysates. Since PAI-1 is rapidly secreted upon synthesis, the induction by PAI-1
may only be reflected in secreted PAI-1. The secreted PAI-1 binds to the
substratum on tissue culture dishes and saturates the binding sites on the
substratum. The cell-lysates were prepared by scraping adherent cells. The
lysates generated in this manner include both intracellular and substrate-bound
extracellular PAI-1 and hence may not show a true representation of changes in
protein expression. The Western blot showed specific inhibition of the PAI-1 band
with CytB treatment, whereas the other non-specific bands seen on the blot
remained constant. This suggests that CytB is not affecting the secretion of PAI-1
in which case an increased intracellular PAI-1 would have been seen. However,
since the cell scrapings contain both intracellular and the substratum-bound PAI-
1, which is generally saturated at the cell density used in this study, these
differences were masked on cell-scrappings. CytB treatment blocked PAI-1
expression in both cell-lysates and conditioned media suggesting that the
inhibition was at the level of protein synthesis or earlier at the mRNA level.
Subsequently, the conditioned cell culture media containing the secreted proteins
was used to detect the changes in PAI-1 protein expression. The changes in PAI-
1 protein expression upon treatment with agents that increase or decrease actin
polymerization were consistent with the changes in PAI-1 mRNA expression
under similar treatment conditions.
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122
The changes in actin cytoskeleton upon treatment under the experimental
conditions examined above were visualized by staining with Texas-red labeled
phalloidin, an agent that binds polymerized F-actin fibers (Chapter 5). Treatment
with Y-27632 and CytB showed loss of abundant stress fibers seen under control
conditions. Simultaneous immunostaining of the same cells for PAI-1 antigen
showed inhibition of PAI-1 expression in cells with less F-actin staining. This
experiment confirmed simultaneous decrease in F-actin content and PAI-1
expression upon treatment with inhibitors of actin polymerization.
Stabilization of actin cytoskeleton on TGFp-induced PAI-1 expression
If the hypothesis that the changes in actin cytoskeleton regulate PAI-1
expression is true and decreased stress fiber content of the cells blocks PAI-1
expression, then an increase in stress fibers should increase PAI-1 expression.
Jasplakinolide stabilizes the stress fibers thereby increasing the cellular stress
fiber content. Jasplakinolide increased basal PAI-1 expression and augmented
the stimulatory effect of TGF(3 on PAI-1 mRNA and protein expression (Chapter
5). Treating the cells with agents that increase the stress fiber content tested the
possibility that enhanced actin cytoskeletal assembly has an effect opposite to
that of disassembly on PAI-1 expression and essentially rules out the possibility
that the observed effects of actin depolymerization are artifacts of the use of
pharmacologic inhibitors The increase in stress fiber content caused by JAS
could not be visualized since it binds to the F-actin fibers and therefore interferes
with binding of phalloidin, the agent that helps to visualize F-actin [175]
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123
To ensure that the increase in PAI-1 expression upon JAS treatment was
associated with the stabilization of actin cytoskeleton, the cells were pre-treated
with inhibitors of actin polymerization prior to JAS treatment (Chapter 5). Co
treatment with actin depolymerizing agents such as CytB, Toxin B and Y-27632
blocked the stress fiber formation and reversed the effects of Jasplakinolide on
PAI-1 expression. Thus, the changes in actin polymerization correlate with
changes of PAI-1 expression. Taken together, these results indicate that Rho
modulates the ability of TGFp to regulate PAI-1 expression by controlling the
state of actin polymerization and that the actin cytoskeleton mediated changes in
TGFp-induced PAI-1 expression depend upon the degree of its organization.
The findings of this study are based on pharmacological agents. The
effects of pharmacological inhibitors may show non-specific effects. However,
multiple agents using unique mechanisms of action showed a similar effect
suggesting that the effects are specific to the treatment effect. Still, the use of
constitutively active or dominant negative mutants may provide further
confirmation of the observations.
The opposing effects of actin depolymerization and stabilization on PAI-1
expression are consistent with similar effects on a1 (I) collagen, a component of
ECM, by Schnaper et al [87], They showed inhibition of TGFp-mediated
expression of a1 (I) collagen by actin depolymerization [87], In the same study
they showed that stabilization of stress fibers with JAS antagonized the inhibitory
effect of actin depolymerization. These results also show that the degree of
organization of actin cytoskeleton correlates with net ECM deposition. Since
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124
collagen and PAI-1 both contribute to ECM accumulation, it is plausible that both
the genes are regulated by similar or coordinated mechanisms. Studies have
shown that Rho-kinase inhibition also inhibits PAI-1 expression in response to
other agonists such as Ang II [81]. This is consistent with the hypothesis that
changes in actin polymerization may act as a final common pathway that
modulates the effect of multiple extracellular stimuli on gene expression.
The effect of actin cytoskeleton on PAI-1 promoter activity
Once the role of actin cytoskeleton in regulation of PAI-1 expression was
confirmed, the subsequent studies aimed to determine the mechanism of this
regulation. Since PAI-1 expression is mainly regulated at the level of
transcription, the effects of actin cytoskeleton on PAI-1 transcription were
examined using human PAI-1 promoter (Chapter 6).
The mechanism of PAI-1 mRNA regulation by cytoskeleton was
investigated by examining the effects of actin depolymerization on PAI-1
promoter activity. First, an 1100 bp human PAI-1 promoter containing major
regulatory elements was cloned into a luciferase vector. The identity of the
cloned promoter was confirmed by sequencing. Due to difficulties in transfecting
primary cultured human mesangial cells, initial experiments were performed in rat
mesangial cells. The rat and human PAI-1 promoter contain similar regulatory
elements, albeit in different numbers. Dual luciferase assay system containing
firefly luciferase under the control of PAI-1 promoter and renilla luciferase under
minimal thymidine kinase promoter was used to transfect rat mesangial cells.
The renilla luciferase was used to normalize for transfection efficiencies. In the
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initial time-course experiment with TGFp, no changes in renilla luciferase were
obtained upon different treatments. Normalized PAI-1 values show that TGFp
increases PAI-1 promoter activity marginally in rat mesangial cells. The efforts to
reproduce similar experiment in human mesangial cells failed to show consistent
counts for renilla luciferase activity. Consequently, luciferase values from human
mesangial cells were normalized to the protein concentration of the sample. The
luciferase values were represented as luciferase count/pg protein. Studies with
human mesangial cells show ~250% increase in human mesangial cells. This
effect is consistent with previous studies with the same PAI-1 promoter [112].
This increase is also consistent with the increase in PAI-1 mRNA and protein
expression seen upon TGFp treatment. Since the fold-increase in PAI-1 promoter
and mRNA are similar, the regulation of mRNA seems to be at the transcriptional
level. These findings are consistent with previous reports suggesting PAI-1
mRNA is mainly regulated at the level of transcription [136, 137], Treatment with
CytB blocked basal as well as TGFp-induced PAI-1 promoter activity suggesting
critical requirement of organized actin cytoskeleton for PAI-1 expression.
Although it is not completely known how changes in actin polymerization
regulate PAI-1 promoter activity, there are several possible mechanisms that can
explain this regulation. Since PAI-1 promoter is rich in regulatory elements, it is
possible that actin depolymerization alters the binding of transcriptional factors to
the regulatory elements in PAI-1 promoter. Alternatively, the binding to yet
uncharacterized PAI-1 promoter elements may be altered by actin
depolymerization. As described previously in the thesis, there are potential
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126
similarities in the regulation of PAI-1 and a-SMA. The promoter region of a-SMA
contains CArG elements, which are sensitive to changes in actin polymerization
[176], Hautmann et al have shown cooperation between TGFP-responsive
elements and CArG elements for TGFp-induced expression of a-SMA [177],
Although, CArG elements have been shown only in uPA promoter so far, other
yet unidentified cytoskeleton-responsive elements may drive PAI-1 promoter in
concert with TGFp-response elements [178], Another possible mechanism may
involve Nuclear Factor Yin Yang 1 (YY1). In myofibroblasts YY1 represses
expression of a-SMA and TGFP-responsive genes such as PAI-1 [179]. Ellis et al
have shown that YY-1 competes with serum response factor (SRF) for binding to
serum response elements (SRE) in the a-SMA promoter. F-actin inhibits this
repressive effect of YY1 on a-SMA expression, thereby increasing a-SMA
expression [180]. Conversely, G-actin inhibits a-SMA expression by facilitating
repression by YY1. Similarly, YY1 competes with SMAD for binding to the PAI-1
promoter and blocks TGFp-induction of PAI-1 promoter [179]. Although, it is a
potential mechanism of regulation of PAI-1 expression by changes in actin
cytoskeleton, no direct evidence linking actin cytoskeleton and YY1-binding to
PAI-1 promoter has been shown so far. In future studies, this can be examined
by performing an electrophoretic gel mobility-shift assay on the nuclear extracts
from the cells treated with actin depolymerizing or stabilizing agents. Given the
facilitation of YY1 binding to promoter elements by G-actin, an increase in G-
actin content upon actin depolymerization is expected to result in the
displacement of SMAD binding to SMAD-binding sites by YY1, and vice versa by
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127
increase in F-actin content. Alternatively chromatin immunoprecipitation (ChIP)
assay can be used to identify interaction of YY1 or other potential regulatory
factors to PAI-1 promoter elements [181].
The effect of actin cytoskeleton on plasminogen activators
Having confirmed the regulation of PAI-1 expression by the actin
cytoskeleton, the regulation of other members of plasminogen activators system
was examined next. The effects of TGFp and actin cytoskeleton on tPA and uPA
expression were examined in the same set of samples that were used to study
PAI-1 expression.
An eight hour treatment with TGF(3 caused a small but statistically
significant inhibition of tPA mRNA expression (Chapter 6). This effect is
consistent with the role of TGF[5 as a profibrotic growth factor. Previously,
however, Baricos et al did not see any significant effect of TGF(3 on tPA protein in
cultured human mesangial cells at 72 hours [89]. The effect of TGF[3 observed on
tPA mRNA expression in the current study was small and remains to be
confirmed at the protein level. This small effect could represent the variability
among different mesangial cell isolates or the effect of differences between the
treatment periods. At the same time, inhibition of actin polymerization increased
basal tPA mRNA expression and reversed the inhibition exerted by TGF(3. This
finding is particularly interesting in that it shows coordinated regulation of the two
genes by the actin cytoskeleton. Simultaneous inhibition of PAI-1 expression and
upregulation of tPA allows tPA to act unopposed by PAI-1 resulting in a fibrolytic
state. These results are in agreement with previous studies showing increased
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128
tPA expression and fibrolytic activity by simvastatin, an HMG CoA reductase
inhibitor that inhibits Rho activation in human vascular smooth muscle cells,
endothelial cells and peritoneal mesothelial cells [173, 182, 183], These studies
provide indirect evidence that inhibition of Rho activation increases tPA
expression. The current study provides direct evidence that inhibition of Rho-
mediated actin polymerization increases tPA expression in human mesangial
cells and that there is a coordinated regulation of the plasminogen activation
system by the actin cytoskeleton. If the effect seen at the mRNA levels is
sustained functionally, it favors increased breakdown of ECM by tPA upon
treatment with actin depolymerizing agents, a potential utility in breaking down
ECM from sclerosed glomeruli of diseased kidneys.
The coordinated regulation of PAI-1 and tPA may result from the extensive
homology between 5’ -flanking regions of PAI-1 and tPA genes [115], Bosma et
al observed only six gaps during alignment of 521 positions of 5’ sense strand of
PAI-1 (non-coding) with 5’ antisense strand of tPA (coding). The presence of
extensive nucleotide homology, although on opposite strands, may allow
common regulatory factors to drive the expression of PAI-1 and tPA coordinately.
Accordingly, changes in actin cytoskeleton may change the binding of regulatory
factor to PAI-1 and tPA coordinately. In fact, a coordinated increase in the
expression of expression of PAI-1 and tPA has been shown in vivo in association
with stress, surgery and myocardial infarction; and in vitro by endothelial cells in
response to thrombine, histamine and glucocorticoids [115].
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129
On the other hand, uPA expression was increased by TGF(3 (Chapter 7).
The increase seen in uPA by TGFp is consistent with a similar increase shown
by Baricos et al. [89]. However, they showed that although TGFp increased both
uPA and PAI-1 expression, the net effect was inhibition of plasminogen activation
by PAI-1, suggesting that the increase in PAI-1 overcomes the increase in uPA in
human mesangial cells. Therefore, the TGFp-mediated increase in uPA mRNA
may not be functionally significant. In the current study, the net effect of TGFp on
ECM turn over was not examined. The effects of actin depolymerization on uPA
mRNA did not show statistical significance possibly because of too much data
variability.
Human mesangial cells produce smaller amounts of uPA and may not
respond consistently to TGFp-treatment [155]. Alternatively, increasing the
sensitivity of the mRNA detection protocol may minimize the data variation.
Overall, the observation that the actin cytoskeleton regulates tPA and PAI-1 but
not uPA in mesangial cells is consistent with the conclusions of Baricos et. al.
that tPA has a more important role in ECM turnover. They reported that loss of
uPA had no effect on plasminogen activation in mesangial cells from uPA knock
out mice. In contrast, tPA null mesangial cells showed defective plasminogen
activation [61],
The tPA and uPA proteins could not be detected from the conditioned
media using Western blot analysis despite multiple optimization attempts. The
amounts of tPA and uPA expression are much smaller as compared to PAI-1.
Using conditioned media from cultured human mesangial cells, Baricos et al
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130
showed a large molar excess of PAI-1 (1.2 ± 0.1 x 10'9 M) over uPA (1.2 ± 0.1 x
10'12 M) and tPA (0.19 ± 0.04 x 10'9M) [155]. The expression levels for PAI-1
mRNA (0.01 pg range) in the current study were also at least 10 fold higher than
those for tPA and uPA (0.001 pg range). These low levels of tPA and uPA protein
expression may be below the level of detection for Western blot analysis. In
future, more sensitive detection methods such as ELISA may be helpful in
detecting tPA and uPA from conditioned media.
Taken together, the results of tPA and uPA analysis show that TGF(3
inhibited tPA mRNA and increased uPA mRNA expression. Actin
depolymerization increased tPA expression under basal and TGF(3-treated
conditions. As discussed earlier, the PAI-1 expression is inhibited under similar
culture conditions. The coordinated regulation of tPA and PAI-1 by actin
cytoskeleton could be useful therapeutically in countering fibrosis.
In summary, this study supports the concept that actin cytoskeleton
can modulate gene expression by regulating intracellular signal transduction. It
provides initial evidence that the inhibition of actin polymerization may
coordinately block the expression of profibrotic genes (such as PAI-1) and
increase the expression of anti-fibrotic genes (such as tPa and uPA). Agents that
inhibit actin polymerization can be potentially beneficial in the treatment of
sclerotic diseases.
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131
CHAPTER 9
CONCLUSIONS AND FUTURE DIRECTIONS
Conclusions
In summary, TGFp increased PAI-1 expression in human mesangial cells.
Depolymerization of actin cytoskeleton using inhibitors of Rho and Rho-kinase
inhibited PAI-1 expression. The effects of Rho-inhibition were mimicked by direct
inhibition of actin cytoskeleton suggesting that the effects of Rho-inhibition were
mediated through actin cytoskeleton. On the contrary, stabilization of actin
cytoskeleton increased basal and TGFp-mediated PAI-1 expression. Thus bi
directional changes in the organization of actin cytoskeleton were followed by
changes in the PAI-1 expression suggesting modulation of gene expression by
the actin cytoskeleton. Inhibition of actin polymerization also blocked PAI-1
promoter activity suggesting transcriptional regulation of PAI-1 expression by the
actin cytoskeleton. In contrast to the inhibition of PAI-1 expression, tPA
expression was increased by te actin depolymerization by both direct inhibitors
and inhibitors of Rho-signaling. Thus, changes in actin cytoskeleton mediate the
effects of Rho-GTPases in modulating PAI-1 and tPA expression in response to
TGFp. The opposite regulation of PAI-1 and tPA suggests a coordinated gene
regulation by the actin cytoskeleton whereby they may control ECM degradation.
The results of the current studies with PAI-1 and tPA combined with the previous
work on a-SMA and hypertrophy suggest that changes in organization of the
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132
actin cytoskeleton regulate myofibroblast differentiation and ECM accumulation
by coordinately modulating expression of multiple genes.
Among the number of interesting questions that arise from these
observations, two particularly interesting ones were addressed first to explore
future directions. (1) What intracellular signaling pathways are regulated by
changes in actin polymerization during the modulation of TGFp-induced PAI-1
expression? (2) Can agents that inhibit actin polymerization, including inhibitors
of Rho, protect against fibrosis by inhibiting PAI-1 expression in vivo in animal
models? The first question was the logical next step in characterizing the
mechanism of cytoskeleton mediated gene regulation. The second question
addressed the clinical usefulness of the findings of this study.
Role of MAPK in TGFP-induced PAI-1 expression
Introduction
Results discussed in chapters 3 through 7 showed that the Rho-mediated
changes in actin cytoskeleton modulate induction of PAI-1 in response to
extracellular stimulus such as TGFp. The intracellular signaling pathways
involved in this regulation are not known. SMAD proteins, being the primary
intracellular mediators of TGFp, are potential candidates. However Tsuchida et al
recently showed that SMAD4, essential in SMAD-dependent target genes
responses, regulates only the early (2 hours) TGFp-mediated PAI-1 response
and not the sustained PAI-1 response in mesangial cells [184], Thus, it appears
that non-SMAD intracellular signaling pathways may also regulate TGFp-induced
PAI-1 expression. This observation is supported by findings that the effects of
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133
SMAD and mitogen-activated protein kinases (MAPKs), a group of serine-
threonine kinases consisting of three sub-families of intracellular signal
transducers, are additive in achieving maximum TGFp-dependent responses in
human mesangial cells [185]. TGFp-induced PAI-1 expression has been shown
to require MAPK in a cell-type specific manner [186-190], TGF(3-mediated PAI-1
expression requires extracellular signal regulated kinase (ERK) in astrocytes and
renal epithelial cells [188, 189], p38 in hepatocytes and fibroblasts [186, 190],
and c-jun-NH2 terminal kinase (JNK) in MvlLu epithelial cells [187], The
requirement of MAPK for TGF-P mediated PAI-1 expression has not been
examined yet in human mesangial cells.
Recently, Schnaper et al have showed that TGFp-induction of collagen
gene expression requires ERK activation [191]. Also, actin depolymerization
blocks TGFp-induced collagen expression [87]. Taken together these studies
suggest that actin depolymerization blocks TGFp-induced collagen expression by
inhibiting ERK activation. Similarly, TGFp may be activating MAPK to induce PAI-
1 in an actin cytoskeleton dependent manner. The hypothesis that the actin
cytoskeleton modulates TGFp-induced PAI-1 expression by modulating MAPK
activation was proposed. As a first step towards examining this hypothesis, the
requirement of MAPKs in TGFp-induced PAI-1 expression was examined in
human mesangial cells.
Working hypothesis
TGFp-induced PAI-1 expression in human mesangial cells requires
activation of MAPK pathway in a cytoskeleton dependent manner.
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134
Experimental model
To examine the requirement of MAPK in TGFP-mediated PAI-1
expression, pharmacological inhibitors of MAPK were used block MAPK
signaling. The efficacies of reported effective concentrations of MAPK inhibitors
were confirmed in human mesangial cells in the lab prior to the use. The
literature indicated that the effective doses were 30 pM for PD98059, the ERK
inhibitor [192], 5 pM for SB203580, the p38 inhibitor [193] and 10 pM for
SP600125, the JNK inhibitor [194], These kinase inhibitors were relatively
specific at the doses used in the study. PD98059 and SB203580 were found
specific inhibitors of ERK and p38 respectively with minimal inhibition of other
kinases at doses higher than those used in this study [195], SP600125 was
found >20-fold selective inhibitor of JNK at IC5o of 10 pM, the same dose as used
in this study [196].
To examine if one or more of the three MAPKs were required for TGFp-
induced PAI-1 activation in human mesangial cells, serum-starved mesangial
cells were pre-treated with MAPK inhibitors followed by TGFp-treatment. The
effects on PAI-1 mRNA and protein expression were examined.
Results
Since TGFP activates different MAPK modules depending on the cell type
and the target gene, the requirement of each of the three MAPK for TGFp
mediated PAI-1 expression in human mesangial cells was examined first. Serum-
starved human mesangial cells were pre-treated for one-hour with 30 pM
PD98029, 5 pM SB203580 and 10 pM SP600125 to inhibit ERK, p38 and JNK
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135
respectively. Cells were then treated with 10 ng/ml TGFp for 8 hours (mRNA
analysis) or 24 hours (protein analysis). Real-time PCR analysis showed that
inhibition of ERK, p38 and JNK changed PAI-1 mRNA expression in serum-free
media to 94.6 ± 10.8%, 87.2 ± 6.6% and 82.7 ± 14.0% of untreated control
respectively. None of the effects were statistically significant. However, ERK and
JNK inhibition blocked TGFp-induced PAI-1 expression to 64.7 ± 14.7% (p=0.06)
and 52.7 ± 4.7% (p<0.005) of TGFp-treated control respectively. Inhibition of p38
increased PAI-1 expression to 114.9 ± 5.7% of TGFp-treated control (p=0.05)
(Fig. 32).
Western blot analysis of conditioned media (n=3) showed that ERK and
JNK inhibition inhibited basal PAI-1 expression to 70.6 ± 13.2% and 64.3 ±
14.5%of untreated control respectively (p<0.05). Inhibition of p38 had no
significant effect on basal or TGFp-induced The PAI-1 protein expression (96.6 ±
12.4%). However, ERK and JNK inhibition blocked TGFp-induced PAI-1
expression to 49.5 ± 12.6% (p<0.005) and 44.4 ± 12.6% (p<0.001) of TGFp-
treated control respectively. Inhibition of p38 changed PAI-1 expression to
90.7 ± 5.0% of TGFp-treated control (Fig. 33).
From these results both ERK and JNK appear to be required for TGFp-
induced PAI-1 expression whereas p38 is not required for basal or TGFp-induced
PAI-1 expression.
Discussion
The main focus of this study was to identify which, if any, of the MAPK
was being activated by TGFp for PAI-1 regulation. Inhibitors of ERK and JNK
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136
— TGFp
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Fig. 32. Effect of MAPK inhibition on PAI-1 mRNA expression. Serum- starved human mesangial cells were pre-treated for one-hour with 30 pM PD98029, 5 pM SB203580 and 10 pM SP600125 to inhibit ERK, p38 and JNK respectively. Cells were then treated with 10 ng/ml TGFp for 8 hours followed by quantitative real-time PCR analysis of mRNA. The vertical bars and the error bars represent the mean value for PAI-1 mRNA and SEM respectively. The value for untreated control is set at 100%. *: p<0.005 compared to S-, f : p=0.06 compared to TGFp, t t : p=0.05 compared to TGFp, t t t : P<0.05 compared to TGFp (n=3).
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137
PAI-1---- ^
TGFp - - + + - - + + - - + + - - + +
s- PD98059 SB203580 SP600125
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Fig. 33. Effect of MAPK inhibition on PAI-1 protein expression. Serum- starved human mesangial cells were pre-treated for one-hour with 30 pM PD98029, 5 pM SB203580 and 10 pM SP600125 to inhibit ERK, p38 and JNK respectively. Cells were then treated with 10 ng/ml TGFp for 24 hours followed by Western blot analysis of conditioned media (n=3). Repersentative blot is shown in panel A. Panel B shows graphical representation of three separate experiments. The vertical bars and the error bars represent the mean value for PAI-1 protein and SEM respectively. The value for untreated control is set at 100%. *: p<0.05 compared to S-, **: p<0.005 compared to S-, f . p<0.01 compared to TGF(3, t f : p<0.005 compared to TGFp.
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138
blocked TGFp-induced PAI-1 mRNA and protein expression suggesting their
involvement in TGFP signaling. The requirement of ERK is consistent with
studies showing involvement of ERK in Angiotensin, vascular endothelial growth
factor and platelet derived growth factor induced PAI-1 expression [81, 197, 198],
Involvement of JNK is consistent with regulation of thrombin-induced PAI-1
expression by JNK [199], Overall, the findings are also similar to the results of
Schnaper et al showing activation of ERK and JNK but not p38 by TGFp in
human mesangial cells [191]. The requirements for ERK and JNK may result
from their roles in regulating AP-1 dependent transcription of PAI-1. In fact, a
study published during the preparation of this thesis showed a requirement of
MAPK/AP-1 activation for TGFp-induced PAI-1 expression in rat mesangial cells
[200]. This published result confirmed the original hypothesis proposed in this
lab.
The treatment with ERK and JNK inhibitors in serum-free media blocked
basal PAI-1 protein expression but did not change PAI-1 mRNA expression. This
finding suggests that under basal condition, post-transcriptional regulation of PAI-
1 may contribute to the reduction in protein levels by ERK and JNK inhibitors.
Conversely, inhibition of p38 resulted in a small but significant increase in TGFP-
induced PAI-1 mRNA expression. The increase may result from increased
activity of ERK and JNK upon p38 inhibition as shown by Ohashi et al [201].
In summary, the results confirm a requirement for ERK and JNK in TGFp-
induced PAI-1 expression in human mesangial cells. However, the specificity of
the effects of pharmacological inhibitors has to be ultimately confirmed using
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139
dominant negative mutants of each of the three MAPKs. Although the study is
focused mainly on identifying the role of MAPK as the intracellular mediator of
TGFp signaling, it is appreciated that TGFp signaling may involve other
intracellular signaling molecules such as protein kinase A (PKA) and protein
kinase C (PKC) for the regulation of PAI-1 expression. The activation of ERK and
JNK upon TGFp-treatment can be examined to show a direct evidence for the
role of MAPK in TGFp signaling. The role of actin cytoskeleton in modulation of
ERK and JNK activation can be studied by examining the changes in ERK and
JNK activation upon inhibiting or stabilizing the actin cytoskeleton. The
confirmation of the role of actin cytoskeleton in modulating MAPK activation in
TGFp-induced PAI-1 expression will explain the mechanism of action of
cytoskeleton modifying agents. Overall, the elucidation of specific mechanism of
gene regulation by the actin cytoskeleton will provide avenue for future research
into identifying more specific targets of actin cytoskeleton in the regulation of
intracellular signaling and gene expression.
Effect of Rho-kinase inhibition on sclerosis in chronic mesangiosclerotic
glomerulonephritis
Introduction
The results of cell culture experiments showed coordinated regulation of
two members of plasminogen activator system, PAI-1 and tPA, by the Rho-
mediated changes in actin cytoskeleton (Chapters 3 through 7). The functional
significance of these changes in plasminogen activator system was determined
next by examining the effect on ECM degradation in vivo.
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140
Regulation of ECM turnover is one of the major functions of the
plasminogen activator system [56]. Increased intraglomerular deposition of ECM
proteins resulting in glomerulosclerosis is a major feature of chronic renal
diseases. [202], Increased ECM deposition may result from inhibition of ECM
breakdown by TGFp-induced PAI-1 expression in chronic glomerulosclerotic
renal diseases [59]. The in vitro results of this study show that inhibition of Rho-
mediated actin polymerization inhibits TGFp-induced PAI-1 expression. In vivo
inhibition of ROCK by Y-27632 is also shown to protect against tubulointerstitial
fibrosis in a unilateral ureteral obstruction model [104], These observations
suggested the possibility that if Rho-kinase inhibitors had similar inhibitory effect
on PAI-1 expression in vivo, it could protect against ECM deposition and
glomerulosclerosis.
Working Hypothesis
Rho-kinase activation increases expression of PAI-1 compared to tPA and
uPA, and thus causes mesangial matrix expansion. Treatment of rats with Rho-
kinase inhibitor, HA-1077, will block these changes and provide a protective
effect against glomerulosclerosis
Experimental model
A single injection of anti-Thy 1.1 antibody, that reacts with a Thy 1-like
antigen on the surface of glomerular mesangial cells, leads to a disease
characterized by mesangial proliferation and, deposition of collagen and fibrin in
mesangium [203]. However, this injury is self-limited and resolves as early as 6
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141
weeks [204], Repeated injections of anti-Thy 1.1 antibody have been reported to
produce a more severe form of glomerulonephritis with unresolved
glomerulosclerosis akin to the lesions seen in chronic glomerulonephritis [202,
205], This model system was used to examine effectiveness of ROCK-inhibitor
in prevention and treatment of ECM accumulation in chronic renal diseases.
In order to cause mesangiaosclerotic glomerulonephritis rats were given
four weekly injections of Thy 1.1 antiserum (ATS) containing anti-Thy 1.1
antibody as described in the chapter 2. Each ATS injection was followed by
injection of goat serum to provide complement for fixation of the antibody. Also,
after four weekly injections, the animals were observed/treated for six more
weeks to produce progressive injury [206]. The Rho-kinase inhibitor HA-1077
was used in this study to treat the animals because of its specificity at the dose
used in the study, water-solubility for ease of administration and lower expense.
Moreover, it is already under clinical trials for the use in human patients suffering
from stroke suggesting its potential use in humans [207, 208], The
glomerulosclerotic injury was visualized by histology and immunohistochemistry.
Twenty-four hour urinary protein excretion and serum creatinine levels were
measured as indicators of glomerular injury.
Results
Histopathologic grading of glomerular ECM deposition and injury
The histological sections were stained by Masson trichrome method. The
slides were coded before the analysis. Sclerosis was graded on a scale of 0 for
normal to 3+ for diffusely increased trichrome-positive (blue) mesangial matrix
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142
material. Scores of 2+ and 3+ correspond to focally positive or intermediate
staining intensity of trichrome-positive glomeruli. A practicing renal pathologist
performed grading of trichrome-stained sections. The code was broken only after
completion of the grading. The average score of control rats was 0.8 ± 0.3 (s.e.)
while the group with ATS administration had average score of 1.7 ± 0.6. Rats
treated with HA-1077 had an average score of 1.0 both whether the therapy was
initiated before (s.e. 0.5) or after (s.e. 0.3) the first ATS injection (Table 5). While
these results suggest that the therapy with HA-1077 may have been beneficial,
none of these differences achieved statistical significance at a confidence level of
p < 0.05.
The sections were also stained for smooth muscle a-actin (a-SMA) to
examine myofibroblast differentiation in mesangial cells. For each animal, 100
glomeruli were sequentially analyzed at 40X objective. The glomeruli showing
clear non-hilar staining for a-SMA were counted as positive. The results were
similar to those seen with trichrome staining. The percentage of positive
glomeruli were: 0.8% ± 0.5% in normal control rats, 3.8% ± 2% in ATS-treated
rats and, 2.5% ± 0.6% in rats treated with HA-1077 before and after the ATS
injection (Table 5). Again, none of these results showed any statistically
significant difference between treated and untreated groups.
Effect on urinary protein excretion
Urinary protein results at the end of the study were consistent with production of
mild glomerular injury (Table 5). The control rats excreted 14.6 ± 1.7 mg protein/
24 h, ATS-treated rats excreted 35.0 ± 11.0 mg protein/ 24 h (p<0.05).
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143
Fig. 34. Effect of Rho-kinase inhibition on sclerosis in a rat model of chronic glomerulonephritis. Animals were divided into four groups as follows: Group 1: Normal saline injection alone (A), Group 2: ATS-complement injection(B), Group 3: ATS-complement+ HA-1077 treatment started 1 day before the first injection (C), Group 4: ATS-complement+HA-1077 treatment started 1 week after the first injection (D). Trichrome staining of representative glomeruli show increased mesangial sclerosis in group 2 (B) and, a relative decrease in groups 3(C) and 4 (D) suggesting a protective effect of Rho-kinase inhibition. The arrowheads mark mesangial ECM deposition stained blue by trichrome staining.
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144
Table 5. Effect of Rho-kinase inhibitor on experimental glomerulonephritis
Group 24-hour urinary protein
(mg)
Serumcreatinine(mg/dL)
Histologicalgrading
(0-3)
a-SMA staining
(% positive)
1.Control 14.6 ± 1.7 1.18 ± 0.16 0.8 ±0.3 0.8 ±0.5
2. Disease, no treatment
35.0 ±11.0 (p< 0.05) 1.41 ±0.2 1.7 ±0.6 3.8 ±2.0
3. Disease, Pre-treatment
19.0 ±2.1 (p=0.09) 1.07 ±0.14 1.0 ±0.5 2.5 ±0.6
4. Disease, post-treatment
24.42 ± 3.9 (p=0.12) 1.21 ±0.18 1.0 ±0.3 2.5 ±0.6
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145
Rats started on therapy with HA-1077 before the first ATS injection excreted 19.0
±2.1 mg protein/24 h (p=0.092 as compared to untreated group), whereas rats
started on therapy one week after the first injection had 24.42 ± 3.9 mg
protein/24 h (p=0.193 as compared to untreated group). SDS-PAGE analysis of
urinary proteins showed that albumin was the major urinary protein.
Effect on serum creatinine levels
Serum creatinine measurement at the end of the study followed the same
trends as urinary proteins, but again failed to reach statistical significance for the
treatment effect. These values were: control 1.18 ± 0.16 mg/dl, ATS only 1.41 ±
0.2 mg/dl, HA-1077 started before ATS; 1.07 ± 0.14 mg/dl; and, HA-1077 started
after ATS; 1.21 ± 0.18 mg/dl (Table 5).
Determination of drug efficacy
HA-1077 solutions used in the study effectively inhibited actin
polymerization in cultured human mesangial cells throughout the treatment
period suggesting that the drug was stable in water.
There were no significant differences between groups in kidney to body
weight ratio, blood pressure, daily water consumption and daily urine output.
Discussion
The results of the histologic scoring, a-SMA staining, urinary protein and
serum creatinine values all suggest the same conclusion, that the Rho-kinase
inhibitor, HA-1077 had a beneficial effect on glomerular injury in this chronic
model of mesangial sclerotic glomerulonephritis (Table 5). Although none of
these results individually achieved statistical significance, these studies provide
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146
initial evidence that a Rho-kinase inhibitor could be useful in the treatment of
glomerulosclerosis. In future optimization of the disease model may yield
statistically significant results. At this stage, this chronic model is difficult to
reproduce (personal communication between Dr. Glass, the principle
investigator, and other investigators in the field).
Several factors may contribute to the inability of the results to achieve
statistically significant differences between the study groups. First, the degree of
mesangial sclerosis was mild even in the most severely affected rats. Thus a
model with more severe mesangial injury will be necessary. Border et al. allowed
18 weeks (instead of 10 weeks as used in this study) for the progression of the
disease in their study [206]. Alternatively, more experimental models with more
invasive procedures such as surgical renal ablation could be used to produce
severe injury [209, 210]. Second, a larger study with more animals in each group
may help the trends seen in the result achieve a statistical significance. Third, the
effect of treatment was inconsistent, making it difficult to grade the injury even by
an experienced pathologist. Although the drugs used in these studies were active
and effective in blocking stress fiber formation in cell culture, their in vivo
effective dose could not be assessed. Optimization of therapeutic dose in this
particular model system could potentially give more consistent therapeutic
effects.
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147
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APPENDIX
PERMISSION OF THE AMERICAL PHYSIOLOGICAL SOCIETY
From: "Patel, Keyur MD" <[email protected]>To: '"pripka@ the-aps.org<[email protected]>Date: 11 /30/2005 5:57:14 AMSubject: Request for permission to use a figure for thesis preparation
Dear Sir/Madam,
I am a graduate student at Eastern Virgnia Medical School, (Norfolk,Virginia, USA)currently preparing my written thesis on " Modulation of TGF-beta induced PAI-1 expression by changes in actin polymerization in human mesangial ceils". I have to present a schematic diagram of PAI-1 promoter elements. I found "Figure 3: The PAI-1 promoter and consensus sequences" from the following articel by Binder et al. very useful."Binder, B.R., et al., Plasminogen activator inhibitor 1: physiological and pathophysiological roles. News Physiol Sci, 2002. 17: p. 56-61." PUBMED id: 11909993.
I have created a modified diagram based on the figure 3 of the article to show information pertinent to my thesis. I am hereby requesting a kind permission to use the modified (not the original) diagram for the thesis.Please find attached the original and modified diagram for your review.Also, I am acknowldeding at several places in my thesis that the diagram was modified from an original publication by Binder et al and giving a reference for the same.
Following is the figure legend I am planning to use with the modified figure pending your approval."Figure 21: Schematic representation of PAI-1 promoter.-RE, response element; TGFβ, transforming growth factor: VLDL, very low density lipoprotein; CTF, CCAAt box binding transcription factor; NF1, nuclear factor 1; PMA, phorbol 12-myristate 13-acetate; AP, activator protein; TRE, tetradecanoyl phorbol acetate response element; GRE, glucocorticoid response element. Adopted with modification from Binder et al [Binder, 2002 ]."
I will really appreciate your prompt and favorable consideration of my request. Your approval will help me immensely in completing my thesis in time for submission in December.
Sincerely,
Keyur Patel, MD (Ph.D. Candidate)
THE AMERICAN PHYSIOLOGICAL SOCIETY 9650 Rockvllk Plkt- BHhesda, \1D 20814-3991
Permission is granted for use of the material specified above provided tbe publication is credited as the source, including the words ’‘used with permission.’’
Haj l a j w g g M 'oPHbliralioiis Manager & Executive Editor
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CC: "Patel, Keyur MD" <KPatel1 @ LIJ.EDU>
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VITA
Name: Keyur Pravinchandra Patel
Title: Graduate StudentDepartment of Pathology and Anatomy Eastern Virginia Medical School 700 W Olney Rd Norfolk, VA 23507
Education: 1999-present Ph.D. Candidate,Biomedical Sciences Ph.D. Program(Cell Biology and Molecular Pathogenesis Track)Eastern Virginia medical school, Norfolk, VA, USA 1993-1999 M.B.B.S. (Bachelor of Medicine, Bachelor of Surgery), Smt. N.H.L. municipal medical college, Ahmedabad, India
Awards: Young Pathologist Fellowship, American Society ofClinical Pathologists, 2004Best Graduate Student Poster, Cardiovascular Focal Research Group, Annual Research Day, Eastern Virginia Medical School, 2003
Publications:-Patel K., Harding P., Haney, L.B., and Glass W.F.II (2003) Regulation of the mesangial cell myofibroblast phenotype by actin polymerization. Journal of Cellular Physiology 195(3), 435-445- Vlahou A., Schellhammer P.F., Mendrinos S., Patel K., Kondylis F.I., Gong L., Nasim S., and Wright G.L.Jr. (2001) Development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine. American Journal of Pathology 158(4), 1491-502Oral Presentation:- Keyur P. Patel, Pamela Harding, William F. Glass II (2002) Regulation of (a- SMA Expression and Hypertrophy by Changes in Actin Polymerization in Mesangial Cells. Annual Virginia Nephrology Retreat, Charlottesville, VA Poster Presentations:- Keyur P. Patel. Pamela Harding. William F. Glass II (2004) Changes in actin polymerization regulate plasminogen activator inhibitor type-1 (PAI-1) expression in human mesangial cells. Experimental Biology, Washington, DC- Keyur P. Patel, Pamela Harding, William F. Glass II (2002) Regulation of (a- SMA expression and hypertrophy by changes in actin polymerization in mesangial cells. American Society of Nephrology Renal Week, Philadelphia, PA- Keyur P. Patel, Pamela Harding, Lisa B. Haney, Ping Kong, William F. Glass II (2001) Regulation of smooth muscle a-Smooth Muscle Actin expression by tyrosine kinases and actin polymerization in human mesangial cells. World Congress of Nephrology Renal Week, San Francisco, CA
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