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Regulation of the versican gene : implications for vascular health and disease Rahmani, Maziar 2007

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REGULATION OF THE VERSICAN GENE: IMPLICATIONS FOR VASCULAR HEALTH AND DISEASE  by  MAZIAR RAHMANI M.D., Babol University of Medical Sciences, Babol, Iran 1996  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES  (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA December 2007 Maziar Rahmani, 2007  ABSTRACT Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix and hence plays a central role in tissue morphogenesis and a number of pathologic processes. My main goal has been to investigate the mechanisms of versican gene regulation, focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans- factors. This thesis puts forth new knowledge regarding transcriptional regulation of the human versican gene. In chapter III, I present the cloning of a 752-bp fragment of the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments in the PGL3luciferase reporter plasmid. Furthermore, I identify three potential enhancer and two repressor regions in this promoter. I also demonstrate that both cAMP and C/EBPf3 enhanced and repressed versican transcription in HeLa cells and rat aortic smooth muscle cells (SMC), respectively, suggesting that versican transcription is differentially regulated by the respective mediator and transcription factor in epithelial cells and SMC. In chapter IV, I reveal the role of PI3K/PKB/GSK-30 signaling pathway in regulating versican promoter activity and transcription. Furthermore, I identify that the 0-catenin/TCF-4 transcription factor complex, one of the downstream targets of GSK-3[3, mediates versican promoter activity and transcription. In chapter V, I identify that variations in C-terminal regions of TCF family members determine their repressor or enhancer properties on Wnt target genes. Furthermore, I show that curcumin is a strong inhibitor of the P-catenin/TCF-p300 mediated gene expression. In chapter VI, I demonstrate that the androgen receptor trans-activates versican transcription in prostate cancer cells. Furthermore, I show cross-talk between the androgen receptor and 13-catenin in regulating versican transcription in prostate stromal fibroblasts. Overall, this study charts previously uncharacterized promoter elements, transcription factors, and signal transduction pathways involved in regulation of the versican gene. ii  TABLE OF CONTENTS  Abstract ^  ii  List of Tables ^  xi  List of Figures ^  xii  Abbreviations ^  xv  Dedication ^  xviii  Acknowledgements ^  xix  Co-Authorship Statement ^  xxii  CHAPTER I BACKGROUND ^ 1.1. Vascular functions and diseases ^  1 1  I.1.1. Structure of a normal large artery and extracellular matrix ^ 1 1.1.1.1. Proteoglycans in the vessel wall ^ 1.1.2. Atheromatous diseases ^ 1.1.2.1. Response-to-retention hypothesis-implications for vascular diseases ^ 1.2. Versican — structure and function ^  4 7  7 10  1.2.1. The versican gene, protein and glycosaminoglycan chains structure ^ 10 1.2.2. Function of versican in health and diseases ^ 1.3. Regulation of versican ^ 1.3.1. Signal transduction pathways ^  13 15 15  1.3.1.1. Receptor tyrosine kinase-mediated versican expression ^ 15 1.3.1.2. The PI3K—PKB pathway and versican gene regulation ^ 16 1.3.1.3. Glycogen synthase kinase-3 and versican gene regulation ^ 17 1.3.1.3.1. Activation and inactivation of GSK-3 ^ 21 iii  ^  1.3.1.4. Wnt signaling and versican gene regulation ^ 22 1.3.1.4.1. Wnt signaling and GSK-3 ^  24  1.3.2. Transcription factors involved in versican gene regulation ^ 25 1.3.2.1. f3-catenin/TCF ^  25  1.3.2.2. Other factors ^  26  1.4. Canonical and non-canonical Wnt signaling pathway ^ 1.4.1. Wnt signaling and cellular development in cardiac and vascular systems ^  27  29  1.4.2. Wnt, vascular regeneration, and vascular injury ^ 30 I.S. f3-catenin - binding partners and roles in transcription ^  32  1.5.1. Binding choices at the 13-catenin protein interaction hub ^ 32 1.6. T-cell factor family of DNA-binding proteins ^  34  1.6.1. Structure and protein domain functions of TCF proteins ^ 36 1.6.1.1. DNA-binding and promoter recognition ^ 36 1.6.1.2. Context-dependent activation and repression domains ^ 38 1.6.2. Protein-protein interaction ^  40  1.6.2.1. 13-catenin/Armadillo ^  40  1.6.2.2. Groucho-related genes/Transducin-likeenhancer-of-split (Grg/TLE) ^  40  1.6.2.3. C-terminal binding protein ^  41  1.6.2.4. CREB-binding protein/p300 ^  42  1.6.3. Post-translational modifications ^ 1.6.3.1. Phosphorylation and acetylation ^ 1.7. Androgens and androgen receptors in prostate ^  iv  43 43 47  1.7.1. Androgen and androgen receptor in normal prostate development and maintenance of prostate epithelia ^  48  1.7.2. Versican and prostate cancer ^  49  1.8. References ^  51  CHAPTER II RATIONALE, HYPOTHESIS, AND SPECIFIC AIMS ^ 69 11.1. Rationale ^  69  11.2. Overarching hypothesis ^  70  11.3. Specific aims ^  71  11.4. References ^  73  CHAPTER III IDENTIFICATION OF POSITIVE AND NEGATIVE REGULATORY ELEMENTS OF THE HUMAN VERSICAN GENE PROMOTER: ROLE OF C/EBP AND CREB ON VERSICAN TRANSCRIPTION ^ 74 111.1. Summary ^  74  111.2. Introduction and rationale ^  76  111.3. Experimental procedures ^  80  111.3.1. Alignment and database-assisted human versican promoter analysis ^ 80 111.3.2. Primer design, polymerase chain reaction amplification, and generation of promoter luciferase reporter and deletion constructs ^ 80 111.3.3. Tissue culture ^  83  111.3.4. Plasmid transfection of primary cells and cell lines ^ 83 111.3.5. Dual luciferase reporter and other promoter assays ^ 83 111.3.6. f3-galactosidase assay ^  84  111.3.7. Protein quantification ^  85  111.3.8. Statistical analysis ^  85  111.4. Results ^  85  111.4.1. Identification of potential regulatory elements within the human versican promoter ^  85  111.4.2. Versican promoter construct design ^  86  111.4.3. Characterization of repressor and enhancer elements in human versican promoter ^  87  111.4.4. Differential effect of cAMP inducers, Forskolin and (Bu)2cAMP, on the versican promoter in HeLa and SMC ^ 89 111.4.5. Identification of cAMP-response elements in the human versican promoter ^  89  111.4.6. Involvement of C/EBP family members in the constitutive and cAMP-mediated expression of human versican promoter activity ^  90  111.4.7. Activation of versican promoter by exogenous CREB ^ 91 111.5. Discussion ^  92  111.6. Conclusions ^  105  111.7. References ^  121  CHAPTER IV REGULATION OF THE VERSICAN PROMOTER BY THE JICATENIN/TCF COMPLEX IN VASCULAR SMOOTH MUSCLE CELLS ^ 129 IV.1. Summary ^  129  IV.2. Introduction and rationale ^  129  IV.3. Experimental procedures ^  133  IV.3.1. Isolation and primary culture of rat aortic SMC ^ 133 IV.3.2. Generation of promoter reporter, mutant, and deletion constructs ^ 134 IV.3.3. Plasmid constructs ^  134  IV.3.4. Transfection and luciferase activity assays ^  135  IV.3.5. Immunoblotting ^  135  vi  IV.3.6. RNA extraction and cDNA synthesis ^  136  1V.3.7. SYBR-green quantitative real-time reverse transcriptasepolymerase chain reaction ^  136  IV.3.8. Electrophoretic mobility shift assay ^  137  IV.4. Results ^  138  IV.4.1. The 3 phosphoinositides-dependent signaling mediates versican transcription in vascular SMC ^  138  IV.4.2. PKB is a downstream effector of PI3K in serum stimulation of versican transcription in SMC ^  140  IV.4.3. Cytoplasmic accumulation of P-catenin via GSK-313 inhibition stimulates transcription of versican in SMC ^ 141 IV.4.4. LEF/TCF DNA binding is involved in I3-catenin-induced versican transcription ^  141  IV.4.5. The versican promoter is activated by the 13-catenin-TCF complex ^ 142 IV.4.6. TCF-4 binds to the versican LEF/TCF site ^  144  IV.5. Discussion ^  144  IV.6. Conclusions ^  148  IV.7. References ^  163  CHAPTER V ISOFORMS OF LEF/TCF TRANSCRIPTION FACTORS DIFFERENTIALLY CONTROL TRANCRIPTION OF WNT TATRGET GENES ^ 169 V.1. Summary ^  169  V.2. Introduction and rationale ^  170  V.3. Experimental procedures ^  173  V.3.1. Cell culture and reagents ^  173  V.3.2. Plasmid constructs ^  174  V.3.3. Transfection and luciferase activity assays ^  174  vii  175  V.4. Results ^  V.4.1. 13-catenin and p300 are different in their ability to regulate synthetic TCF reporter (pTOPFLASHx8-Luc) than endogenous Wnt-response promoters, versican-Luc 176 V.4.2. TCF isoforms with various C-terminal lengths differ in their ability to support Wnt/O-catenin/p300-mediated activation of the Wnt-target promoters ^  177  V.4.3. Histone deacetylase inhibitor TSA potentates the induction of Wnt- response genes in a Wnt-dependent but not p300-independent manner ^  179  V.4.4. HDAC inhibitor, TSA, induced Wnt-responsive versican gene in a TCF isoform-independent manner ^  181  V.5. Discussion ^  182  V.6. Conclusions ^  195  V.7. References ^  206  CHAPTER VI ANDROGEN RECEPTOR REGULATION OF THE VERSICAN GENE THROUGH AN ANDROGEN RESPONSE ELEMENT IN THE PROXIMAL PROMOTER ^  214  VI.1. Summary ^  214  VI.2. Introduction and rationale ^  215  VI.3. Experimental procedures ^  215  VI.3.1. Tissue culture ^  218  VI.3.2. Oligonucleotides ^  218  VI.3.3. Electrophoretic mobility shift assay ^  218  VI.3.4. Probe generation for DMS in vitro footprinting ^ 219 VI.3.5. DMS in vitro footprinting: methylation protection analysis ^ 219 VI.3.6. Western blotting ^  219  VI.3.7. Plasmid construction ^  220  viii  VI.3.8. Short hairpin RNA against AR ^  220  VI.3.9. Transfections and luciferase assay ^  220  VI.3.10. Database analyses ^  221  VI.3.11. Immunohistochemical analysis ^  221  VI.3.12. Statistical analysis ^  222  VI.4. Results ^  ii  VI.4.1. Versican proximal promoter luciferase reporter vector responds to steroid stimulation ^  222  VI.4.2. Database searches reveal two potential SRE candidates within the versican proximal promoter ^  223  VI.4.3. Methylation protection reveals the interaction of the AR DNA-binding domain to SRE-2 of the versican promoter ^ 223 VI.4.4. AR binds to versican SRE sites in vitro ^  224  VI.4.5. Mutation of SRE-2 interferes with androgen stimulation of the versican promoter activity ^  224  VI.4.6. Knock-down of AR expression reduces ligand-dependent and ligand-independent versican promoter activity^ 225 VL4.7. Cross-talk between AR and 13-catenin/TCF signaling is essential for optimal transactivation of Wnt- and AR-responsive promoters in prostate fibroblast cells ^ 225 VI.4.8. Versican immunolocalizes to the stroma of prostate gland and increases in human prostate cancer ^ 227 VI.5. Discussion ^  228  VL6. Conclusions ^  232  VI.7. References ^  244  ix  CHAPTER VII CONCLUDING REMARKS AND FUTURE DIRECTIONS ^ 249 APPENDIX I LIST OF PUBLICATIONS, PRESENTATIONS, AND AWARDS ^ 259  LIST OF TABLES Table 1^Oligonucleotides ^  xi  120  LIST OF FIGURES Figure I-I. The main components of the vascular extracellular matrix ^ 2 Figure 1-2. Schematic representative of major components of the intimal extracellular matrix ^  5  Figure 1-3.^Apolipoproteins, oxidized lipids and versican co-localize in arteries from human heart allografts ^ 8 Figure 1-4. Pathogenic concept of atherosclerotic vascular diseases — known or proposed roles of proteoglycans and lipids ^ 9 Figure 1-5.^The versican gene and protein structure ^ Figure 1-6.  12  Schematic representation of growth factor/PI3K/PKB signaling and partial list of downstream molecules ^  18  Figure 1-7.  Regulators of GSK-313 and its role in cellular function ^  20  Figure 1-8.  Schematic representation of Wnt/13-catenin/TCF signaling ^ 23  Figure 1-9.  Binding choices at the 13-catenin protein interaction hub ^ 33  Figure 1-10.  General structure of TCF proteins and binding partners ^ 37  Figure I-11.  Androgen action in the prostate ^  47  Figure III-1. Sequence map, potential and known transcription factor binding sites within the 632 to +118 by region of the human versican gene ^ 108 Figure 111-2. Schematic representation of identified and potential transcription factor-binding sites within the versican 5' flanking sequence and regions encompassed by construct generation 110 Figure 111-3. Human versican reporter luciferase and serial deletion construct design ^ 111 Figure 111-4. Deletion analysis identified enhancer and repressor regions in the -632 to +118 by of the human versican promoter ^ 113 Figure III-5. cAMP responsiveness of human versican promoter construct ^ 114 Figure 111-6. Regulatory regions in the human versican promoter mediate cAMP-induced inhibition of versican promoter activity in RASMC ^ 116 Figure 111-7. C/EBP a and 13 isoforms significantly enhance the versican promoter activity of the luciferase reporter ^ xii  117  Figure 111-8. cAMP converts the enhancement of versican promoter activity by C/EB113 to repression in RASMC, but not in HeLa cells ^ 118 Figure 111-9. CREB enhances versican promoter activity through cAMP dependent and independent mechanisms in RASMC ^ 119 Figure IV-1. PI3K is involved in versican promoter activity and transcription in SMC ^ 149 Figure IV-2. PTEN inhibits versican promoter activity in PC3 cells ^ 151 Figure IV-3. Overexpression of dominant negative PKB and constitutively active PKB blocks and augments induction of versican promoter reporter ^ 152 Figure IV-4. GSK-3f3 inhibition stimulates transcription of versican in SMC ^ 154 Figure IV-5. The LEF/TCF binding sequence at position -492 is crucial for activation of the versican promoter by the f3-catenin-TCF complex ^ 156 Figure IV-6. The versican promoter is activated by13-catenin/TCF complex ^ 159 Figure IV-7. EMSA of a potential TCF-4 binding site in the versican promoter ^ 161 Figure IV-8. Hypothetical model of versican promoter regulation via PI3K/PKB signaling and f3-catenin/TCF transcription factor complex ^ 162 Figure V-1.  Transcriptional regulatory domains of LEF/TCF isoforms and schematic representation of natural and chimeric TCF isoforms employed in this study ^  198  Figure V-2.  f3-catenin and p300 differ in their ability to regulate pTOPFLASHx8-Luc reporter and endogenous Wnt-response promoter, versican-Luc ^ 199  Figure V-3.  TCF isoforms with various C-terminal lengths differ in their ability to support Wnt-3a/p300-mediated activation of the pTOPFLASHx8-Luc reporter ^ 201  Figure V-4.  TCF isoforms differentially regulate f3-catenin/p300-mediated activation of the Wnt-response promoter, versican-Luc ^  202  Figure V-5.  Histone deacetylase inhibitor, TSA, potentiates the effect of Wntbut not p300-mediated induction of Wnt-response genes ^ 203  Figure V-6.  HDAC inhibitor, TSA, induces the Wnt-responsive versican gene in a TCF isoform-independent manner ^  205  Figure VI-1. Androgens activate versican promoter luciferase reporter in LNCaP prostate cancer cells and HeLa cells engineered to express androgen receptor ^ 234  Figure VI-2. Sequence analysis of the versican gene promoter and exon 1 ^ 236 Figure VI-3. Identification of in vitro androgen receptor binding sites in the versican promoter by Dimethylsulfate Methylation-Protection (MeP) footprinting ^ 237 Figure VI-4. Determination of the binding specificity of His-AR2 protected region ^ 238 Figure VI-5. The steroid response element binding sequence at position +75 is crucial for AR ligand-dependent activation of the versican promoter ^ 239 Figure VI-6. Cross-talk between 13-catenin and AR signaling is required for AR regulation of Wnt- and AR-responsive promoter reporters in prostate stromal fibroblast cells ^ 241 Figure VI-7. Stromal versican staining intensity in prostate tissues demonstrated by immunohistochemistry ^ 243 Figure VII-1. Signal transduction pathways and transcriptional control of the versican gene ^  xiv  250  ABBREVIATIONS Abbreviation AI AJs Ang APC AR ARE ARLBD Arm ARNT f3BD p-TrCP1 bFGF BPH bZIP 13-gal CaMK cAMP CA-PKB CREB CKI CBP C/EBP C/Edn CRE CS CSPG CSS CtBP DHT DMEM DMSO DN-PKB ANTCF-4  Definition androgen independence adherens junctions angiotensin adenomatous polyposis coli androgen receptor androgen-response elements androgen receptor ligand binding domain Armadillo aryl hydrocarbon receptor nuclear translocator 13-catenin binding domain 13-transducin repeat-containing protein 1 (beta-TrCP1) basic fibroblast growth factor prostatic hyperplasia basic region leucine zipper 13 galactosidase Ca2±/calmodulin-dependent kinase cyclic adenosine monophosphate constitutively activated mutant of PKB1 vector cAMP-responsive element-binding protein casein kinase I CREB-binding protein CCAAT/enhancer binding protein C/EBP dominant negative CREB response element newborn calf serum chondroitin sulfate proteoglycans charcoal stripped serum C-terminal binding protein dihydrotestosterone Dulbecco's modified Eagle's medium dimethyl sulfoxide dominant negative mutant of PKB1 vector TCF-4 dominant negative mutant -  -  DSPG DTT Dvl ECM EC EGFR EMSA ERK E2F FBS FRP FSH Fz GAG GFP GRE Grg/TLE GS GSK HAT HEK293 HDAC HDACis HIF HeLa His-AR-DBD HMG HSF HSPG IFN IGF-1 IL-1 IRF KSPG LAD LDL LEF/TCF LH LNCaP LPL LRP5/6 MAPK MAZ MCDB-131 MeP  dermatan sulfate proteoglycans dithiothreitol Dishevelled extracellular matrix endothelial cells epidermal growth factor receptor electromobility shift assays extracellular regulated kinase family of transcription factors in higher eukaryotes fetal bovine serum frizzled-related proteins follicle stimulating hormone Frizzled glycosaminoglycans green fluorescent protein glucocorticoid response element Groucho related genes/transducin-like-enhancer-of-split glycogen synthase glycogen synthase kinase histone acetyltransferase human embryonic kidney-293 histone deacetylase inhibitors of HDAC hypoxia inducible factor The cell line was derived from cervical cancer cells taken from Henrietta Lacks, who died from her cancer in 1951 His tagged androgen receptor DNA binding domain high-mobility group heat shock factors heparan sulfate proteoglycans Interferon Insulin-like growth factor 1 Interleukin-1 interferon regulatory factors keratan sulfate proteoglycans left anterior descending low-density lipoprotein lymphoid enhancer factor/T-cell factor leutinizing hormone Androgen-sensitive human prostate carcinoma cells derived from the lymph node metastasis from a 50-year-old Caucasian male Lipoprotein lipase LDL receptor-related protein 6 mitogen activated protein kinase MYC-associated zinc finger protein Molecular and cellular developmental biology 131 Methylation protection -  -  xvi  MITF MMP mTOR MZF NEFA NF-KB NLK PAI PC3 PCP PCR PDGF PI3K PKA PKB PLA2 PG PKC PSA PSA-Luc PTEN RASMC RTK sFRP siRNA SMC SRE TCRa TSA TGF-131 TLR VEGF VCN-632-Luc Wnt  microphthalmia-associated transcription factor matrix metalloproteinase mammalian target of rapamycin myeloid zinc finger nonesterified fatty acids nuclear factor (NF)-KB Nemo-like kinase plasminogen activator inhibitor The prostate cancer cell line was derived from a brain metastasis planar cell polarity polymerase chain reaction platelet derived growth factor phosphatidylinositol 3-kinase protein kinase A protein kinase B phospholipase A2 proteoglycans protein kinase C prostate-specific antigen prostate-specific antigen-Luciferase reporter vector phosphatase and tensin homolog deleted on chromosome 10 rat aortic smooth muscle cells receptor tyrosine kinase secreted frizzled related proteins short interfering RNA smooth muscle cells steroid receptor element T-cell receptor alpha Trichostatin A transforming growth factor-f31 Toll-like receptors vascular endothelial growth factor luciferase reporter vector contains -632/+118 by of the human versican promoter region wingless  xvii  DEDICATION  A very special dedication of this piece of myself to the two people who meant the most to me... my Mom, Mahrokh, and my Dad, Mohsen. Sadly, he left this earthly realm in 1997.  To my Mom and Dad, who found their happiness in my life's happiness. Who were my biggest champions and cheerleaders. Who, over the years, embodied the spirit of compassion and caring towards others. Who were always there with the advice when I needed it. Who worked hard to make sure we had what we needed. But most of all, who worked hard to ensure we knew they loved us and supported us, even when they may not have agreed with the directions we took.  Dad^ I will miss your spirit, support and love in my life as I continue serving as a scientist and physician around the world. You always believed in me, supported me and your last words were, "I love you and your mom, and I am so proud of you." I am so thankful my last words to you were, "I love you, Dad!"  xviii  ACKNOWLEDGMENTS During the course of my graduate career there have been many people who have offered me advice and encouragement that has helped me along the way. I would like to thank several people for their help not only in the work illustrated within the pages of this thesis, but for their overall contribution to my experience as a graduate student during years at University of British Columbia. No words can grasp the admiration and respect I hold for my mentor Dr. Bruce McManus. Bruce helped me get through all the frustrating and difficult moments of graduate school through his steady example, constant encouragement, and true faith in my abilities. Bruce is a patient and optimistic man who provided an excellent learning environment with his scientific insight, guidance, advice and lots of laughter. During the time I've spent in Bruce's lab, I have accumulated so many memories and stories that I can't help but laugh when I think back. Honestly, Bruce made coming to the lab a pleasant experience and I owe him many thanks for being my mentor. I wish also to acknowledge two of my previous mentors Dr. Fereidoun Azizi from Endocrine Research Centre of Shaheed Beheshti University of Medical Sciences and Dr. Ahmad Khaleghnejad from Babol University of Medical Sciences in Iran. They gave me the great opportunity to work in research very early in my career, in which their continuous encouragement throughout those years had made a challenging and often difficult journey rewarding and enjoyable. Their roles have been the critical basis for the qualities that I hope to acquire and profit from in my future endeavourers in medicine. I would also like to offer my thanks to the members of my thesis committee; Drs. Shizu Hayashi, Christopher Overall, Paul Rennie, Cheryl Wellington, David Walker, and Decheng Yang for their insightful discussions of my project, for all of their guidance and spur of the  xi x  moment meetings, and for making my committee meetings and defense very positive experiences. I would like to thank the staff and faculty of the Department of Pathology and Laboratory Medicine, especially graduate advisor, Dr. David Walker, and Ms. Penny Woo, for always being patient and understanding in handling the numerous administrative problems I encountered over the years. My thanks extend also to our precious collaborators: Drs. Jason Read and Dr. Paul Rennie from The Prostate Center at Vancouver General, University of British Columbia for their contribution to the work presented in this thesis. Many thanks to several scientists from around the world providing me the plasmids and reagents that without their generous gifts the completion of this thesis was impossible. I want to extend my immense thanks to Elizabeth Walker for her work and ideas, and her willingness to keep our lab functioning. Heartfelt thanks to all past and current members of Dr. McManus lab and The iCAPTURE Centre at St. Paul's Hospital for making the work environment a pleasurable one, for their advice, technical assistance and friendship. I cannot thank Dr. Decheng Yang, Dr. Aikun Wang and Ms. Zongshu Luo enough for sharing their expertise and knowledge with me in the beginning to familiarize me with cell and molecular biology techniques necessary to achieve my goals presented in this thesis. I also want to thank Dr. Paul McDonald for his technical and scientific contributions to me when I first joined the lab. Especially, I would like to thank my dear friends: Brian Wong, Caroline Cheung, Bobby Yanagawa, Hubert Walinski, Paul Cheung, and John Lai for sharing my happiness and sadness and for their encouragement and support; and students of our laboratory specially Parveer Pannue, Jon Carthy, Seti Boroomand, Lisa Ang, and Julie Ng for their friendship and valuable technical assistance. I am also forever grateful to Dr. Peter Pare and Dr. Bruce McManus for allowing me to be an investigator in a multi-disciplinary and multi-institutional research study led by Dr. Pare at  xx  the iCAPTURE Centre (UBC): "Gene-Environment Interactions in Circulatory and Pulmonary Diseases". This program greatly improved my research skills and self-confidence, brought me many memorable moments, and certainly contributed to my love for science and good times as a graduate student. Dr. Pare's dynamism and optimism are contagious and valuable, and I truly enjoyed working with him and his group apart from my thesis-related research in the past several years. I whole-heartedly thank my dear wife, Sima, and my darling daughter, Nazgol, for all of their love and support through these years, and for the peace, which comes from the ability to exchange ideas and frustrations about a day's work. Lastly, I thank my sisters Marjan and Mona for their encouragement. I am eternally forever grateful to my awe-inspiring mother, Mahrokh, and father, Mohsen, for their character, values, and work ethic they have installed in me, and for their countless sacrifices throughout the years to ensure my success, for which I hope I am able to obtain. And I dedicate this to the memory of my father, Mohsen -1 loved to hear from him to call me son go far as it takes and I am behind you. The work presented in this thesis was supported in part by a Grant-in-Aid from the Heart and Stroke Foundation of British Columbia and Yukon, Doctoral Research Awards from the Heart and Stroke Foundation of Canada, Graduate Fellowship of University of British Columbia, and Doctoral Award from Ministry of Health and Medical Education of Iran.  xxi  Co-Authorship Statement  Chapter IV is a slightly modified version of a paper published in The Journal of Biological Chemistry [Rahmani M, Read JT, Carthy JM, McDonald PC, Wong BW, Esfandiarei M, Si X, Luo Z, Luo H, Rennie PS, McManus BM. Regulation of the versican promoter by the 13-catenin-T-cell factor complex in vascular smooth muscle cells. J Biol Chem. 2005;280(13):13019-28]. The paper was co-authored with other researchers. Overall guidance and directions for the research was provided by me. I designed and performed all experiments unless mentioned below. I also collected and analyzed all data and wrote the manuscript. Jason T. Read assisted in preparation of the Figure IV-7. Jon M. Carthy aided in the experiment of Figure IV-4. Paul C. McDonald prepared the primary rat aortic SMC. Brian W. Wong, Mitra Esfandiarei, Xiaoning Si, and Zongshu Luo prepared some plasmids. Bruce M. McManus, Paul S. Rennie and Honglin Luo made intellectual contribution to the design and final editing of the manuscript. Chapter VI is a slightly modified version of a paper published in The Journal of Biological Chemistry [Read JT, Rahmani M, Boroomand S, Allahverdian S, McManus BM, Rennie PS. Androgen receptor regulation of the versican gene through an androgen response element in the proximal promoter. J Biol Chem. 2007;282(44):31954-63.]. This manuscript is the results of equal contribution of Jason Read and me. Preparation of the manuscript involved effort by both co-first authors (Jason Read and Maziar Rahmani). Initial writing and development work was carried out by me. Writing and editing work was done by both authors, with my initial focus on writing and Jason's on editing. Paul S. Rennie and Bruce M. McManus made intellectual contribution to the design and final editing of the manuscript.  CHAPTER I — BACKGROUND  I.1. Vascular functions and diseases I.1.1. Structure of a normal large artery and extracellular matrix A large artery consists of three morphologically distinct layers. The intima, the innermost layer; the media, the middle layer, mostly consists of smooth muscle cells (SMC); and the adventitia, the outer layer, consists of connective tissues with interspersed fibroblasts and SMC. It is not all that long ago when the generally accepted views on the extracellular matrix (ECM) suggested an entirely passive role for this ubiquitous component of any tissue or organ in the body. In the last two decades it has become clear that the ECM is, on the contrary, an extremely dynamic structure - dynamic because, like almost any other structure of the human body the ECM is subject to constant renewal; dynamic also because the ECM is a structure that serves an architectural role during fetal development and during tissue repair; dynamic, finally because the matrix function is extremely interactive: matrix molecules are deposited by adjacent parenchymal cells and at the same time provide cues that modulate the functional activity of these cells (1, 2). Also in disease processes the ECM plays a crucial role. The matrix is essential in wound healing but excessive matrix deposition in chronic inflammatory or degenerative diseases such as atherosclerotic diseases can lead to organ dysfunction (1, 2). The vascular ECM is a reinforced composite of collagen and elastic fibers embedded in a viscoelastic gel constituted by proteoglycans (PG), hyaluronan, glycoproteins, and water (Figure I-1). These components interact through entanglement and cross-linking to form a biomechanically active polymer network that imparts tensile strength, elastic recoil, compressibility, and viscoelasticity to the vascular wall. In addition, this network interacts with  1  Vascular Extracellular Matrix ^1  Collagens I, III, IV, V, VI, VIII  Elastin microfibrils  Glycoproteins  Elastin, Fibrillin, Emilin  (  Interactive Glycoproteins Fibronectin, Tenascin Thrombospondin, Osteopontin  Proteoglycans Versican, Decorin, Biglycan, Lumican, Perlecan  Figure 1-1. The main components of the vascular extracellular matrix.  2  vascular cells and participates in the regulation of cell adhesion, migration, and proliferation during vascular development and disease. This regulation involves molecular interactions that govern the attachment of vascular cells to specific ECM components, detachment of cells from these components and molecular rearrangements in the ECM that allow cells to change their shape during division and migration. Furthermore, components of the ECM bind plasma proteins, growth factors, cytokines, and enzymes, and these interactions modulate arterial wall metabolism. Thus, the vascular ECM not only maintains vascular wall structure, it also regulates key events in vascular physiology (1, 2). The composition of the ECM is controlled by the coordinate and differential regulation of synthesis and turnover of each of the components. Such differential regulation creates differences in the composition of the vascular ECM during vascular development, between different vascular beds, and in different forms of vascular disease (Figure I-1). For example each layer of the vascular wall (i.e., intima, media, and adventitia) has a different ECM composition. An ECM rich in fibrillar collagen as is found in the adventitia, will impart stiffness and rigidity, whereas a layer enriched in PG and hyaluronan, as found in the intima, is more viscoelastic and compressible (1, 2). Maintaining the appropriate balance of the components in each layer is critical for maintaining vascular wall integrity and resisting rupture and hemorrhage. Additionally, an ECM composition that forms a "loose" and hydrated network enriched in attachment proteins promotes vascular cell adhesion, proliferation, and migration in development and early stages of vascular disease, whereas a dense and fibrous ECM typifies more differentiated vascular tissue and advanced vascular lesions (1, 2).  3  1.1.1.1. Proteoglycans in the vessel wall Proteoglycans are unusual molecules that are characterized by the presence of long, unbranched, highly polyanionic, polymeric side-chains, called glycosaminoglycans (GAG) covalently attached to a core protein. The molecular revolution has enhanced our understanding of PG through the cloning of core protein genes and more recently through the cloning of many genes for GAG assembly enzymes (3). Depending upon the composition of the GAG chains, PG can be classified as heparan sulfate PG (HSPG), chondroitin sulfate PG (CSPG), dermatan sulfate PG (DSPG) or keratan sulfate PG (KSPG). Of these, the CSPG are the most abundant type of PG expressed in mammalian tissues. The ECM CSPG include aggrecan, versican, neurocan, brevican, neuroglycan D, the receptor-type protein tyrosine phosphatase and its splice variant phosphacan (4). The major CSPG in the matrix of the mammalian arterial wall is versican, a member of the hyalectan gene family, often accompanied by decorin and biglycan, members of a separate gene family, the small leucine-rich PG (Figure 1-2) (5). Vascular PG can be found in four locations: (a) in the interstitial ECM, (b) as part of specialized ECM structures such as basement membranes, (c) as part of cell membranes, and (d) intracellularly. Proteoglycans found in each of these locations tend to share common structural features that in part determine their role in these tissue compartments. In terms of volume, not weight, the major component of the intima of large arteries is the PG. They form a tridimentional network that fills approximately 60% of the space between the endothelial cells (EC) and the internal elastic lamina. The PG layer of the intima becomes more prominent with physiological intimal thickening at branching points and early during atherogenesis (1). The major types of PG identified in blood vessels include large (1000 kDa) CSPG such as versican (6, 7), small (-120-300 kDa) leucine-rich DSPG such as decorin and biglycan (8),  4  Syndecan Endothelium  Glypican . Perlecan Decorin  Collagen  Biglycan  Versican  Elastin fiber  Internal elastic lamina  Smooth muscle cells (media)  Figure 1-2. Schematic representative of major components of the intimal extracellular matrix. The vascular ECM is a composite of collagen and elastic fibers embedded in a viscoelastic gel consisting of PG, hyaluronan, glycoproteins, and water. One of the major PG in the vessel wall is a large CSPG called versican. Versican interacts with variety of ECM components and modulates their functions. Furthermore, it can bind, retain and modify lipoproteins that permeate the vessel wall. There are abundant evidences that this versatile PG is an important participant in intimal SMC migration, proliferation, adhesion and vascular remodeling. Our laboratory and others have demonstrated prominent expression of versican in various forms of atherosclerotic diseases.  5  KSPG such as lumican (9), and HSPG such as perlecan and other basement membrane PG (10). All of these PG associate with different components of the vascular ECM and influence the properties of these components. For example, decorin is located along collagen fibrils and regulates collagen fiber diameter and organization (11). Perlecan inserts into basement membranes and contributes to the permeability characteristics of this structure, serves as substrate for vascular cells (12), and retains growth factors involved in vascular remodeling (13). Versican is present throughout the interstitial space of the vascular ECM (1, 14) and interacts with hyaluronan and link proteins (1, 14, 15) to fill the ECM space not occupied by the fibrous components of the ECM. These complexes create a reversibly compressible compartment in the vascular ECM and provide a swelling pressure within the ECM that is offset by the collagen fibrils (16). These counterbalancing forces in part allow the blood vessel to resist deformity created by the pulsatile pressure of the circulatory system. The distribution of PG throughout the blood vessel wall is variable. For example, the intima is particularly enriched in PG (1, 14) with lower amounts in the media and adventitia. Versican/hyaluronan complexes and biglycan are prominent in the intima and media, and decorin is concentrated in the collagen-containing adventitia. Perlecan is present in basement membranes throughout both the intima and media layers. It has been recently reported that there is a selective enhanced deposition of lumican in the intima of the atherosclerosis-prone internal carotid artery compared with the intima of the atherosclerosis-resistant internal thoracic artery (17). Proteoglycans are also present on the surface of vascular cells (18). These PG may be inserted directly into vascular cell membranes via hydrophobic sequences in the core proteins, as is the case for the HSPG of the syndecan family (18). Other cell surface HSPG associate with vascular cell membranes by phosphatidylinositol linkages and compromise a separate family of  6  membrane PG termed glypicans (19). Membrane PG serve a variety of vascular functions: as attachment proteins for enzymes involved in lipid metabolism (6, 20) and blood coagulation (21); as binding proteins for the attachment of vascular cells to their ECM (18); and as binding proteins for growth factors and cytokines (18). Figure 1-3 shows presence and co-localization of apolipoproteins, oxidatively modified lipids and versican in an artery from allograft vasculopathy.  1.1.2. Atheromatous diseases 1.1.2.1. Response-to-retention hypothesis — implications for vascular diseases Several lines of evidence indicate that the key initiating event in early atherogenesis is the subendothelial retention of atherogenic lipoproteins (6). Once retained, atherogenic lipoproteins undergo several modifications with important biologic consequences. The earliest modification is lipoprotein aggregation. Aggregation may occur when retained low-density lipoprotein (LDL) is oxidized extensively or digested by several enzymes that are known to be present within the arterial wall. Oxidative and enzymatic modifications of retained lipoproteins lead to the generation of biologically active molecules, such as lysophosphatidyl choline and ceramide that have been shown in vitro to provoke endothelial expression of cell adhesion molecules, monocyte chemotaxis, and SMC proliferation. Importantly, aggregated LDL is avidly taken up by cultured macrophages and SMC, leading directly to the formation of foam cells. Altogether, these processes promote local inflammation, enhanced lipoprotein retention, lesion progression, and, ultimately, clinical events (Figure 1-4) (6). Work from several laboratories, including our own, has implicated CSPG of the arterial wall in lipoprotein retention (1, 22). Retained lipoproteins in prelesional tissue, early  7  Figure 1-3. Apolipoproteins, oxidized lipids and versican co-localize in arteries from human heart allografts. (a-b) Photomicrographs of Oil Red 0-stained lipid droplets in lesions from allograft coronary arteries. (a) Several fine lipid droplets are seen within the intima. In addition, numerous medium size lipid droplets are observed within endothelial cells at the luminal surface (arrowheads) (x100). (b) Section of allograft coronary artery double-stained, histochemically with Oil Red 0, and immunohistochemically with muscle-specific alpha-SM actin antibody. Lipid droplets are associated extracellularly and intracellularly with smooth muscle cells (arrowhead) (x320). (c-t) Photomicographs of histologic sections from the LAD coronary artery of a 50-year-old male recipient (18-year-old male donor) 300 days post-transplant showing an intermediate-sized intimal lesion of allograft vasculopathy. The vessel is stained histochemically with Movat's pentachrome at low power (c) and high power (d). The vessel is stained immunohistochemically for versican (e) and apolipoprotein E (f), or factor VIII-related antigen (inset). The lesion shows characteristic concentric intimal thickening and colocalization of versican with apolipoprotein E. (g-h) Photomicrographs of a mild lesion from the LAD coronary artery of a 45-year-old male recipient (20-year-old male donor) at 210 days post-transplant and with 16% luminal narrowing demonstrating colocalization of versican and oxidatively modified lipoproteins. The vessels was stained for versican (g), alpha-SM actin (h), malondialdehyde-modified LDL (MDA-LDL) (i). Original magnifications: x6.6 (c); x50 (d-t); x50 (insets). Scale bars: 30pm (c); lOpm (d - 1); 10 gm (inset); 40 mm (g-i). Adapted from Rahmani et al. Transplant vascular disease: Role of lipids and proteoglycans. The Canadian Journal of Cardiology 2004;20:58B-65B. 8  Endothelial hairy  I  Role of HSPGs in vascular homeostasis and disease HSPGS  Lipoproteins  fvtonocytes  rf  fl  EC  41111111110____)  00 3  / 0  4-a  00 Macrophagetfoam cell  CSPGs  7  sPLA2 & LPL  5&6  4-c  Aggregationlrusion of lipoproteins Cytokines & ^ growth factors  (^  Figure 1-4. Pathogenic concept of atherosclerotic vascular diseases — known or proposed roles of proteoglycans and lipids. (1) Endothelial injury is mediated by cardiovascular risk factors such as hypertension, diabetes/insulin resistance, dyslipidemia, obesity, homocysteinemia, infection, genetic predisposition to atherosclerosis, and drug toxicity. (2) Roles of HSPG include anticoagulant properties, anti-platelet aggregation/adhesion, reduction of blood viscosity and lipid metabolism through binding apolipoproteins, as well as LPL. Reduced HSPG may not only affect endothelial barrier function, but also increase retention of atherogenic lipoproteins [LDL and Lp(a)] and circulating monocytes. (3) Atherogenic apoB- and E-containing lipoproteins cross the endothelial barrier, where they encounter and are retained by vascular PG. (4) The retention of lipoproteins renders them susceptible to oxidative modification. Oxidized lipoproteins can stimulate many biological processes involved in atherogenesis. These processes include (a) the expression of monocyte adhesion molecules, chemotactic factors, and colony-stimulating factors, the effect of which is to cause circulating monocytes to bind to EC, enter the intima and undergo differentiation and activation, (b) stimulation of the expression of PG with increased glycosaminoglycan chain length, which can result in the further retention of unoxidized lipoproteins, and (c) stimulation of expression of cytokines and growth factors by SMC that have migrated to the intima from the media. In addition, (5) apoB-containing lipoproteins and secretary PLA2, through their interaction with arterial PG, may facilitate enzymatic hydrolysis of lipoprotein phospholipids. NEFAs, oxidized NEFAs, and lysophosphatidylcholine (lyso-PC) bind albumin or remain associated with modified lipoproteins. This interaction can induce aggregation and fusion of the lipoproteins, processes that are enhanced by intimal PG. As well, (6) LPL bound to the components of ECM of the arterial intima can lead to retention of LDL in these structures by acting as a molecular bridge. Retention increases the residence time of LDL in the intimal matrix, thus allowing the particles to be modified more extensively than they would otherwise have been. Finally, (7) interactions among lipoproteins, PG, SMC, and macrophages lead to lipoprotein retention and foam cell formation. apo, apolipoprotein; CSPG, chondroitin sulfate proteoglycans; EC, endothelial cells; ECM, extracellular matrix; HSPG, heparan sulfate proteoglycans; Lp(a), lipoprotein (a); LPL, lipoprotein lipase; LDL, low-density lipoprotein; lyso-PC, lysophosphatidylcholine; NEFAs, nonesterified fatty acids; PG, proteoglycans; sPLA2, secretory phospholipase A2; SMC, smooth muscle cells. Adapted from Rahmani et al. Transplant vascular disease: Role of lipids and proteoglycans. The Canadian Journal of Cardiology 2004;20:58B-65B. 9  atherosclerotic lesions, and advanced plaques are closely associated with arterial PG, mainly CSPG. Further, the binding of LDL to PG enhances its aggregation and its susceptibility to oxidation or enzymatic digestion. Most intriguingly, preliminary reports indicate that mice transgenic for site-directed mutants of apoB (23) or PG-binding-defective LDL (24), engineered to abolish the binding of LDL to arterial CSPG, have markedly reduced lipoprotein retention and reduced early lesion development than mice expressing wild-type control LDL or PG. Thus, lipoprotein retention is a major mechanism in early atherogenesis and a major mechanism of early LDL retention depends on binding to arterial wall CSPG. For these reasons, CSPG as a general class of molecules are commonly considered to be atherogenic.  1.2. Versican — structure and function Versican belongs to the family of large aggregating PG that has been named hyalectans (3) or lecticans (25). Versican is the most versatile hyalectin in regard to its structure and tissue distribution. Recent data emerging from in vitro and in vivo studies suggests that versican modulates cell adhesion, proliferation, and migration, and hence plays a central role in tissue development and maintenance as well as in a number of pathologic processes (1, 14, 15, 26-29).  1.2.1. The versican gene, protein and glycosaminoglycan chains structure Naso et al. (30) identified the transcription start site(s) for versican gene. They predicted the likely site of transcription initiation to be about 16 by downstream of the TATA box (30). Transient expression assays of 876 by (-632/+240) of the human versican gene in a CAT reporter vector in HeLa cells and IMR-90 embryonic lung fibroblasts have shown significant CAT expression (30). Furthermore, three additional constructs, which include the 220 by of exon one  10  plus 445, 209, or 30 by upstream of the transcription start site, respectively, were assayed in HeLa cells. The initial deletion construct (-445 bp) yields a 2-fold increase in activity when compared to the full 872-bp construct. This implies the presence of repressor or silencing elements in the deleted region. The next deletion construct (-209 bp) involved numerous potential cis-elements and showed 50% of the promoter activity as compared to the full 876 by construct. A final deletion construct, which includes only 30 by upstream of the transcription start site, had slightly less activity than the construct which included an additional 179 bp. These results indicate that the human versican 5'-flanking sequence contains promoter, enhancer, and repressor elements able to drive CAT expression in cells derived from epithelial or mesenchymal tissues. The versican splice-variants are encoded by a single gene localized on chromosome 5q 12-14 in the human (31) and on chromosome 13 in the mouse genome (32). The organization of the gene tightly follows the domain structure of the core protein (33, 34) (Figure 1-5). Both the human and mouse gene extend over 90-100 kb and are divided into 15 exons. The GAG attachment domains, GAG-a and GAG-P, are each encoded by a giant exon of 3 and 5.3 kb size in the human and 2.9 and 5.2 kb in the mouse gene, respectively. Alternative mRNA-splicing of these exons gives rise to transcripts of about 12 kb size for the VO isoform, 9 kb for the versican V1 isoform, 6.5 for the versican V2 isoform, and 3 kb for the smallest V3 variant (Figure 1-5). Since the 3' untranslated region contains several polyadenylation signals (three in the human and four in the mouse gene), the different splice-variants appear as multiple bands closely spaced on Northern blots. To date, the entire primary structure of human (35, 36), murine (37), bovine (38), and chick (39) versican mRNA have been sequenced from cDNA clones. Several versican core  11  A Chromosome+ 5  90kb  ski ktt  ni  911 311 213 2  34  HP/JEIR GAG.  a  GAG.p ^ELM ^rix003 ^VCCLI  vo 12 kb vi  kb  V2  6_5 kb  V3  3 kb  Figure 1-5. The human versican gene and protein structure. A, The versican gene extends over a 90 kb on the chromosome 5q12-14. The single gene is composed of 15 exons, including two large exons encoding for the GAG chain binding domains. B, Alternative splicing produces versican transcripts of VO, V1, V2 and V3, respectively. The differential splicing occurs in the central portion of the molecule, producing isoforms with altered GAGbinding potential. VO represents the full-length protein, containing both GAG-a and GAG-I3 (depicted in red and blue for GAG-a and GAG-(3). VI and V2 have GAG-a and GAG-13 spliced out, respectively, while V3 lacks both GAG-binding regions. Differential splicing leads to versican core proteins of variable molecular weight and GAG-binding capacity and leads to potential differences in the functions of these isoforms. HABR, hyaluronan binding region; E, epidermal growth factor repeats; L, lectin binding domain; C, complement regulatory region. 12  proteins have been identified (Figure 1-5). The structural diversity originates from alternative splicing processes, which generate four splice-variants of human (36, 40), mouse (37, 40) and bovine versican (38) as discussed above. Six versican isoforms may exist in the chicken (41). All versican splice forms include a link-protein-like structure with an immunoglobulin-like loop and a tandem-repeat domain at the N-terminus, and a set of two Epidermal Growth Factor-like elements, a C-type lectin domain and a complement regulatory domain at the C-terminus (Figure 1-5). The differences among the versican splice-variants are found in the central portion of the core proteins. In versican VO, two chondroitin sulfate-carrying segments, named GAG-a and GAG-13, are present, whereas the smaller V1 and V2 isoforms lack the GAG-a or the GAG-f3 domain, respectively. There are no GAG-carrying modules in versican V3. Based on consensus sequence examination, it is estimated that the number of potential chondroitin sulfate attachment sites is 17-23 in human VO, 12-15 in versican V1,and 5-8 in versican V2 (36). Due to absence of both central domains, versican V3 is most likely devoid of GAG side chains and therefore may not be a PG (40).  1.2.2. Function of versican in health and diseases Versican is one of the main components of the ECM where it provides hygroscopic properties to create a loose and hydrated matrix that is necessary to support key events in development and disease. Through direct or indirect interactions with cells and molecules, versican is able to regulate cell adhesion and survival, cell proliferation, cell migration and ECM assembly [reviewed in (42)]. Versican interacts with its binding partners through its N- and Cterminal globular regions as well as its central GAG-binding region [reviewed in (15)]. It binds to ECM components such as hyaluronan, type I collagen, tenascin-R, fibulin-1 and -2, fibrillin-1,  13  fibronectin, P- and L-selectin, and chemokines (15). Versican also binds to the cell surface proteins CD44, integrin 131, epidermal growth factor receptor, and P-selectin glycoprotein ligand1 (15). These multiple binding interactions play important roles in cell and tissue behaviour. Versican expression is modulated in several cardiovascular diseases (1, 14). Through its interaction with hyaluronan and other ECM partners, versican creates pericellular matrices that are required for arterial SMC proliferation and migration. It is also prominent in advanced lesions of atherosclerosis, at the borders of lipid-filled necrotic cores as well as at the plaquethrombus interface, suggesting roles in lipid accumulation, inflammation, and thrombosis. Versican influences the assembly of ECM and controls elastic fiber fibrillogenesis, which is of fundamental importance in ECM remodeling during vascular disease. Taken together, these reports underscore the significant importance of this PG in vascular health and disease particularly in atherosclerotic disorders (1, 14). Epithelial-mesenchymal interactions play pivotal roles in the organ morphogenesis as well as establishment and progression of many diseases including cancer. During the hair cycle, the epithelium and the mesenchyme are regulated by a distinct set of molecular signals that are unique for each distinct phase of the hair cycle. The signaling exchange between the follicular epithelium and the mesenchyme is modulated by PG, such as versican, which may significantly enhance or reduce the biological activities of secreted growth stimulators (43, 44). In the central and peripheral nervous system, versican is expressed by glial cells and is implicated in the regulation of cell adhesion, migration, pattern formation, and regeneration (45). Versican, along with other CSPG, is up-regulated following central nervous system injury and has been shown to exhibit inhibitory effects on neurite outgrowth in vitro (29). Therefore, it is  14  likely that the increased expression of these molecules contributes to the non-permissive nature of the glial scar. However, the relative contribution of versican remains to be determined. In addition, aggrecan and versican, two members of large CSPG, along with their binding partner hyaluronan, likely provide tendon tissues with a high capacity to resist compressive and tensile forces associated with loading and mobilization (27, 46). Given the distinct and pivotal role of versican in development and disease, interventions which target the versican molecule and/or the cells which produce it will comprise an important field of future investigations for discovery of suitable molecular drug targets.  1.3. Regulation of versican 1.3.1. Signal transduction pathways  Host signaling pathways that are involved in the regulation of versican are not well understood although preliminary studies conducted in recent years have begun to elucidate the intracellular signal transduction pathways required for versican expression in different cells and tissues.  1.3.1.1. Receptor tyrosine kinase-mediated versican expression In vitro experiments using monkey arterial SMC have shown that platelet derived growth  factor (PDGF)-stimulated versican core protein expression and GAG chain elongations are regulated by distinct signaling pathways. It has been demonstrated that the tyrosine kinase inhibitor, genistein, reversibly inhibits PDGF-stimulated versican mRNA and core protein expression in a dose-dependent manner in arterial SMC. In contrast, genistein does not affect the increase in GAG chain elongation that is induced by PDGF. These findings suggest that versican  15  is regulated by a versatile mechanism as different aspects of its biosynthesis are differentially regulated (47). Inhibition studies also examined the signal transduction pathways involved in the angiotensin (Ang) II-mediated increase in versican gene expression in rat vascular SMC (48). The concentration- and time-dependent increase in mRNA levels of Ang II receptor-expressing cells in response to Ang II was inhibited by the Ang II receptor antagonist losartan, the epidermal growth factor receptor inhibitor AG1478 and the MAP kinase inhibitor PD98059. This increase was not inhibited by the PKC inhibitors chelerythrine and staurosporine, indicating that Ang II-mediated stimulation of SMC versican expression is regulated by receptor tyrosin kinase (RTK)-dependent mechanisms (48). The idea that RTK-dependent pathways are involved in stimulation of versican core protein expression by both PDGF and Ang II suggests the existence of a common pathway for growth factor-mediated upregulation of versican expression by SMC.  1.3.1.2. The PI3K—PKB pathway and versican gene regulation We have recently shown that phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling through inhibition of glycogen synthase kinase-313 (GSK-3(i) and subsequently nuclear accumulation of f3-catenin enhances versican transcription in SMC (refer to chapter IV of this thesis) (49). Our interest in this pathway has been driven by its frequent aberrant activation in various diseases and its impact on cell survival decisions (50). There is abundant evidence to suggest potential roles for versican in various biological functions including cell proliferation and survival (51). The analogous function of versican and PI3K/PKB signaling encouraged us to investigate the influence of PI3K/PKB signaling on control of versican expression level. Phosphoinositide-3 kinases are a family of evolutionary conserved lipid kinases that mediate many cellular responses in both physiologic and pathophysiologic states (52). Class I  16  PI3K can be activated by either receptor tyrosine kinase/cytokine receptor activation (class IA) or G-protein-coupled receptors (class IB). Once activated PI3Ks generate phosphatidylinositols (3,4,5)P3 leading to the recruitment and activation of PKB and PDK1, which then activate a range of downstream targets including GSK-3(3, mammalian target of rapamycin (mTOR), p70S6 kinase, endothelial nitric oxide synthase and several anti-apoptotic effectors (Figure 1-6). PI3Ka and c are expressed in cardiomyocytes, fibroblasts, EC and vascular SMC where they modulate cell survival/apoptosis, hypertrophy, contractility, metabolism and mechanotransduction. Evidences suggest that the PI3K signaling pathway is involved in a wide variety of diseases including cardiac hypertrophy, heart failure, preconditioning and hypertension (52).  1.3.1.3. Glycogen Synthase Kinase-3 and versican gene regulation As explained above, I showed that phosphorylation and inhibition of GSK-313 and subsequently nuclear accumulation of 13-catenin enhances versican transcription in SMC (refer to chapter IV of this thesis) (49). Glycogen synthase kinase-3 is an unusual protein serine/threonine kinase that is active under resting conditions and is inactivated upon cell stimulation. The two mammalian isoforms, GSK-3a and 13, play largely overlapping roles and have been implicated in a variety of human pathologies, including Type II diabetes, Alzheimer's disease, bipolar disorder, cardiovascular diseases and cancer (53-56). Glycogen synthase kinase-3 was originally identified as one of several protein kinases capable of phosphorylating the rate-limiting enzyme of glycogen deposition, glycogen synthase (GS) (57). In peripheral tissues, insulin modulates glycogen metabolism through a co-ordinated increase in glucose transport and glycogen synthesis. Insulin stimulates glycogen synthesis by  17  Growth factor  P T308  S473  Akt (inactive)  Akt (active)  Cull Survival  Gell Cyule  Bad Fork/load IKKct FLIP  E 2F p21 MDM2 hTEIZT  G lure  Protein  Metabolism  Synthes1S  031(3 GLUT4  rnTOR S6K1 4E-BP1  Figure 1-6. Schematic representation of growth factor/PI 3-kinase/PKB signaling and partial list of downstream molecules. PKB is activated by growth factors or cytokines on their respective receptor tyrosine kinase (RTK) in a PI3K- dependent manner, and phosphorylation of two residues by PDK1 (T308) and PDK2 (S473) is required for its full activation. Downstream target molecules are grouped according to their function. Note that these downstream molecules include both direct PKB substrates and indirect downstream effectors. For instance activation of PKB inhibits the pro-apoptotic protein Bad by phosphorylation. PKB can also activate NF-KB by direct interaction with IkEi kinase (IKK), resulting in release of NF-KB from the block by the inhibitor Ibo. The anti-apoptotic effects of NF-KB are mediated by changes in gene expression. Inactivation of Fox transcription factors by PKB may result in cell cycle progression and in inhibition of apoptosis. Inhibition of glycogen GSK-3 by PKB-dependent phosphorylation has also been linked to cell cycle progression and stress resistance. Adapted from Ichiro Shoji and Kenneth Walsh. Role of Act Signaling in Vascular Homeostasis and Angiogenesis. Circ Res. 2002;90:1243-1250. 18  activating GS through dephosphorylation of GS, particularly at the sites targeted by GSK-3 (58). This action of insulin is mediated by simultaneously inhibiting GSK-3 kinase activity and by activating one of the glycogen-associated forms of protein phosphatase 1. It was shown that the insulin-mediated inhibition of both isoforms of GSK-3 (a and (3) was mediated through a phosphorylation-dependent mechanism, with the phosphorylation of each isoform occurring at a serine (S) residue in the N-terminal lobe of the protein kinase (S21 for GSK-3a and S9 for GSK3(3). Several protein kinases can phosphorylate these residues, namely the p70 ribosomal S6 kinase, p90 ribosomal S6 kinase, protein kinase A and protein kinase C (Figure 1-7). However, insulin mediated phosphorylation and inhibition of GSK-3 in vivo is primarily mediated through activation of the PI3K/PKB pathway (59). It appears that GSK-3 exists in a constitutively active conformation in resting cells, and that inhibition of GSK-3 activity (through serine phosphorylation) is a means by which extracellular stimuli regulate this protein kinase. However, there are additional means of regulating GSK-3 activity distinct from phosphorylation e.g. subcellular localization, binding to scaffold proteins, etc. Although GSK-30 was initially described for its function to inhibit glycogen synthesis through phosphorylation of GS (57) it has been revealed that GSK-3f3 regulates a wide range of cellular functions, including metabolism, gene expression, and cytoskeletal integrity (Figure 1-7) (60). GSK-313 is also involved in a variety of disease processes, such as tumorigenesis and the development of Alzheimer's disease (61, 62). GSK-3—catalyzed phosphorylation of some substrates, such as the axin—adenomatous polyposis coli (APC)—f3-catenin complex, may not require priming phosphorylation (63). GSK-30 is localized predominantly in the cytoplasm but is also found in the nucleus.  19  ILK  7,1 PKA PKC LiCL p  RSK  p 70,S6K  TK  GSK3 -13 Cytoskeleton Development  )  Apoptosis  Protein Translation  Cell Cycle Gene Expression Metabolism  Figure 1-7. Regulators of GSK-3D and its role in cellular function. GSK-313 is tightly controlled by a variety of regulators and is centrally involved in diverse processes related to cell growth and death. ILK, integrin linked kinase; LiC1, lithium chloride; PKA indicates protein kinase A; PKB, protein kinase B; PKC, protein kinase C; p9ORSK, p90 ribosomal S6 kinase; p70S6K, p70 S6 kinase-1; and TK, tyrosine kinase; Wnt, wingless proteins.  20  1.3.1.3.1. Activation and inactivation of GSK-3 An important characteristic of GSK-3 is the fact that it is catalytically active in cells even under unstimulated conditions. Thus, total cellular activities of GSK-3 may be predominantly regulated at the level of protein expression. Interestingly, however, phosphorylation of GSK-3 at Tyr216 can further increase its kinase activity (64) Because GSK-3(3 negatively regulates downstream signaling mechanisms, inactivation of GSK-313 in fact stimulates many cellular functions by removing the negative constraint imposed by GSK-313. The activity of GSK-313 is regulated by multiple mechanisms (Figure 1-7). Most importantly, inactivation of GSK-313 is induced by phosphorylation via upstream protein kinases (63). The phosphorylation sites that lead to the inactivation of GSK-3 have been identified as Ser21 for GSK-3a and as Ser9 for GSK-30 (65-67). PKB is one of the most thoroughly studied of the kinases that have been identified as upstream regulators of GSK-313. Because PKB is a major kinase downstream from PI3K, many stimuli that activate PI3K inhibit GSK-313 through PKB. Activation of the Wnt signaling pathway (see below) is another important mechanism to inhibit activity of GSK-3(3. In the presence of Wnt, Dishevelled (Dvl) disrupt interaction between GSK-3 and axin, thereby leading to inactivation of GSK-3 via mechanisms distinct from the phosphorylation of GSK-3 at the residue targeted by insulin (68). Although both insulin and Wnt pathways inactivate GSK-313, they regulate distinct targets: insulin induces an increased activity of GS but has no influence on the protein level of f3-catenin. In contrast, Wnt increases the cytosolic pool of (3-catenin but not GS activity (69). It has been suggested that GSK-313 phosphorylates (3-catenin only when it is sequestered by the axin-APC complex (70, 71). Besides endogenous regulators of GSK-313, several compounds directly inhibit kinase activities of GSK3(3. LiC1 is the most commonly used inhibitor of GSK-3(3 (72). SB-216763 and SB-415286,  21  structurally distinct maleimides, are potent and selective cell-permeable inhibitors of GSK-313 (73).  1.3.1.4. Wnt signaling and versican gene regulation The canonical Wnt—wingless signaling pathway (Figure 1-8) regulates various biologic processes including early embryogenesis and neoplasia by increasing the stability and transcriptional activity of a key mediator, 13-catenin (74). In the absence of Wnt ligand, GSK-313 promotes the phosphorylation of f3-catenin at key serine/threonine residues, targeting it for degradation through the ubiquitin—ligase pathway (75). Canonical Wnt signaling requires the Frizzled (Fz) and LDL receptor-related protein 5/6 (LRP5/6) coreceptors and leads, by a poorly understood process, to the activation of Dvl. Dishevelled can inhibit the activity of the so-called 0-catenin destruction complex; a complex of proteins including APC, Axin, and GSK-30 that otherwise phosphorylates P-catenin, thus preventing its destruction by the ubiquitin-proteasome pathway. These and other mechanisms allow Wnt ligands to both stabilize 13-catenin and promote its entry into the nucleus where it recruits transactivators to high-mobility group (HMG) box DNA binding proteins of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family (Figure 1-8) (74). There is a recent report that the versican gene was identified as a target gene of Wnt signaling using microarray technology to analyze human embryonic carcinoma cells stimulated with active Wnt protein (76). The promoters of nearly all the target genes identified, including the versican promoter, harbour putative TCF binding sites. Kishimoto et al. (77) also showed that culture of the cells expressing a secreted Wnt-3a protein maintain the expression of green fluorescent protein placed under the control of a fragment of the human versican promoter. Because our findings also suggest that versican transcription is predominantly mediated by the  22  Figure 1-8. Schematic representation of Wnt / 13-catenin / TCF signaling. In cells devoid of a Wnt and/or growth factor-mediated signals, [3-catenin is held by the GSK-313/APC/axinconductin complex, resulting eventually in degradation of I3-catenin. TCF target genes are repressed by TCF interacting with nuclear co-repressors. Upon Wnt signaling (through Dishevelled) and/or growth factor-mediated signals (such as PI3K/PKB) GSK-313 inactivates, resulting in disruption of the quarternary complex and f3-catenin build-up. A transcriptionally active complex with TCF is formed that leads to target genes transcription. The abbreviations used are: APC, adenomatosis polyposis coli; 0-cat, 13-catenin; Dvl, Dishevelled; Fz, Frizzled receptor; GSK-313, glycogen synthase kinase-313; ILK, integrin link kinase; LRP5-6, LDL receptor-related protein (LRP) 5-6; Pg, plakoglobin; Pl3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; RTK, receptor tyrosine kinase; LEF/TCF, lymphoid enhancer factor/T cell factor; Ub, ubiquitin; Wnt, wingless.  23  GSK-3(3/(3-catenin pathway in SMC (49), it is likely that Wnt/O-catenin signaling mediates versican expression in normal or disease states.  1.3.1.4.1. Wnt signaling and GSK-3  The Wnts, a family of secreted glycoprotein ligands, are essential for proper embryonic development due to their role in the regulation of cellular proliferation, differentiation, motility and polarity (78). One way that Wnts can mediate a response is through the stabilization of a specific pool of f3-catenin that is usually targeted for degradation. Stabilization of this 13-catenin results in its accumulation in the cytosol and ultimately the nucleus, where it binds architectural transcription factors of the LEF/TCF family to activate transcription of Wnt target genes (Figure 1-8) (79). GSK-3 plays a central role in this canonical Wnt signal transduction pathway, since its phosphorylation of 13-catenin on key residues is required for f3-catenin's ubiquitination and proteasomal degradation in 'resting' cells (Figure 1-8). Conversely, upon Wnt treatment, GSK-3 must be prevented from phosphorylatingr3-catenin to bypass ther3-catenin degradation machinery. Phosphorylation of f3-catenin by GSK-3 occurs in a multiprotein complex containing, in part, axin, APC and p-catenin (80). Mutations in axin, APC and 13-catenin have been linked to numerous types of human cancer, including those of the breast, skin, liver and colon (81). Although once thought to be one of the few unprimed substrates for GSK-3,13-catenin has been shown to be primed at S45 by casein kinase I (CKI), allowing efficient subsequent serial phosphorylations by GSK-3 on residues T41, S37 and S33 (82). It is the phosphorylation of f3catenin on S33 and S37 that appears to be essential for 13-catenin's recognition by the ubiquitin ligaser3-transducin repeat-containing protein (f3-TrCP)-1, which targets it for subsequent  24  degradation (83). Mutations of f3-catenin at the residues phosphorylated by CKI or GSK-3 sites have been found in numerous types of cancer, including colorectal cancer, melanoma, hepatocellular and ovarian carcinoma (81). Axin and APC are also GSK-3 substrates. The phosphorylation of axin by GSK-3 has been reported to increase axin's stability and affinity for f3-catenin (84). It has been proposed that the mechanism of I3-catenin stabilization in Wnt signaling might depend more on the regulation of axin stability rather than that of 13-catenin itself, since it appears that axin might be the limiting factor regulating (3-catenin degradation (85). The phosphorylation of APC by GSK-3 also increases APC binding to f3-catenin (86). The Wnt-induced mechanism precluding GSK-3's phosphorylation of (3-catenin remains elusive. Wnt treatment of human embryonic kidney-293 (HEK293) cells, which was effective in stabilizing (3-catenin, caused a reduction in GSK-3 activity as measured by immunoprecipitating endogenous GSK-3 and assaying its activity in vitro (69). This reduction in activity was not as a result of the S9/21 phosphorylation. Insulin treatment, although causing a reduction in GSK-3 activity and increasing S9 phosphorylation, did not affect 13-catenin levels in the same cells. Thus it appears that the GSK-3 involved in f3catenin regulation is 'insulated' from other signaling pathways and is regulated in a unique manner.  1.3.2. Transcription factors involved in versican gene regulation 1.3.2.1. p-catenin/TCF (3-catenin is a 92-kDa protein, which links E-cadherin to a-catenin and the actin microfilament network of the cytoskeleton (Figure 1-8) (87). However, another important function of (3-catenin is its transcriptional activity in the nucleus. Conventional Wnt signaling  25  causes 13-catenin accumulation in a complex with the transcription factor LEF/TCF that regulates target gene expression as described in the section on Wnt signaling (Figure 1-8). Briefly, the key factors in P-catenin signaling are its stabilization and accumulation in the cytoplasm, whereupon it translocates to the nucleus and mediates target gene transcription. We have recently shown that phosphorylation and inhibition of GSK-313 by PKB and subsequent 0-catenin/TCF complex formation is essential for activation of versican transcription (refer to chapter IV of the thesis) (49).  1.3.2.2. Other factors Yoon et al. (88) have recently performed oligonucleotide-array gene expression analysis of human cells with wild-type p53 (p53 +/+), heterozygote (p53 +/-), and homozygote (p53 -/-) alleles. They demonstrated that the versican gene is highly expressed in p53 +/+ but expressed at lower levels in p53 +/— cells, and thus is a direct target of p53. They also confirmed their microarray results by performing quantitative analysis of versican mRNA. They also identified the putative p53 response element in the 5' portion of the first intron, 424 by from the end of the untranslated exon 1. Luciferase assays showed that the versican promoter construct was activated more than 200-fold by cotransfection with wild type p53 in p53-null cells compared with the control plasmid. Mutant p53 did not activate the versican promoter. Furthermore, these investigators showed the interaction of p53 protein and the versican p53 binding site using shift and supershift assays. These experiments demonstrate a p53-dependent induction of the versican gene. I have recently reviewed the functional importance of p53-mediated versican regulation (89).  26  1.4. Canonical and non-canonical Wnt signaling pathway Above I have explained the evidence supports the role of Wnt signaling on regulation of versican gene. Furthermore, I described the interaction of GSK-30 and Wnt signaling. Here I explain the Wnt signaling pathways in further details as this signal transduction pathway has been one of the main focuses of my investigations in this dissertation (chapter IV-VI). Wnt proteins, derived from Drosophilia Wingless and the mouse Int-1 genes, represent a large family of secreted cysteine-rich glycosylated proteins. This novel family of proteins are intimately involved in cellular signaling pathways that play a role in a variety of processes that involve embryonic cell patterning, proliferation, differentiation, orientation, adhesion, survival, and apoptosis (90-92). Until recently, nineteen of the twenty-four Wnt genes that express Wnt proteins have been identified in the human. In addition, more than eighty target genes of Wnt signaling pathways also have been demonstrated in human, mouse, Drosophilia, Xenopus, and Zebrafish. This representation encompasses several cellular populations, such as neurons, cardiomyocytes, EC, cancer cells, and pre-adipocytes. Wnt binds to Fz transmembrane receptors on the cell surface to activate downstream signaling events. These involve at least three intracellular signaling pathways with two that are considered of particular importance. The canonical pathway discussed above involving Wntl, Wnt3a, and Wnt8 controls target gene transcription through f3-catenin-dependent pathways. Another pathway pertains to intracellular calcium (Ca 2+ ) release which is termed the noncanonical or Wnt/Ca 2+ pathway consisting primarily of Wnt-4, Wnt-5a, and Wnt-11 that functions through non O-catenin-dependent pathways, such as the planar cell polarity (PCP) pathway (93) and the Wnt-Ca 2+-dependent pathways (94). Recent work has illustrated that eleven members of the Fz transmembrane receptors have been identified in the human and  27  mouse genomes (92). If one examines the Wnt signaling transduction pathway, it becomes evident that these pathways play critical roles during embryonic, non-vertebrate and vertebrate development as well as tumorigenesis (95, 96). Multiple studies have shown the importance of Wnt transduction pathway in controlling the pattern of the body axis as well as the development and maturation of the central nervous system (97), cardiovascular system (98-100), and the limbs (101). During embryological development, alternations of the Wnt pathway can lead to abnormal morphogenesis in animal models (102, 103) and congenital defects in humans (104, 105). In mature tissues, the Wnt pathway is involved in the self-renewal of stem cells and may be responsible for the maintenance of many normal tissues (106, 107). Studies have revealed that dysfunction of the Wnt pathway can lead to neurodegenerative disorders, such as Alzheimer's disease (108, 109) and heart failure (110, 111). Upon binding to either the Fz receptor or a receptor complex consisting of Fz and LRP5/6, Wnt protein can activate one of three different signaling cascades. These cascades include the canonical Wnt signaling pathway (93, 112), the Wnt/PCP pathway (93, 112), or the Wnt/Ca2+ pathway (93, 94). Each of pathways, although distinct, appears to be transduced initially through Dvl, a cytoplasmic multifunctional phosphoprotein (113). Yet, the ultimate response to Wnt interaction will most probably depend upon the cellular context at that time (114). In mammals, the Dvl protein family members contains Dvl-1, Dvl-2, Dvl-3 in all organs (115). At the level of Dvl, the Wnt signaling pathway can be separated along one of three different cascades that are dependent upon the three highly conserved domains of Dvl. As a result, Dvl is a key transducer of the Wnt signal that acts at the plasma membrane or in the cytoplasm in all three Wnt signaling pathways. However, new work has suggested that Dvl also  28  acts within the nucleus and nuclear location of Dvl is essential for its function in the Wnt signaling pathway (116). I described in the previous sections that Wnt signaling pathway is the main mechanism that activates canonical Wnt signaling. The canonical Wnt signaling pathway also is activated by several other cellular mechanisms. The shifting of proteins from the cadherin-bound pool to the cytoplasmic pool can increase the amount of available freer3-catenin for the activation of target genes. Several receptor tyrosine kinases can phosphorylate tyrosine residues of the 0-catenin and cadherin-catenin complex to allow 13-catenin to become dissociated from the complex and increase the amount of P-catenin in the cytoplasm for subsequent translocation to the nucleus (114). Furthermore, surface receptors, such as epidermal growth factor receptor, c-RON and cErbB2, can then stimulate the canonical Wnt signaling pathway (117). In addition, the insulinlike growth factor (IGF) causes tyrosine phosphorylation and 0-catenin stabilization (118). Integrin-linked kinase also can activate the canonical Wnt signaling pathway through the inhibition of GSK-30 and cAMP-responsive element (CREB)-binding protein (CBP)-dependent pathway (119).  1.4.1. Wnt signaling and cellular development in cardiac and vascular systems Studies of the Wnt pathway have focused largely on very early development and on tumorigenesis. Recent observations point to a role for Wnt signaling in vessel development and pathology. Although not yet investigated systematically, several Wnt ligands have been demonstrated to be expressed in the cells of blood vessels in vivo and in vitro, including Wnt-2, 5a, -7a and -10b. Mice deficient for Wnt-2 display vascular abnormalities including defective placental vasculature. Wnt receptors, Fz, are also expressed by vascular cells in culture and in  29  situ [reviewed in (120)] . Of the 10 murine Fz identified to date, Fz-1, -2, -3, and -5 have been  demonstrated in EC and vascular SMC; mice deficient for Fz-5 display vascular abnormalities and are embryonic lethal. Two soluble, naturally occurring Wnt antagonists, frizzled-related proteins (FRP)-1 and -3, are also expressed by vascular cells (120). Stabilization of the downstream signaling component 13-catenin in blood vessels has been demonstrated in several developmental and pathologic states, further supporting the idea that Wnt signaling plays an important regulatory role in the vasculature (120).  1.4.2. Wnt, vascular regeneration, and vascular injury The Wnt pathway modulates angiogenesis, a process that consists of new capillary formation from pre-existing vessels into an avascular area (121). Several Wnt ligands, such as Wnt-2, Wnt-5a, Wnt-7a, and Wnt-10b, are expressed endogenously in EC and vascular SMC. More importantly, Wnt receptors that involve Fz-1, Fz-2, Fz-3, and Fz-5 are also expressed in these cell populations for Wnt to exert a direct biological effect (120). It has been demonstrated that mice deficient in Wnt-2 and Fz-5 display vascular abnormalities that include defective placental vasculature as well as embryonic lethal mutations (120), suggesting that the Wnt pathway is essential for vessel development. In addition to vascular development, the Wnt pathway participates in the remodeling of vascular structure and the regulation of apoptosis during vascular injury. Using a rat aorta balloon injury model, the Fz receptor genes have been shown to be transiently down-regulated as early as one hour following injury (122). Yet, Frzb-1, a secreted protein that acts as an antagonist of Wnt signaling, can be increased and appears to coincide with the arrest of aortic SMC proliferation (122). Similarly, the secreted protein FrzA can be elevated in EC during traumatic  30  manipulation and subsequently block the proliferation of EC (123). FrzA, a member of the group of secreted frizzled related proteins (sFRP) that is expressed in the cardiovascular system, has been shown to antagonize the Wnt signaling pathway (123). It has recently reported that adenoviral FrzA-treated mice in a unilateral hindlimb ischemia showed a decrease in cell proliferation, capillary density, and blood flow recovery and a reduced expression of cyclin and cdk activity in the ischemic muscle (124). These evidences suggest that an impairment of the Wnt pathway, via FrzA overexpression, controlled proliferation and neovascularization after muscle ischemia. It has been recently demonstrated that LRP6, one of the Wnt co-receptors, regulates proliferation and survival through the Wnt cascade in SMC (125). In regards to the vascular inflammation it has been demonstrated that Wnt and NF-K13 signaling cross-talk with each other. E3-ligase, P-TrCP1, was a rate-determining mediator that regulates the ubiquitinmediated degradation of IKBa. Degradation of Ix13 is an essential step in nuclear factor (NF)-KB activation, a signaling pathway that regulates expression of numerous components of the immune system. A recent study suggests that Wnt signaling regulates P-TrCP1 leading to NF-KB activation in SMC. These findings together point to the crosstalk between the Wnt cascade and NF-xB signaling in SMC through the E3-ligase, P-TrCP1 (125). Cardiovascular calcification, specifically vascular and valvular calcification, is progressive and associated with arterial and valvular stiffness and increased cardiovascular mortality. The pathogenesis of cardiovascular calcification is complex and includes factors that promote calcification and others that inhibit calcification. It has been recently demonstrated that diabetic LDLR-/- mice on high fat diet show vascular calcification by paracrine Wnt activation in adjacent adventitial myofibroblasts, suggesting a potential Wnt paracrine osteogenic signal in cardiovascular calcification (126).  31  1.5. p-catenin-bindin g partners and roles in transcription 1.5.1. Binding choices at the f-catenin protein interaction hub 13-catenin provided one of the first examples of a dynamic protein interaction hub (Figure 1-9). In Drosophila, the pioneering work of Christiane Nusslein-Volhard and Eric Wieschaus identified 13-catenin as a key effector of Wnt signaling (127). In this role, it functions in the nucleus, linking TCF with other transcriptional regulators, including Legless (Bcl-9) and Pygopus, to activate transcription of Wnt target genes [reviewed in (128)]. Meanwhile, Rolf Kemler, Masatoshi Takeichi, Barry Gumbiner and colleagues identified 13-catenin as a component of adherens junctions (AJs) (129-131). There, 0-catenin links cadherin adhesion receptors to a-catenin, which in turn links to the cytoskeleton [reviewed in (132)]. Finally, Paul Polakis, Burt Vogelstein, Kenneth Kinzler, Walter Birchmeier, Hans Clevers and colleagues identified f3-catenin in a third guise (133-135). In the cytoplasm, 13-catenin interacts with the APC and axin proteins; they recruit GSK-3 and CK Ito form a destruction complex that phosphorylates 0-catenin and targets it to the proteasome. The interactions of 0-catenin with adhesion, transcription and destruction complexes ensure proper tissue architecture and cell-fate decisions during normal development. Dysregulated interaction decisions can be oncogenic, as failure to destroy cytoplasmic 13-catenin plays a role in most colon cancers, as well as in other human tumors. The complex life of f3catenin raises many questions. How does 13-catenin choose among its possible partners, do different complexes compete for a limiting pool of f3-catenin, and can it move from one complex to another? Indeed, at least one partner in each complex binds to the central Armadillo (Arm) repeats of 13-catenin (Figure 1-9). Axin, APC, E-cadherin, TCF and the C-terminus of 0-catenin bind to overlapping, although non-identical, sites in this Arm repeat region (136-141). Thus, there looms the potential for competitive binding interactions like all protein—protein  32  Free f3--c L:110:17  Adhesion complex  Destruction complex  Proteasome  Microtubule complex  B.  Transcription  J  complex Air  I3-catenin: N-terminus  12 Armadillo  .0"  Intramolecular Repeats binding  3 4 5 6  C-terminus  9 10 11 12  Cad  Figure 1-9. Binding choices at the p-catenin protein interaction hub. (A) In adhesion complexes (blue), f3-catenin (Peat; red) links cadherin (Cad) to a-catenin (acat), which in turn binds to actin (gray). In the cytoplasm, f3-catenin is funneled into a destruction pathway (purple). Here, it interacts with APC and axin, which recruit GSK-3 and casein kinase I (CKI) to form a destruction complex that targets 13-catenin for destruction by means of the proteasome. However, 13-catenin is also a key effector of the Wnt signaling pathway (green). Wnt signaling inhibits the destruction complex, allowing free 13-catenin to accumulate in the cytoplasm. This 13-catenin can enter the nucleus, where it links TCF with other transcriptional regulators, including Legless (Bc1-9) and Pygopus, to activate transcription of Wnt target genes. Additionally, I3-catenin—APC complexes interact with the microtubule cytoskeleton (bottom left). (B) Axin, APC, TCF, E-cadherin, and the C-terminus of 13-catenin bind to overlapping, although non-identical, sites in the central Armadillo (Arm) repeat region off3-catenin, whereas a-catenin binds to the N-terminal domain off3-catenin. CBP/p300 also binds to both C-terminal tail and Arm repeats. Adapted from Tony et al. Decisions, decisions: pcatenin chooses between adhesion and transcription. TRENDS in Cell Biology 2005;15:234-237.  33  interactions, 13-catenin interaction choices are likely based on protein interaction affinities and the concentrations of [3-catenin and its partners. When Drosophila 13-catenin is limiting, it preferentially associates with AJs (142), suggesting that this affinity might be the highest. In current models, as 13-catenin is synthesized, it is assembled into AJs, and any cytoplasmic (3catenin is captured by the destruction complex and destroyed, when Wnt signaling is absent. In this case, no 13-catenin is available to activate transcription. Wnt signaling inhibits the destruction complex by an unknown mechanism and, as a result, the level of free cytoplasmic 13-catenin rises, 13-catenin enters the nucleus and binds to TCF to initiate transcription of Wnt-responsive genes [reviewed in (92)]. In this model, the transcriptional activity of[3-catenin hinges on stabilization of cytoplasmic (3-catenin, without a need to change the affinities of specific protein interactions. These mechanisms induce structural changes in 13-catenin that help dictate whether 13-catenin interacts with adhesion or transcription complexes.  1.6. T cell factor family of DNA binding proteins -  -  Regardless of whether the underlying cause of activation of Wnt/13-catenin signal transduction cascade, the critical outcome is the inappropriate activation of genes which control critical cellular functions i.e., survival, proliferation, and cell fate. The culprits of the processes are heterodimeric transcription factor complexes formed by a transactivating component, [3catenin, and a DNA-binding subunit, a member of the TCF family of proteins. Often, TCFs are simply perceived as carriers for 13-catenin, but actually they carry out more complex functions in gene regulation. In Wnt signaling processes they behave as binary switches, both repressing and activating target genes [reviewed in (143, 144)]. Additionally, outside Wnt/13-catenin signaling, they act as architectural factors. T-cell factor function and activity is modulated by numerous  34  protein interaction partners in addition to 13-catenin. Various post-translational modifications affect the subcellular distribution of TCFs and their interactions with DNA and proteins. A multitude of TCF isoforms with different gene regulatory potential is generated by dual promoter usage and alternative splicing (143, 144). TCF genes are subject to mutation and deregulation in cancer cells and TCF family members possess qualities of tumor suppressors as well as tumor promoters (143, 144). And yet, despite all the progress made, the full range of the functional diversity and the regulatory complexity of TCF proteins are just beginning to emerge. In mammals, there are four TCF family members; TCF-1, LEF-1, TCF-3 and TCF-4 (143, 144). The interaction with one of these guides 13-catenin to specific Wnt target genes and thereby enhances their transcription. As ultimate nuclear effectors of the Wnt/P-catenin signal transduction cascade, TCF family members carry out important and crucial functions in both cell cycle control as well as fate decisions and differentiation. Although the initial findings which uncover the interaction between 13-catenin and TCF proteins suggested that they cooperate with 13-catenin in the stimulatory branch of the Wnt/13-catenin pathway, many further studies established a more complicated and diverse picture of TCF functions (145). In fact, as implied by their names, the two family members TCF-1 and LEF-1 also participate in Wnt and 0-catenin independent gene regulatory processes and this was known long before it was realized that TCFs interact with P-catenin [reviewed in (146)]. Even in the context of Wnt signaling TCF proteins are not only required for the activation of Wnt target genes, but also for their transcriptional repression in the absence of Wnt stimuli (145). T-cell factor family members act in a cell-context and promoter-specific manner in which they fulfill partially redundant but also specific tasks (147-150). In addition, they are part of both positive and negative regulatory feedback loops which dampen and enhance Wnt signals (151-153).  35  1.6.1. Structure and protein domain functions of TCF proteins The principle structure of a prototypic TCF dedicated to Wnt/13-catenin signal transduction is depicted schematically in Figure I-10. The N-terminal part contains a short amino acid stretch that mediates the interaction with 13-catenin and related proteins. The adjacent section harbors binding sites for transcriptional coregulators, and has additional modulating effects on transcativation (153). This part abuts the HMG domain which is responsible for sequencespecific DNA binding of TCFs (154). The C-terminal parts of TCFs which are highly variable in length, provide contacts for coactivator proteins and again alter the transctivation properties of TCFs (147, 149, 155, 156). Although a similar overall organization of TCF proteins cannot be denied, TCFs possess a high degree of structural variability which arises from alternative splicing and the usage of different promoters. Not surprisingly, there are reports suggesting that different TCF family members and their isoforms fulfill noninterchangable functions in transcriptional control processes (147, 149, 150).  1.6.1.1. DNA-binding and promoter recognition The TCF proteins belong to a group of DNA-binding transcription factors which have a signature motif containing a single copy of the so-called HMG domain (Figure I-10) [reviewed in (154)]. Single HMG domain proteins like TCFs bind in a sequence specific manner (154). Originally, the consensus sequence for TCF-binding elements recognized by LEF-1 and TCF-1 was a rather loosely defined 8-bp motif 5-A/T A/T CAAAGG-3 (157). Based on a binding site selection assay with TCF-1, a recent study suggested an extended recognition sequence 5AGATCAAAGGG-3 (158).  36  DNA-binding Consensus Motif  A.  1  ^  400-600 as Variable Length C-Terminus  B. Co-activator  MITF  Co-repressor  HBP Figure 1-10. General structure of TCF proteins and binding partners. A, TCF structural domains. TCF proteins are composed of an N-terminal domain of approximately 300 amino acids, an 80-amino acid HMG domain, an adjacent nuclear localization signal (N LS), and a C-terminal tail which is highly variable in length. The binding domain for 13-catenin ((3BD) is a short amino acid sequence motif close to the very N terminus. The HMG domain of TCF mediates sequence-specific DNA binding to the consensus recognition motif indicated. Several domains located at the very N terminus and within the central part of the N terminus contribute to context-dependent transcriptional activation by TCF proteins. The C-terminal tail regions of certain TCF isoforms (TCF1E and TCF4E) harbor an additional promoter-specific activation domain. Transcriptional repression mediated by TCF depends mainly on a region within the N-terminal half of the proteins. TCF3 and TCF4 variants with long C-terminal tails contain additional repressing elements composed of two short peptide motifs at their C-termini. Most of these functional domains correlate with interaction sites for coactivators and corepressors of TCF. B, Interaction partners of TCF. Listed are known interaction partners of TCF for which the binding sites in TCF have been mapped and for which the functional importance of the interaction is known. Adapted from Hecht, A. (2004) Members of the T-cell factor family of DNA-binding proteins and their roles in tumorogenesis. In Gossen, M., Kaufmann, j. and Triezenberg, S.J. (eds.), Transcription Factors. Springer-Verlag, Berlin, Vol. 166, pp. 123-165.  37  The E-isoforms of TCF proteins harbor another block of positively charged amino acids, the so-called CRARF domain in their extended C termini (159). The CRARF domain, named after a characteristic stretch of cysteine and arginine residues, is evolutionary conserved and the highly similar sequences are present in mammalian TCF-1E and TCF-4E isoforms, as well as in transcription factors not related to TCFs (147, 157). The presence or absence of the CRARF domain has been shown to influence the DNA-binding properties of TCF-4E (149). Even though HMG domains in TCF proteins are nearly identical in amino acid sequence (157) and TCF proteins are thought to interact with the same DNA sequences, displaying only minor differences in affinity (160), this raises the possibility that regions outside of the HMG domain contribute to specificity of target gene recognition of TCF proteins. TCF proteins are known to interact with other sequence-specific DNA-binding factors, for example microphthalmia-associated transcription factor (MITF) or the Smad proteins (161, 162). At least under experimental conditions, the interaction with Smads allowed the formation of a TCF-promoter complex even upon mutational inactivation of the cognate TCF-binding sites in the Xenopus Twin promoter (161). Hence, heterodimerization with Smads or MITF could enable TCFs to recognize certain promoters even if these lack a cognate TCF binding elements, a mechanism that could alter the spectrum of potential TCF target genes considerably.  1.6.1.2. Context-dependent activation and repression domains  DNA-bound TCF proteins are functionally neutral on their own. Depending on the particular circumstances, however, TCF proteins can exert both activating and repressing functions. Transcriptional activation and repression can be assigned to defined sequence elements within TCFs (Figure I-10). For Wnt/13-catenin regulated TCF target genes, the very N  38  terminus is of critical importance (145, 163). At a subset of Wnt-regulated promoters, the CRARF-domain specifically present at the C termini of TCF-1E and TCF-4E additionally functions as an obligatory but context-dependent transcriptional activation domain (147, 149). In mammals, the CRARF domain allows for selective activation of the (3-catenin/TCF-responsive LEF-1 promoter by TCF-1E and the Wnt-responsive Cdxl promoter by TCF-4E (147, 149). A further contribution to promoter activation can be made by amino acid sequences encoded by an alternatively spliced exon upstream of the HMG domain (160, 164). The N-terminal half of LEF1 harbors yet another transactivation domain, which functions specifically in the context of the T-cell receptor (TCR)a enhancer but not at Wnt-inducible promoters (165). In the case of sequences derived from the alternatively spliced exon, the molecular basis for their activation potential is not known but the other activation domains coincide with interaction domains for well-known binding partners of TCFs: f3-catenin, ALY and p300 (Figure I-10) suggesting that these domains function by mediating protein-protein interactions and cofactor recruitment. A similar scenario is probably true for the repression domains in TCFs which were mapped to a region upstream of the HMG domain and to two short sequence motifs at the C termini of the TCF-3E and TCF-4E isoforms (Figure I-10). While the former interacts with Grg/TLE corepressors (166), the latter interacts with C-terminal binding protein (CtBP) (155, 156). Which activation or repression domains are used is probably determined by the exact promoter architecture and the molecular framework provided by the various transcription factors and their cofactors which assemble into higher order structures at a given regulatory element.  39  1.6.2. Protein-protein interaction 1.6.2.1. p-catenin/Armadillo The event which propelled TCF proteins into center stage was the discovery that LEF-1 and XTCF-3 interacted with the multifunctional protein P-catenin (145, 167). The reason why this sparked such enormous interest is that the formation of this heterodimeric protein complex closed a gap in the signal transduction pathway of Wnt growth factors and provided an explanation for how signal could be relayed into the nucleus. As further studies showed, (3catenin appears to function as a mobile transcriptional activation domain which is guided to its target genes by exploiting the sequence specific DNA-binding capacity of TCFs (143, 145). By now it is clearly established that 13-catenin and its Drosophila ortholog, armadillo, as well as more distant relatives in diverse species including nematods all bind to an N-terminal domain in TCFs spanning approximately amino acids 10-55 (Figure I-10). Among the key (3catenin interacting partners are TCFs, cadherins, APC and axin which appear to interact competitively with the same or closely overlapping sections of the Arm repeat region of [3catenin (137, 138, 168). Slight structural differences of P-catenin binding domain ((3BD) from different TCFs appear to exist, but the overall structure of the PBD in TCFs is similar to the corresponding 0-catenin-interacting domains in E-cadherin and APC (137, 138, 168).  1.6.2.2. Groucho-related genes/Transducin-like-enhancer-of-split (Grg/TLE) TCFs have the potential to act as binary switches which can impose either a repressed or an activated state on a promoter. In both cases, though, functional properties of the targeted promoter are determined by specific cofactors associated with TCFs. The primary binding partners employed in promoter inactivation are transcriptional corepressors which are encoded  40  by a group of genes called " Groucho-related genes" in the mouse or "transducin-like-enhancerof-split" in humans or Grg/TLE [reviewed in (169)]. Grg/TLE proteins are required in many developmental programs where they act as DNA nonbinding repressor proteins which are brought to their target promoters through interactions with a wide range of diverse transcription factors (169). TCFs were shown to interact with Grg/TLE biochemically and genetically (153, 166). There is no selectivity among TCFs and Grg/TLE family members, i.e., all TCFs interact with all Grg/TLE proteins (Figure I-10) (166). The interaction is mediated by a region in TCFs located immediately upstream of the HMG domain and by the Gln-rich N-terminal domain in Grg/TLE (170). Their activity probably requires an additional biochemical interaction with histone deacetylase (HDAC) (171). A more recent study on the molecular basis of the switch from transcriptional repression by Grg/TLE to activation during Wnt signaling by 0-catenin has been demonstrated that 13-catenin displaces Grg/TLE from LEF/TCF by binding to a site on LEF1 that includes sequences overlapping the Grg/TLE-binding site (172).  1.6.2.3. C-terminal binding protein Whereas all TCFs are endowed with the ability to interact with Grg/TLE corepressors, TCF-3 and TCF-4 splice variants with long C-terminal extensions additionally interact with the CtBP (155, 156). CtBP is an evolutionarily conserved nuclear phosphoprotein [reviewed in (173)]. In mammals, two highly related genes encoding CtBP1 and CtBP2 exist and both proteins were shown to bind to TCF-4 (156). Together with Groucho and its relatives, CtBP represents the major transcriptional corepressor activity with important roles in developmental processes in vertebrates and invertebrates. The mechanism of repression is not yet clear but both HDAC-dependent and independent means could be used. CtBP binds to a characteristic amino  41  acid sequence motif `PLDLS' and closely related variants thereof. Two copies of this motif are present in TCF-3 (PLSLT and PLSLV) and TCF-4 (PLSLS and PLSLV) (Figure I-10). Recent studies in human tissue culture cells showed, however, that CtBP mediates a HDAC-dependent repression of the axin 2/conductin gene (156). TCF proteins hence may utilize the Grg/TLE and CtBP corepressors in a highly context-dependent, albeit sometimes redundant, manner.  1.6.2.4. CREB-binding protein/p300 The CBP and the related p300 are bimodal transcriptional coactivators which can modulate transcription processes by acetylating histones and nonhistone transcription factors or by bridging transcriptional activators to components of the basal transcription machinery (174). In D. melanogaster, dCBP has been reported to be a negative regulator of Wnt signaling, whereas in vertebrates, p300 and CBP are essential coactivators of 13-catenin which are necessary for the activation of Wnt targets (175-179). LEF-1 is thought to be acetylated by dCBP which was claimed to interact with the LEF-1 HMG domain (179) (Figure I-10). p300 was also shown to interact with a region containing the CRARF domain at the C terminus of the TCF-4E isoform but acetylation of TCF-4E was not investigated (149). Whether other TCF family members also interact with CBP or p300 and which domains in TCFs are capable of interacting with p300/CBP is not yet fully investigated. Nonetheless, it appears that p300 and CBP are among the interaction partners of TCF proteins, but binding of TCFs to p300/CBP may be mediated by multiple TCF domains. Moreover, the functional consequences of these interactions appear to vary between species and perhaps also between TCFs.  42  1.6.3. Post-translational modifications 1.6.3.1. Phosphorylation and acetlyation Protein phosphorylation is one of the most widespread post-translational modifications and — not surprisingly — several different kinases are known to target TCFs thereby changing their biochemical properties and consequently their biological functions. The generation of these asymmetries requires the suppression of Popl (C. elegans ortholog of mammalian TCF) activity in the anterior sister cell which is achieved by Lit-1, a mitogen-activated protein kinase-related to the Nemo and Nemo-like kinase (NLK) in D. melanogaster and vertebrates (180, 181). At the molecular level it has been shown that Lit-1 phosphorylates Popl and that the action of Lit-1 correlates with a relocation of Pop-1 from the nucleus to the cytoplasm (181). In vertebrates, NLK phosphorylates the TCF family members mLEF-1, XTCF-3 and hTCF-4 (182). In the case of hTCF-4, phosphorylation by NLK reduces its DNA-binding affinity (182). Aside from NLK there are three additional kinases which have been implicated in regulating TCF activity. As a component of the P-catenin destruction complex GSK-30 plays a central role in the transmission of Wnt signals. In extracts from Xenopus embryos GSK-30 was additionally found to be associated with XTCF-3 and recombinant GSK-30 phosphorylates XTCF-3 in vitro (183). This phosphorylation results in decreased complexation of XTCF-3 and P-catenin. Because TCFs compete with APC for binding to J3-catenin, a reduced binding capacity of TCF-3 would render 13-catenin more readily susceptible to being shuttled into the destruction complex by APC. Interestingly, the effect of GSK-30-mediated phosphorylation of XTCF-3 may be counteracted by another kinase, namely CK Ie. CK Ie, which like GSK-30 has multiple functions in Wnt signal transduction and other cellular processes, also associates with XTCF-3 (183). In contrast to GSK-3I3, however, phosphorylation of XTCF-3 by CK I promotes complex  43  formation off3-catenin and XTCF-3 and thereby protects f3-catenin from degradation. Phosphorylation nonetheless appears to be an important and versatile regulatory tool which serves to control major functional properties of TCF proteins: their intracellular distribution, their DNA-binding properties and the interaction with their transcriptional coactivator (3-catenin. A link between protein modification through acetylation and transcriptional control was first revealed by the observation that transcriptionally active chromosomal regions are enriched for hyperacetylated histones. Today, numerous other nonhistone proteins are known to be acetylated and a variety of protein functions such as protein-protein interactions, protein —DNA binding and subcellular localization can be influenced by acetylation [reviewed in (184)]. Two groups of antagonistically acting enzymatic activities control protein acetylation. Acetylases transfer the acetyl group from acetyl-coenzyme A to the NH2 groups of lysine side chains whereas deacetylases catalyze the removal of the acetyl moiety (184). Acetylation has been linked to various aspects of TCF functions albeit at present most of the information relating to acetylation of TCFs has been obtained from studies with invertebrates. There are some observations which indicate that perhaps not all findings can be extended to the mammalian system but currently it cannot be strictly ruled out that acetylation and acetylases play a similar role in mammalian cells. Therefore, the two examples for acetylation of TCF family members in invertebrates are listed below. The first case was reported in D. melanogaster, where the acetylase dCBP was found to interact with HMG box of pangolin/dTCF (179). Genetic analyses suggested an inhibitory role of dCBP in Wnt signaling processes. A potential molecular explanation for this effect was provided by the finding that dCBP acetylates lysine  K25  in the 13-catenin binding site of dTCF, thereby weakening the  interaction between f3-catenin/armadillo and dTCF (179). To date, however, there is no evidence  44  for a similar function of CBP or p300 in vertebrates. Rather, both CBP and p300 serve as important coactivators of 0-catenin and TCFs in Wnt signaling (149, 175-178). The second recent report about acetylation of a TCF protein is from C. elegans. Here, the TCF family member Popl is acetylated by CBP/p300 at residues K185, K187 and K188, but apparently not at the N-terminus (185). This again is in conflict with the findings in the Drosophila system. In addition, acetylation of Popl has a positive regulatory function because it is required for its nuclear localization and retention (185). Unfortunately, it is doubtful whether the findings of Gay and coauthors can be extrapolated to vertebrate TCFs, because the main Popl acetylation target K185 is not conserved in the mammalian orthologs of Popl. While it thus remains largely unclear to what extent TCF proteins themselves are targets for acetylation in vertebrates, TCFs nonetheless utilize acetylases and deacetylases to execute their regulatory functions in mammalian cells. A novel activation domain present specifically in the "E" isoforms of TCF-1 and TCF-4 has been shown to interact with p300 in vitro and in cellular extracts (149). It is likely that this interaction is important for the context-dependent activation of certain promoters by TCF-1 and TCF-4. The mechanism underlying p300 activity in this context is unknown but it could involve acetylation of TCFs. This is suggested by the observation that in TCF-4 each of the two binding sites for the transcriptional repressor CtBP is flanked by a lysine residue (173). In the case of El A, which also binds both p300 and CtBP, a similarly configured lysine near the CtBP-binding site is reversibly acetylated by p300/CBP and this acetylation modulates the interaction between ElA and CtBP (186). By extrapolation, acetylation of the C terminus of certain TCF splice variants might be required for promoter activation by weakening the interaction with the CtBP repressor proteins.  45  In addition to this stimulatory interaction between TCF-4 and the p300 acetylase, TCFs can also interact with a deacetylase to repress transcription. LEF-1 has been found in a complex with HDAX1 (187). The interaction may occur either directly or indirectly through Grg/TLE repressors which are known to bind HDAC1. Inhibition of HDAC1 by TSA enhances Wnt/Pcatenin target gene expression and alters the acetylation state near Wnt/f3-catenin regulated promoters (187). This indicates that association of LEF-1 and HDAC1 contributes to the transcriptional repression of target genes in the absence of a Wnt stimulus through a chromatinbased mechanism. However, chromatin may not be the only target of LEF-1-associated HDAC 1. In a recent report, HDAC 1 overexpression counteracted LEF-1-dependent nuclear accumulation and retention of 13-catenin (188). This is clearly reminiscent of the role of acetylation/deacetylation of Popl in subcellular localization of C.elegans. Unfortunately, however, it was not analyzed whether it was only 0-catenin which failed to accumulate in the nucleus in the presence of excess HDAC1, or whether the subcellular distribution of LEF-1 was affected as well. This would be expected if the Popl paradigm was applicable to LEF-1 and mammalian cells.  1.7. Androgens and androgen receptors in prostate Androgens, acting through the androgen receptor (AR), are required for prostate development and normal prostate function (189). Androgen action can be considered to function through an axis involving the testicular synthesis of testosterone, its transport to target tissues, and the conversion by 5a-reductase to the more active metabolite, 5a-dihydrotestosterone (DHT). Testosterone and DHT exert their biological effects through binding to AR and inducing AR transcriptional activity (Figure I-11). The androgen-induced transcriptional activation of AR  46  T/DHT  TGF13 I IL-6 / IGF1  (R4 v  (^  ..............^ ... .........  .....  ^  Smad/Akt/MAPK Pathway  P  Cytoplasm  Pol II comple  ARE ARE  I TATA  Figure I-11. Androgen action in the prostate. Testosterone (T) and DIET bind to androgen receptor (AR) and promote the association of AR coregulators (ARAs). AR then translocates to the nucleus and binds to AREs in the promoter regions of target genes to induce cell proliferation and apoptosis. Other signal transduction pathways, such as those involving TGFf3, IL-6, and IGF-I, can also enhance AR activity via phosphorylation of AR and/or ARAs. ARAs, androgen receptor coregulators; ARE, androgen response element; Hsp, Heat shock protein; R, membrane receptor; P, protein phosphorylation; PSA; prostatic specific antigen. Adapted from Heinlein CA and Chang C. Androgen receptor in prostate cancer. Endocr Rev. 2004;25:276-308. 47  is modulated by the interaction of AR with coregulators and by phosphorylation of AR and AR coregulators in response to growth factors (189). AR and the modulators of AR activity remain important in prostate cancer. Approximately 80-90% of prostate cancers are dependent on androgen at initial diagnosis, and endocrine therapy of prostate cancer is directed toward the reduction of serum androgens and inhibition of AR (190). However, androgen ablation therapy ultimately fails, and prostate cancer progresses to a hormone refractory state. AR is expressed throughout prostate cancer progression and persists in the majority of patients with hormone refractory disease (191).  1.7.1. Androgen and androgen receptor in normal prostate development and maintenance of prostate epithelia The prenatal development of the prostate is dependent on androgen, particularly on DHT. After the development of the prostate, androgens continue to function in promoting the survival of the secretory epithelia, the primary cell type thought to be transformed in prostate adenocarcinoma (192). In the normal prostate, the rate of cell death is 1-2% per day, which is balanced by a 1-2% rate of proliferation (193). The reduction of serum and prostatic DHT levels by castration results in a loss of 70% of the prostate secretory epithelial cells due to apoptosis in adult male rats, but the basal epithelia and stromal cell populations are relatively unaffected (194). In the intact rat prostate, the secretory epithelial cells show strong AR immunoreactivity, whereas the majority of basal epithelial cells are AR negative (195), suggesting an explanation for their different sensitivity to androgen. However, AR is also expressed in the prostatic stroma, although castration results in the loss of stromal AR expression (196). The prostatic stroma therefore has the capacity to respond to androgen, but androgen is not required for its survival. In  48  the normal prostate, cellular homeostasis is modulated in part by paracrine growth factor regulation between epithelial and stromal cells (197). A subset of these growth factors, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), can be regulated by androgens and can influence vascular survival (198). It is possible that castration initially alters prostatic growth factor production in the stroma, which contributes to a decrease in vascular function. The resulting reduction in blood flow, combined with an altered growth factor environment and decreased expression of other androgen regulated proteins, may contribute to apoptosis of the secretory epithelia.  1.7.2. Versican and prostate cancer Stromal tissue mediates the induced growth and development of embryonic epithelium into differentiated prostate (199). Prostatic stroma is also necessary in the maintenance of adult prostatic secretory epithelium (200, 201). In addition, stromal-epithelial interactions play a significant role in steroid-influenced prostate carcinogenesis, and in the progression to the hormone-insensitive phenotype (202). Tumor cells must remodel the matrix to facilitate communication and escape control by the microenvironment. Remodeling can also include interactions with "alternative" ECM, leading to cellular proliferation, structural disruption, and circumvention of apoptosis (203). Current findings obtained through a variety of approaches increasingly point to the contribution of stromal components to oncogenic signals that mediate both phenotypic and genomic changes in epithelial cells (204-206). Several reports have shown that versican plays a role in cell adhesion (207), migration (208), proliferation (209), differentiation (210), angiogenesis (211), and resistance to oxidative stress-induced apoptosis (51); all of which are important events in tumor initiation and/or progression. Versican is highly expressed in many malignancies,  49  including prostate cancers (212). Recent studies demonstrated that prostate cells from tumor and benign prostatic hyperplasia (BPH) tissues induce host stromal cells to accumulate versican levels via a paracrine mechanism (213, 214). Further, versican has been shown to be an important modulator of tumor cell attachment to the interstitial stromal matrix of the prostate; a factor likely important in cancer cell motility and local invasion of the prostatic stroma (215). As well it has been suggested that versican may be a useful marker of disease progression in patients with early-stage prostate cancer (212). 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Gay, F., Calvo, D., Lo, M.C., Ceron, J., Maduro, M., Lin, R. and Shi, Y. (2003) Acetylation regulates subcellular localization of the Wnt signaling nuclear effector POP1. Genes Dev, 17, 717-22.  186.  Zhang, Q., Yao, H., Vo, N. and Goodman, R.H. (2000) Acetylation of adenovirus El A regulates binding of the transcriptional corepressor CtBP. Proc Natl Acad Sci USA, 97, 14323-8.  187.  Billin, A.N., Thirlwell, H. and Ayer, D.E. (2000) Beta-catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator. Mol Cell Biol, 20, 6882-90.  65  188.  Henderson, B.R., Galea, M., Schuechner, S. and Leung, L. (2002) Lymphoid enhancer factor-1 blocks adenomatous polyposis coli-mediated nuclear export and degradation of beta-catenin. Regulation by histone deacetylase 1. J Biol Chem, 277, 24258-64.  189.  Heinlein, C.A. and Chang, C. (2004) Androgen receptor in prostate cancer. Endocr Rev, 25, 276-308.  190.  Denis, L.J. and Griffiths, K. (2000) Endocrine treatment in prostate cancer. Semin Surg Oncol, 18, 52-74.  191.  Mohler, J.L., Chen, Y., Hamil, K., Hall, S.H., Cidlowski, J.A., Wilson, E.M., French, F.S. and Sar, M. (1996) Androgen and glucocorticoid receptors in the stroma and epithelium of prostatic hyperplasia and carcinoma. Clin Cancer Res, 2, 889-95.  192.  De Marzo, A.M., Nelson, W.G., Meeker, A.K. and Coffey, D.S. (1998) Stem cell features of benign and malignant prostate epithelial cells. J Urol, 160, 2381-92.  193.  Berges, R.R., Vukanovic, J., Epstein, J.I., CarMichel, M., Cisek, L., Johnson, D.E., Veltri, R.W., Walsh, P.C. and Isaacs, J.T. (1995) Implication of cell kinetic changes during the progression of human prostatic cancer. Clin Cancer Res, 1, 473-80.  194.  English, H.F., Kyprianou, N. and Isaacs, J.T. (1989) Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration. Prostate, 15, 233-50.  195.  Prins, G.S., Birch, L. and Greene, G.L. (1991) Androgen receptor localization in different cell types of the adult rat prostate. Endocrinology, 129, 3187-99.  196.  Prins, G.S. and Birch, L. (1993) Immunocytochemical analysis of androgen receptor along the ducts of the separate rat prostate lobes after androgen withdrawal and replacement. Endocrinology, 132, 169-78.  197.  Russell, P.J., Bennett, S. and Sticker, P. (1998) Growth factor involvement in progression of prostate cancer. Clin Chem, 44, 705-23.  198.  Burchardt, M., Burchardt, T., Chen, M.W., Hayek, O.R., Knight, C., Shabsigh, A., de La Taille, A. and Buttyan, R. (2000) Vascular endothelial growth factor-A expression in the rat ventral prostate gland and the early effects of castration. Prostate, 43, 184-94.  199.  Cunha, G.R., Hayward, S.W. and Wang, Y.Z. (2002) Role of stroma in carcinogenesis of the prostate. Differentiation, 70, 473-85.  200. Cunha, G.R., Reese, B.A. and Sekkingstad, M. (1980) Induction of nuclear androgenbinding sites in epithelium of the embryonic urinary bladder by mesenchyme of the urogenital sinus of embryonic mice. Endocrinology, 107, 1767-70.  66  201.  Cunha, G.R. and Young, P. (1991) Inability of Tfm (testicular feminization) epithelial cells to express androgen-dependent seminal vesicle secretory proteins in chimeric tissue recombinants. Endocrinology, 128, 3293-8.  202.  Condon, M.S. (2005) The role of the stromal microenvironment in prostate cancer. Semin Cancer Biol, 15, 132-7.  203.  Pupa, S.M., Menard, S., Forti, S. and Tagliabue, E. (2002) New insights into the role of extracellular matrix during tumor onset and progression. J Cell Physiol, 192, 259-67.  204.  Tlsty, T.D. and Hein, P.W. (2001) Know thy neighbor: stromal cells can contribute oncogenic signals. Curr Opin Genet Dev, 11, 54-9.  205.  Bhowmick, N.A., Chytil, A., Plieth, D., Gorska, A.E., Dumont, N., Shappell, S., Washington, M.K., Neilson, E.G. and Moses, H.L. (2004) TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science, 303, 848-51.  206.  Bhowmick, N.A., Neilson, E.G. and Moses, H.L. (2004) Stromal fibroblasts in cancer initiation and progression. Nature, 432, 332-7.  207.  Landolt, R.M., Vaughan, L., Winterhalter, K.H. and Zimmermann, D.R. (1995) Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth. Development, 121, 2303-12.  208.  Ang, L.C., Zhang, Y., Cao, L., Yang, B.L., Young, B., Kiani, C., Lee, V., Allan, K. and Yang, B.B. (1999) Versican enhances locomotion of astrocytoma cells and reduces cell adhesion through its G1 domain. J Neuropathol Exp Neurol, 58, 597-605.  209.  Zhang, Y., Cao, L., Yang, B.L. and Yang, B.B. (1998) The G3 domain of versican enhances cell proliferation via epidermial growth factor-like motifs. JBiol Chem, 273, 21342-51.  210.  Wu, Y., Sheng, W., Chen, L., Dong, H., Lee, V., Lu, F., Wong, C.S., Lu, W.Y. and Yang, B.B. (2004) Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth. Mol Biol Cell, 15, 2093-104.  211.  Cattaruzza, S., Schiappacassi, M., Ljungberg-Rose, A., Spessotto, P., Perissinotto, D., Morgelin, M., Mucignat, M.T., Colombatti, A. and Penis, R. (2002) Distribution of PGM/versican variants in human tissues and de novo expression of isoform V3 upon endothelial cell activation, migration, and neoangiogenesis in vitro. JBiol Chem, 277, 47626-35.  212.  Ricciardelli, C., Mayne, K., Sykes, P.J., Raymond, W.A., McCaul, K., Marshall, V.R. and Horsfall, D.J. (1998) Elevated levels of versican but not decorin predict disease progression in early-stage prostate cancer. Clin Cancer Res, 4, 963-71.  67  213.  Sakko, A.J., Ricciardelli, C., Mayne, K., Tilley, W.D., Lebaron, R.G. and Horsfall, D.J. (2001) Versican accumulation in human prostatic fibroblast cultures is enhanced by prostate cancer cell-derived transforming growth factor betal. Cancer Res, 61, 926-30.  214.  Cross, N.A., Chandrasekharan, S., Jokonya, N., Fowles, A., Hamdy, F.C., Buttle, D.J. and Eaton, C.L. (2005) The expression and regulation of ADAMTS-1, -4, -5, -9, and -15, and TIMP-3 by TGFbetal in prostate cells: relevance to the accumulation of versican. Prostate, 63, 269-75.  215. Sakko, A.J., Ricciardelli, C., Mayne, K., Suwiwat, S., LeBaron, R.G., Marshall, V.R., Tilley, W.D. and Horsfall, D.J. (2003) Modulation of prostate cancer cell attachment to matrix by versican. Cancer Res, 63, 4786-91.  68  CHAPTER II — RATIONALE, HYPOTHESIS, AND EXPERIMENTAL AIMS  ILL Rationale Versican, a chondroitin sulfate proteoglycan, is one of the main components of ECM. Versican provides loose and hydrated matrices during key events in development and disease (1, 2). It participates in cell adhesion, proliferation, migration, and angiogenesis, and hence plays a central role in tissue morphogenesis and maintenance as well as in a number of pathologic processes including atherosclerotic vascular diseases, cancer, tendon remodeling, hair follicle cycling, central nervous system injury and neurite outgrowth (1-3). In the context of vascular biology, versican is generally considered to be pro-atherogenic and central to vascular injury and repair events, because of its ability to trap cholesterol-rich lipoproteins in addition to its impact on cell adhesion, survival, proliferation, migration, and ECM assembly (4-6). Versican is a complex molecule with modular core protein domains and GAG side chains and various steps in its synthesis and processing that regulate its constituency. Despite its pivotal role in normal tissue homeostasis and pathogenesis of several diseases, the soluble factors, signaling pathways and transcriptional events that regulate the production of versican in vivo and in vivo are unknown (2). In order to fully appreciate the functional roles of versican as they relate to changing patterns of expression and function in development and disease, in depth knowledge of versican's biosynthetic processing is necessary. Thus, this dissertation focuses, for the first time, on identifying and assessing the functional roles of transcriptional regulatory elements of the versican gene through in  69  vitro methodologies with the ultimate goal of understanding the molecular basis for regulation of the versican gene. The main goals of my doctoral research project have been to investigate the transcriptional regulation of the versican gene, focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans factors. -  Several experimental steps were needed to reach this goal. The first step involved determining the functionally relevant promoter regions of the versican gene by studying transcription activity through the use of luciferase reporter promoter assays. The second step involved confirmation of the functional relevance of different regions through the identification and characterization of specific transcription factors that bind to the versican promoter DNA to affect its expression. Specifically, these studies will include promoter identification and analysis; reporter gene assays of promoter fragments and mutants; in vitro footprinting; DNA element identification; and transcription factor analysis. These investigations will reveal the molecular basis of versican gene regulation at the gene promoter, nucleosome, and signal transduction levels.  11.2. Overarching Hypothesis Versican gene expression is controlled by growth factor-mediated signal transduction pathways and specific down-stream transcription factors which interact with specific cis-acting regulatory elements in the human versican promoter. Such regulation governs the production of provisional wound-repair ECM, as well as the mitogenic phenotype of the cells, thereby having a major influence on tissue response to injury, including repair and remodeling.  70  11.3. Specific Aims 1. To characterize the positive and negative regulatory regions in human versican promoter 2. To identify the role and mechanism of GSK-313 signaling and 13-catenin/TCF transcription factor complex on versican transcription 3. To define the role of LEF/TCF transcription factor isoforms and their cofactors on versican transcription 4. To elucidate mechanism of androgen receptor mediated regulation of versican transcription In Specific Aim 1 (chapter III), I will amplify a 752-bp fragment representing the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments using human genomic DNA. The fragments will be cloned in the PGL3-luciferase plasmid. To discern the enhancer and repressor regulatory regions of the versican promoter, I conduct a series of transfection studies using deletion mutants of the proximal promoter region (632/+118) in epithelial HeLa cells and rat aortic SMC. Further, I will examine the role of cAMP signaling and two families of transcription factors downstream of this signaling, CREB and CCAAT/enhancer binding protein (C/EBP), in versican gene transcription. In Specific Aim 2 (chapter IV), I will determine one of the main signal transduction pathways and its downstream cis-elements and trans-factors governing versican transcription. In particular, I will identify the role of PI3K / PKB and subsequent phosphorylation / inhibition of the GSK-313 signaling pathway in regulating versican expression. Further, I will identify the transcription factor complex and promoter sequence downstream of GSK-30 signaling, P-catenin/TCF-4 transcription factor  71  complex in the versican promoter, mediating versican promoter activity and transcription (7). In specific Aim 3 (chapter V), I will show that variation in structure and expression of different TCF isoforms and their capacity to interact with different coactivators and co-repressors results in differential regulation of versican promoter activity by these TCF isoforms. Finally, in Specific Aim 4 (chapter VI), I will investigate promoter elements and mechanisms of androgen receptor-mediated regulation of the versican gene in prostate cancer cells. I will demonstrate that androgen receptor trans-activates versican expression. Furthermore, I show the cross-talk between androgen receptor and (3-catenin in the regulation of versican transcription in prostate stromal fibroblasts. An understanding of the regulation of versican provides a first step towards the discovery of novel therapies based on endogenous regulatory elements for several disorders where this molecule plays crucial roles in their pathogenesis.  72  11.4. REFERENCES 1.  Wight, T.N. (2002) Versican: a versatile extracellular matrix proteoglycan in cell biology. Curr Opin Cell Biol, 14, 617-23.  2.  Rahmani, M., Wong, B.W., Ang, L., Cheung, C.C., Carthy, J.M., Walinski, H. and McManus, B.M. (2006) Versican: signaling to transcriptional control pathways. Can J Physiol Pharmacol, 84, 77-92.  3.  Wu, Y.J., Lapierre, D., Wu, J., Yee, A.J. and Yang, B.B. (2005) The interaction of versican with its binding partners. Cell Res, 15, 483-94.  4.  Rahmani, M., McDonald, P.C., Wong, B.W. and McManus, B.M. (2004) Transplant vascular disease: Role of lipids and proteoglycans. Can J Cardiol, 20, 58B-65B.  5.  Wight, T.N. and Merrilees, M.J. (2004) Proteoglycans in atherosclerosis and restenosis: key roles for versican. Circ Res, 94, 1158-67.  6.  Rahmani, M., Cruz, R.P., Granville, D.J. and McManus, B.M. (2006) Allograft vasculopathy versus atherosclerosis. Circ Res, 99, 801-15.  7.^Rahmani, M., Read, J.T., Carthy, J.M., McDonald, P.C., Wong, B.W., Esfandiarei, M., Si, X., Luo, Z., Luo, H., Rennie, P.S. et al. (2005) Regulation of the versican promoter by the beta-catenin-T-cell factor complex in vascular smooth muscle cells. J Biol Chem, 280, 13019-28.  73  CHAPTER III — IDENTIFICATION OF POSITIVE AND NEGATIVE REGULATORY ELEMENTS OF HUMAN VERSICAN GENE PROMOTER: ROLE OF C/EBP AND CREB ON VERSICAN TRANSCRIPTION *  III.1. SUMMARY  The main aims of this chapter of my thesis are (1) to identify regulatory elements in the human versican gene that mediate the repression and/or enhancement of versican transcription, and (2) to investigate mechanisms involved in cAMP-mediated regulation of the versican gene. A 752-bp fragment representing the human versican promoter (- 634/+118 bp) and nine stepwise 5' deletion fragments were amplified using human genomic DNA. The fragments were inserted in front of the luciferase reporter gene in the PGL3-luciferase plasmid. To discern the enhancer and repressor regulatory regions of the versican promoter, I conducted a series of transfection studies using deletion mutants of the proximal promoter region in epithelial HeLa cells and rat aortic SMC. Luciferase activity was analyzed following transfection. Transfection of stepwise 5' deletions of the versican promoter constructs identified three potential enhancer (-632 to -584, 397 to -335, and -150 to -90 bp) and two repressor regions (-584 to -397 and -335 to -150 bp). Of particular interest is the presence of C/EBP and CREB binding sites in the human versican promoter sequence. I thus examined the role of cAMP in mediating versican gene transcription, as well as the direct role of C/EBP and CREB transcription factors in this process. HeLa cells and SMC transfected with the versican luciferase construct followed by treatment with cAMP agonists [Forskolin or (Bu)2cAMP)] for 24 hrs were assayed for luciferase activity. Interestingly, cAMP agonists significantly enhanced and inhibited versican promoter activity in epithelial HeLa cells and SMC, respectively. To examine the respective roles of C/EBI13, vectors * A manuscript based on the results of this chapter is in preparation.  74  expressing C/EBP13 were co-transfected with the versican-Luc vector in HeLa and SMC in the presence or absence of cAMP inducers. Constitutive expression of C/EB13 13 resulted in significant induction of versican promoter activity in both HeLa cells and SMC. While (Bu)2cAMP slightly enhanced C/EBP[3-mediated induction of promoter activity in HeLa cells, it converted the C/EBPP-mediated enhancer to a repressor of versican promoter activity in SMC. To further investigate the dependence of C/EBPI3-mediated versican transcription on the binding of C/EB13 13 to the cis-acting elements, HeLa cells and SMC were co-transfected with the versican-Luc, C/EBIT, and C/EBP dominant negative (C/Edn — which lacks the DNA binding domain) expression vectors. Interestingly, C/Edn inhibited C/EBP-induced transcription in HeLa cells. Furthermore, C/Edn released the inhibition of the promoter activity mediated by the combination of (Bu)2cAMP treatment and C/EB1 3 13 expression in SMC. Our results also showed that the CREB expression plasmid significantly enhanced versican promoter activity in the presence and absence of cAMP inducer in both HeLa cells and SMC. In conclusion, I have defined distinct positive and negative regulatory regions in the human versican promoter involved in versican transcription. I have also shown that the combination of treatment with cAMP along with co-transfection with C/EB1 3 13 plasmid enhanced versican promote activity in HeLa cells, but repressed versican transcription in SMC, suggesting that versican transcription is differentially regulated by these mediators and transcription factor in epithelial cells and SMC. Furthermore, I demonstrated that CREB enhanced versican promoter activity through cAMPdependent and -independent mechanisms in HeLa cells and SMC.  75  111.2. INTRODUCTION AND RATIONALE  Previous studies from our laboratory have shown that the aberrant accumulation of PG, particularly versican, is a hallmark of vascular lesions associated with atherosclerosis, restenosis and allograft vasculopathy (1, 2), and is potentially important in the development of myxomatous heart valve lesions (3). In recent work, we have shown that treatment of vascular SMC with growth factors, namely epidermal growth factor (EGF) and insulin like growth factor-I (IGF-I), results in upregulation of versican mRNA and core protein expression (4). Furthermore, there have been reports that several growth factors, cytokines and interleukins mediate transcriptional regulation of the versican gene [reviewed in (5, 6)] . The growth factors PDGF-BB and transforming growth factor (TGF)-01 both upregulate versican mRNA and core protein synthesis in SMC (7). The effects of these growth factors are not specific to SMC, since versican expression is also upregulated by TGF-01 in skin and gingival fibroblasts (8) and EC (9), as well as in prostate stromal fibroblasts (10). Furthermore, evidence suggests that EGF and IGF-I stimulate the expression of versican transcripts in human malignant mesothelioma (11). The positive response of the versican gene to IGF-1 may be cell-type specific, however, as a decrease in versican expression has been reported in osteoblast-like cells in response to this growth factor (12). As expression of the versican gene was found to be regulated at the mRNA level by various cytokine-signaling events, the main focus of my thesis has been to examine the signal transduction pathways and transcriptional elements involved in the regulation of versican expression. Recent data has suggested that gonadotropin hormones, leutinizing hormone (LH) and follicle stimulating hormone (FSH) regulate versican mRNA and protein expression in the mice and rodent ovary (13). These observations suggest that versican is a matrix component of the  76  granulosa layer throughout folliculogenesis and is enriched in remodeling matrices during ovulation and neovascularization of the corpora lutea. Mechanism of the LH- and FSH-receptor action is through ligand binding activation of adenylate cyclase, increased production of cAMP, and activation of protein kinase A (PKA). In work detailed here, I provide further evidence on the specific molecular mechanisms involved in cAMP signaling and its down-stream transcription factors that mediate versican regulation. The regulation of gene transcription by cAMP has been studied extensively, and while there may be several transcription factors capable of mediating this response, the most extensively studied is the CREB. It is well-established that CREB has both constitutive and cAMP-inducible activities, with distinct domains within the protein contributing to these activities. The cAMP-inducible activity of CREB is activated via phosphorylation of a specific serine residue by PKA, and the phosphorylated form of CREB is then able to bind CBP and recruit it to the promoter to provide further bridging to the preinitiation complex. This elegant system has been described in a recent review (14). Interestingly, there is one CREB response element (CRE) in the proximal human versican promoter. Here I demonstrate preliminary evidence that constitutive and cAMP-mediated activation of CREB is involved in the regulation of versican transcription. Evidence supports the hypothesis that members of the C/EBP family also have intrinsic cAMP-inducible activity and are thus capable of being mediators of cAMP responsiveness. While it is rather well-established that C/EBP can mediate cAMP responsiveness by indirect mechanisms, which include (i) enhanced translocation of C/EBP from the cytosol to the nucleus in response to elevated cAMP levels (15), and (ii) enhanced expression of C/EBP in response to cAMP (16), it is not generally appreciated that isoforms of C/EBP possess domains which  77  contain an intrinsic cAMP-inducible activity independent of direct phosphorylation by PKA. This intrinsic activity may only manifest itself within certain promoter contexts due to the involvement of other so-called 'accessory' factors [reviewed in (17)]. My computational search of the human versican promoter suggested the presence of potential binding sites for C/EBP in this regulatory region. These data motivated me to test the hypothesis that cAMP regulates versican transcription through C/EBP. Naso et al. (18) reported that the human versican gene has one transcription start site. Transient expression assays of 876 by (-632/+240) in a CAT reporter vector in HeLa cells and IMR-90 embryonic lung fibroblasts have shown significant CAT expression. I focused my investigation on this proximal regulatory region for several reasons: firstly, evidence indicates that this region of the human versican 5'-flanking sequence contains promoter elements that are able to drive CAT expression in cells derived from epithelial or mesenchymal tissues; secondly, a computational search of this region suggested the presence of potential binding sites for several transcription factors (discussed in this chapter); and finally, my own investigations also identified certain cis-elements and trans-factors in this proximal promoter region that are involved in versican transcriptional regulation (chapter III-VI of this dissertation) (19, 20). Despite the important role of versican in the pathogenesis of several diseases, the soluble factors, signaling pathways and transcriptional events that regulate the production of this PG are unclear. In ongoing and exciting investigations, I have elucidated cis-acting sequences and transfactors in the versican promoter involved in the modulation of versican transcriptional regulation (chapter III-VI). This chapter of my thesis focuses on identifying and assessing the functional roles of transcriptional regulatory regions of the versican gene through in vitro methodologies. I have begun these studies by determining the regions responsible for repressor and enhancer  78  activity of the versican promoter. I have investigated versican promoter activity in transiently transfected epithelial and mesenchymal origin cells. Further, using stepwise 5' deletion mutants of this promoter I have identified repressor and enhancer regulatory elements. In this study I also demonstrate that the human versican gene is up- and down-regulated by cAMP analogs in HeLa epithelial cells and SMC, respectively. By screening the human versican gene promoter, I have identified two putative sites for transcription factors that mediate cAMP-induced gene transcription, CRE and C/EBP sites. Furthermore, I have shown that the constitutive and cAMPmediated CREB activation enhanced versican promoter activity both in HeLa and SMC. In contrast, my data demonstrate that while C/EBP enhanced versican promoter activity in both HeLa cells and SMC, upon addition of the cAMP analog, C/EBP enhanced and repressed versican promoter activity in HeLa cells and SMC, respectively. These data suggest that versican transcription is differentially regulated by these elements in epithelial cells and SMC. I should highlight that the cloning construction of the human versican promoter luciferase vectors and identification of the positive and negative regulatory regions of the human versican promoter presented in this chapter have been the foundation for meticulous identification of ciselements and trans-factors described in the following chapters. Furthermore, the preliminary results of cAMP-, C/EB13 13- and CREB-mediated regulation of versican transcription have been the focus of my recent and ongoing investigations and by no means are a complete story. I have discussed the experimental strategies of the ongoing investigations in the discussion section of this chapter.  79  111.3. EXPERIMENTAL PROCEDURES 111.3.1.^Alignment and database-assisted human versican promoter analysis —  The versican proximal promoter was searched against the MatInspector (http: itwww. gs Ede 'biodv m at i nspector.html) (21) and ConSite (http:/ rkhead.^ki. sei cg bin/ consite) (22) databases to detect potential transcription factor binding sites (Figure III-1).  Putative and experimentally confirmed regulatory elements are shown and the identity of the transcription factors is indicated. The arrow indicates the human versican gene transcription initiation site. Primer design, polymerase chain reaction (PCR) amplification, and generation of promoter luciferase reporter and deletion constructs — Primer design was  performed using the primer design program Oligo 6.0. Primers were synthesized by Nucleic Acid Protein Service Unit of University of British Columbia. Oligonucleotide primers were dissolved in nuclease free water at a stock concentration of 15 uM. An aliquot of primer was diluted to a working concentration of 150 gM. For primer optimization, an initial PCR reaction was performed using the following conditions: 10x buffer, 1mM MgC12, 0.2 µM primer, 2.5 mM dNTPs, 25 cycles using the annealing temperature suggested by the Oligo 6.0 program. Using a picture of the original product as a guide, conditions were optimized by changing one variable at a time, beginning with MgC1 2 . The reaction was repeated using the same conditions, but increasing the MgC12 concentration in 0.5 mM increments up to 4 mM. After identifying the optimal MgC12 conditions, the optimized primer concentration were identified in the same manner, using increments of 0.1 uM to 0.5 uM. Finally, the optimal annealing temperature and cycle number were determined for each particular primer set using the MgC12 and primer concentrations determined by the previous steps. The correct temperature was usually within  80  three degree °C above or below that suggested by Oligo 6.0. The cycle number was important for staying within the exponential range of PCR amplification. Usually, at 30 cycles and beyond, the reaction was saturated, so cycle numbers below 30 were used. A list of generated primers is shown in Table III-1. The wild type human versican promoter sequence, VCN-632-Luc, was constructed by amplifying the -632 bp/+118 by region upstream of the transcription start site of the versican gene and exon 1, and — 632/+620 by and +98/+118 by were used as primers. The sequences of the oligonucleotides used in this study are shown in Table III-1. Deletions in the 5' flanking regions of the versican promoter were constructed by PCR using a pairwise combination of the sense primers with the antisense primer as depicted in Table III-1. To clone the dVCN-584-Luc, dVCN-527-Luc, dVCN-465-Luc, dVCN-397-Luc, dVCN-335-Luc, dVCN-250-Luc, dVCN-150Luc, dVCN-90-Luc, and dVCN+47-Luc constructs the human versican promoter sequence, VCN-632-Luc was used as template DNA for PCR amplification. Following the generation of the DNA fragments by PCR, the PCR product size was confirmed by gel electrophoresis. The PCR product was excised manually from the gel and purified from the agarose gel using a PCR purification kit (Quiagen). Purified PCR products and constructs generated from these products (see below) were sequenced by the Nucleic Acid Protein Service Unit of University of British Columbia to confirm the sequence of versican promoter regions. Gel-purified PCR product (11.tg) was then co-digested with the corresponding restriction enzymes as mentioned above. The restriction enzyme digested product was then isolated by agarose gel electrophoresis and purified by a DNA purification kit (Promega). The pGL3-Luciferase vector (Promega) was linearized with M/u/ and BglII restriction enzymes and joined with Quick T4 DNA Ligase (New England Biolabs) to the amplified gel purified PCR fragments in the following manner (Figure III-1).  81  Promoter DNAs were combined with digested vector DNA (pGL3-Luc) at a ratio of either 5:1 or 10:1 with T4 DNA ligase (2.5 U/gl reaction) (Promega) in 10x ligase buffer at room temperature for at least 90 minutes. Following ligation, the newly generated DNA was used to transform JM109 competent E.coli bacteria (Promega) through a heat shock protocol. Competent cells (100 pl) were placed on ice for 5 minutes until just thawed from their -80° C frozen state. Between 5 and 20 ng of newly generated recombinant DNA was added to the cells and placed on ice for an additional 10 minutes. The cells were then heat shocked at 42 °C for 45 seconds and allowed to recover on ice for 2 minutes. Eight mL of cold LB media was added to the cell/DNA mixture and cells were allowed to grow in a shaking incubator for 60 to 90 minutes at 37 °C. At the end of this period, an aliquot of the cells (150-500 gl) was plated on an LB agar plate containing 50 gg/mL amplicillin and allowed to grow overnight in a 37 °C incubator. After 18-20 hours, individual colonies were isolated with sterilized applicators and grown overnight in test tubes containing 8 mL LB media and 50gg/mL ampicillin. Following 12-16 hours of growth, DNA was isolated from the mini-prep cultures using plasmid isolation mini-prep kits (Quiagen). Verification of transformation was performed by dual restriction enzyme digestion of the purified DNA product followed by 1% agarose gel electrophoresis. If the DNA product was the expected size, then the mini-prep culture was grown to a 800 mL LB media containing 50 µg/mL ampicillin. The final plasmid DNA was isolated from the large volume culture using a maxi-prep system (Quiagen). DNA again restriction endonuclease digested and analyzed by agarose gel electrophoresis. The concentration of the plasmid DNA were measured by spectrophotometry at 260/280 nm. Sequences of the versican promoter regions contained in the DNA constructs were confirmed as above. Transformed bacteria were stored in 10% glycerol/LB media solution at 20° C until future use.  82  111.3.3.^Tissue culture — HeLa cells and A7r5, smooth muscle cells derived from the thoracic aorta of an embryonic BDIX rat, were obtained from the American Type Culture Collection (ATCC). Both cell lines were maintained in DMEM medium (Invitrogen). Media were supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 100 units/ml penicillin/streptomycin. II1.3.4.^Plasmid transfection of primary cells and cell lines — Transfection of several cell types was attempted using different transfection reagents and conditions. The most frequently used transfection reagents included lipofectamine 2000 (Invitrogen TM Life Technologies) and Fugene 6 (Roch). Both of these reagents work through the formation of liposomes, which aid in the transfer of the construct DNA into the cell without causing toxicity. For most experiments 250,00 cells per well were plated in a 24-well tissue culture plates (90% confluence) with the appropriate media and 10% fetal bovine serum (FBS). Plating of cells was performed at least 24 hours before beginning of the transfection. The DNA/liposome complex was created by mixing transfection reagent and DNA construct at a ratio of 1:2 or 1:3 (0.5-2 pg DNA to 2-6 gl Lipofectamine/Fugene per 150 pl media) to Opti-MEM media (Invitrogen TM Life Technologies, media without serum and antibiotics). At the time of the transfection, cells were rinsed in Opti-MEM media and then the media/liposome/DNA complex was added. At 24 hours post-transfection, 10% serum was introduced to the cultures already in Opti-MEM media. III.3.5.^Dual luciferase reporter and other promoter assays — At 24, 48, or 72 hours post-transfection, cells were harvested after removal of media followed by a single wash with PBS. Cells were then lifted with lx passive lysis buffer (Promega) and mechanical pipetting. Following 20 minutes of cell lifting and lysing, cells and lysate were transferred to microfuge tubes and spun for 10 minutes in a tabletop microcentrifuge to collect lysate. 20g1 of  83  each sample lysate was then combined with 100 ill of Luciferase assay reagent or Luciferase Assay Reagent II (Promega) and read in a luminometer. If transfection experiments involved a co-transfection with a plasmid containing a renilla luciferase coding region, a secondary "Stopand Glo" substrate (100 pl) was added to the original Luciferase Assay Reagent mixture. Again, this mixture was read in a luminometer and recorded. Dual reporter assays were used to normalize transfection efficiencies between cell cultures. Two different co-transfection reporter plasmids were tested including renilla luciferase plasmids (pRLTK vector, Promega) and a (3galactosidase containing plasmid (Promega). 11L3.6.^fl-galactosidase assay — For some experiments, a co-transfection was performed using the pGL-3 generated construct and a 0-galactosidase reporter vector (Promega). For the assay of these experiments, the procedure utilized part of the dual luciferase assay with modifications. 24, 48, and 72 hours after transfection, cells were washed with PBS, lysed and lifted with lx passive lysis buffer (Promega), and transferred to microfuge tubes and spun for 10 minutes in a tabletop microcentrifuge to collect lysate. Twenty ill of each sample lysate was then combined with 100 ptl of Luciferase Assay Reagent or Luciferase Assay Reagent II (Promega) and read in a luminometer to determine light production. Lysate (500 was then placed in a 96 well plate and incubated with 2x O-galactosidase substrate in assay buffer (Promega). The samples were then mixed and incubated at 37° C until a faint yellow color developed. The reaction was stopped with the addition of 150 pl of 1M Sodium Citrate buffer (pH 7.4, Promega) and the product's color was measured in a spectrophotometer at 420 nm. Ratios of firefly luciferase divided by 0-galactosidase activity were used to normalize the reporter assay for different transfection efficiencies between cultures.  84  III.3. 7.^Protein quantification — Protein quantification of the cell lysate was  performed using the BioRad BSA protein assay kit. Different protein concentrations were mixed with the protein assay dye. Bovine serum albumin standards were diluted for comparison. Following incubation for 30 minutes 37° C, the absorbance of samples were measured in a spectrophotometer at 595 nm. Based on a standard curve for BSA, the concentration of the protein lysate was calculated. III.3.8.^Statistical analysis — Significant differences between experimental groups  were determined using the Student's t test. Results for cell culture data are expressed as means ± SEM. Calculated P values were 2-sided, and those < 0.05 considered statistically significant.  111.4. RESULTS III.4.1.^Identification of potential regulatory elements within the human versican promoter — In order to define the promoter region of human versican, transcription  factor computer searches were used to identify possible sequences of interest. DNA sequences up to 632 by upstream and 118 by downstream of the transcription start site were initially analyzed using the computer search programs and database Matlnspector and ConSite (21, 22) to identify sequences in the versican gene, which contain motifs for known transcription factor binding sites. Figure III-1 is a diagram summarizing some of the potential transcription factor binding sites that occur within these regions of the human versican regulatory region. Although the database searches generally used a threshold of an 85% homology score, many of these transcription factor binding sites were present with higher homologies. In the Figure 111-2 the numbers that are preceded by a negative sign indicate the length upstream of that sequence from  85  the transcription start site (+1, indicated by the black arrow). Potential transcription factor binding sites include the TATA box and several others indicated in Figure III-1 and 111-2. 111.4.2.^Versican promoter construct design — The next step in the analysis of the  versican promoter involved creating a variety of promoter constructs and testing their ability to drive the expression of a reporter gene. This involved the insertion of the versican promoter regions in front of a luciferase gene in a reporter plasmid. Then various cell lines were transfected with these luciferase reporter vectors to identify important regulatory regions within the versican promoter. Versican expression constructs were designed using the computer assisted primer design program Oligo 6.0. Using PCR to generate DNA from human genomic DNA with engineered restriction sites, the primary area of focus was the first 632 by upstream of transcription start site to the first 118 bp of the exon one (Figure 111-2). Table III-1 represents a list of all of the versican primers designed to generate expression constructs. Contained within this table are the primer sequences as well as the engineered restriction sites that were designed within the PCR primers. These engineered restriction sites served as a mechanism for restriction enzyme cleavage and ligation into the vectors. The specific enzyme sites that were incorporated into the primer design were the same sequences present within the multiple cloning region of the pGL3-basic vector (Figure III-3A). Figure 111-3 and Table III-1 contain the primer titles with expected product size. Each DNA segment was cut from its respective plasmid with the restriction enzyme M/u/ and Bglft and separated by agarose gel electrophoresis (Figure III-3B).  For the generation of constructs, the reporter vector pGL3-basic (Promega) was used (Figure III-3A). This reporter vector is free of any enhancer/promoter elements and served as an ideal host for the PCR generated versican DNA regions in order to identify areas which regulate  86  transcriptional activity. A model of the pGL3-basic vector and the construct assembly can be seen in Figure III-3A. Following the generation of the versican promoter containing constructs, construct size and quality were confirmed by restriction enzyme digestion followed by agarose gel electrophoresis (Figure III-3B). The sequence of the PCR generated construct inserts were confirmed by DNA sequencing performed by UBC Nucleic Acid Protein Service Unit. DNA sequencing results compared to GeneBank sequences confirmed the authenticity of the sequence. III.4.3.^Characterization of repressor and enhancer elements in human versican promoter — The DNA sequences corresponding to the upstream regulatory region of the human  versican gene were cloned in a pGL3-basic luciferase reporter plasmid (as described above) which allowed the detection of minimal transcription elements when transfected in different mammalian cell lines. Furthermore, additional constructs were designed to encompass even smaller regions of interest within the promoter. This dissection of the promoter allowed examining relatively small regions in the versican promoter (-50 bp) to determine whether or not enhancing or suppressing elements were present. The transfection of the cell lines with the promoter constructs identified areas that enhanced or suppressed transcriptional activity (Figure 111-4). The first versican promoter construct used for transfection experiments consisted a 632 segment of human versican promoter just upstream of the transcription start site with the adjacent 118 by of the first exon (-632 to +118). This segment of the promoter was identified as the probable region of interest based on transcription factor analysis performed by the computer programs. The 750 by construct (-632 to +118) was transfected into HeLa cells and SMC utilizing an empty pGL3-basic vector as a negative control. With the 632 by promoter segment,  87  enhanced luciferase activity was seen as compared to the pGL3-basic vector alone in all cell types tested (Figure 111-4). Based on these preliminary findings, further constructs were used in transfecting the above cell lines. Figure III-4A indicates the size of the constructed promoter segments that were confirmed in Figure III-3B and recorded in Table III-1 and their location within the upstream promoter region. The constructs used in the promoter studies included the original 632 by wild-type segment, and 9 additional segments with sizes smaller than that of the original construct. Figure III-4B shows the specific luciferase activity produced by each construct in the HeLa and SMC. The results of 5' serial deletion constructs revealed a dramatic decrease in luciferase activity between the VCN-632-Luc and dVCN-584-Luc constructs in HeLa and SMC. It suggests the presence of enhancer elements in region between -632 to -584 bp. Progressive 5' deletions of regions towards transcription start site showed enhanced promoter activity with highest levels in the plasmid dVCN-397. These results support the presence of repressor elements located in the versican promoter possibly between -584 to -397 bp. Deletion of —50 by between -397 to -335 caused significant reduction in promoter activity. This finding again suggests the presence of enhancer elements in region between -397 to -335. It is possible that with the loss of the first potential repressor region through deletion of the -584 to -397 regions, the effect of the enhancer, -397 to -335 bp, dominated transcriptional activity. The markedly decreased activity observed with deletions beyond -335 to -150 by further corroborates the hypothesized location of a strong enhancer region in -397 to -335 bp. The deletion from -335 to -250 by did not significantly change the promoter activity; however deletion between -250 and -150 by significantly enhanced promoter activity, suggesting the presence of repressor elements between -335 to -150 bp. Further deletion between -150 to -90 by caused significant loss of  88  promoter activity. This result also suggests the presence of enhancer elements in this region. Further deletion between -90 to +47 did not change the promoter activity significantly. In brief, the results of luciferase assay following transfection of two different cell lines, HeLa cells and SMC, with versican promoter serial deletion constructs identified three potential enhancer (-632 to -584, -397 to -335, and -150 to -90) and two repressor regions (-584 to -397 and -335 to -150).  ^111.4.4.^Differential effect of cAMP inducers, Forskolin and (Bu)2cAMP, on the versican promoter in HeLa and SMC — HeLa (Figure III-5A and III-5B) and SMC (Figure III5C and III-5D) were transfected with an equal amount (0.3 gg) of VCN-632-Luc construct. After transfection, the cells were treated with two strong inducers of cAMP, Forskolin and (Bu)2cAMP, for 24 h, and luciferase activity was determined simultaneously for all samples. The results, summarized in Figure 111-5, are presented as the fold increase over the basal activity of the respective promoter and demonstrate about two fold activation of the versican promoter in response to Forskolin in HeLa cells; however (Bu)2cAMP treatment did not significantly alter the promoter activity. In contrast, both cAMP inducers, Forskolin and (Bu)2cAMP, significantly inhibited promoter activity in RASMC. These results suggest that cAMP-mediated versican transcription is cell type dependent.  ^111.4.5.^Identification of cAMP-response elements in human versican promoter — Members of the C/EBP family and CREB have intrinsic cAMP-inducible activity and are thus capable of being mediators of cAMP responsiveness (17). The proximal versican promoter was searched for transcription factor binding sites that would confer cAMP transactivation. A search for consensus C/EBP and CRE in the versican promoter revealed the presence of two potential C/EBP binding sites, TCGTACTC and TCTTTGCTGATTT sequences at positions -438 to -445  89  and -477 to -489 bp, respectively. Furthermore, a CRE, CGGCTCTGAC, at position -32 to -42 by was identified (Figure III-1 and Figure III-2A). To determine the functional significance of consensus C/EBP and CRE sequences and the ability of the 5'-flanking sequence to direct cAMP-mediated repression of versican gene expression in cAMP-cultured SMC, I transfected serial deletions of the versican promoter constructs into the SMC and cultured the cells in cAMP-containing medium, 1 mM (Bu) 2 cAMP, for 24 h as indicated in the Figure 111-6 and described in the figure legend. As shown in Figure 111-6 transfection of cells with the dVCN-584, dVCN-465, and dVCN-250 plasmids followed by incubation with cAMP, resulted in inhibition of luciferase activity of 2.6-, 5.2, and 1.8-fold compared to conditions without cAMP inducer. However, cAMP treatment of cells transfected with dVCN-90 compared to non-treated cells did not significantly alter luciferase activity. The significant inhibition of luciferase activity for dVCN-584 and dVCN-465 suggest that cAMP response region mediate inhibition of versican promoter activity might be located between -584 to -250 bp. This region of the human versican promoter includes two potential binding sites for C/EBP. Furthermore, our result shows that cAMP is not capable of inhibiting luciferase activity due to the dVCN-90 region of the versican promoter. This area also includes a CRE. III.4.6.^Involvement of C/EBP family members in the constitutive and cAMPmediated expression of human versican promoter activity — To examine the respective roles of  C/EBPa and C/EBPI3, plasmids expressing either C/EBPa or C/EB1 3 13 were co-transfected with the wild-type pVCN-632-Luc vector into HeLa cells. As shown in Figure 111-7, the presence of overexpressed C/EBPa and C/EBPf3 significantly increased the basal promoter activity in HeLa cells. To further study the role of C/EBP in the basal and cAMP mediated activity of the versican promoter, HeLa and SMC cells were co-transfected with the VCN-632- Luc vector, C/EB1 3 13, and  90  a C/EBP dominant negative expression vector (C/Edn) in the presence or absence of cAMP inducers Forskolin (HeLa cells) and (Bu)2cAMP (SMC) as indicated in the Figure 111-8. C/Edn has been described as being able to prevent the binding of both C/EBPa and C/EB1 3 13 to their sites (23). Our results show that C/EB13 13 expression vector has constitutive enhancer activity on versican transcription in both cell lines. In contrast, C/Edn expression vector reduced C/EB1 3 13induced versican promoter activity in the absence of cAMP inducers in both cell lines. Interestingly, although cAMP inducers treatment of the cells transfected with C/EB1 3 13 showed the synergism between cAMP and C/EBPf3 in HeLa cells, cAMP converted the enhancement of versican promoter activity by C/EB13 13 to repression in SMC. Taken together, these results indicate that both C/EBPa and C/EBPI3 isoforms are involved in the transcriptional regulation of the versican gene. Furthermore, binding of C/EBP[3 to the cis-elements of the versican promoter is likely involved in constitutive and cAMPmediated enhancement of versican transcription in epithelial HeLa cells. In addition, constitutive and cAMP-mediated C/EBPP-induced enhancement and repression of versican transcription, respectively, also require binding of C/EBI13 to cis-elements in the 5' flanking region of the versican promoter in RASMC. Finally, cAMP converted the constitutive C/EBP-mediated enhancesome transcriptional complex to repressor in the human versican promoter in RASMC. 111.4.7.^Activation of versican promoter by exogenous CREB — Sequence  analysis using TFSEARCH software has identified CREB as the potential candidate binding to the core sequence of the human versican promoter (Figure III-1). Identification of potential CREB binding site to the critical core sequence responsible for the versican promoter activation led me to question whether CREB is sufficient to activate the versican promoter and whether cAMP modulates CREB function on versican gene regulation. To investigate the role of  91  constitutive CREB as well as function of cAMP on CREB-mediated versican promoter activity, I co-transfected the VCN-632-Luc construct and CREB expression vector into HeLa cells or SMC in presence and absence of (Bu)2cAMP (Figure 111-9 and III-9B). Co-transfection of CREB elevated versican promoter activity by nearly two-fold in both HeLa cells and SMC (Figure III9). These results suggest that constitutive expression of CREB protein induce versican promoter activity. To examine the role of cAMP on CREB-mediated regulation of versican promoter, HeLa cells or SMC co-transfected with VCN-632-Luc construct and CREB expression plasmid followed by treatment with (Bu)2cAMP. My result showed that in both cell lines tested cAMP slightly enhanced the function of CREB on versican promoter activity. In brief, these results indicate that the constitutive CREB expression and cAMP-mediated CREB activation enhanced versican promoter activity in both HeLa epithelial cells and SMC.  111.5. DISCUSSION  Upstream or downstream of the promoters are the binding sites for sequence-specific transcription factors that create the pattern of regulated gene expression. Transcription rates can be affected by the binding of a single transcription factor or by a complex series of transcription factor interactions. The combination of enhancers and silencers within a gene's regulatory region affect the expression pattern for the overall gene. As part of my initial work done on the versican gene, deletion analysis was used to study the promoter activity within the first 632 by upstream of the transcription start site to the first 118 by of the exon one. For the deletion analysis, constructs of different lengths were generated through PCR, placed into a promoterless luciferase reporter gene vector, and transfected into HeLa cells and SMC. Luciferase production was used to measure transcription levels. This work was successful in verifying enhancer and repressor  92  regulatory elements in the -632 by to +118 by regions. Our data also indicate that members of the C/EBP family differentially control constitutive and cAMP-dependent transcription of versican in HeLa cells and SMC. Further, a bioinformatic analysis of the 750-nt sequence (-632 by - +118 bp) of the human versican regulatory region was performed by running Matlnspector Professional against the TRANSFAC\ Database 6.0. This analysis demonstrated several important potential binding sites in the proximal promoter and enhancer regions that have been, in part, the basis for investigation of signaling pathways, cis-elements and trans-factors presented in this dissertation. I have also discussed the evidence suggesting the potential importance of some of these transcription factors in the regulation of versican transcription; however they have not been the focus of my investigations in this dissertation.  Functional mapping of the human versican gene promoter region: identification of repressor and enhancer regulatory regions — Understanding the regulation of the basal promoter of genes is prerequisite for understanding normal and pathologic states of the tissue. By understanding what initially turns a gene on and off, it becomes possible to comprehend differences in expression patterns from embryo to adult. Versican expression can be altered by various cytokine-signaling events, autoimmune disease, cancer, cardiovascular diseases, and during development (5, 6). As expression was found to be regulated at the RNA level, I further examined the transcriptional regulation of versican gene. My studies on the versican promoter gene shows that a 632 by upstream of the transcription start site and 118 by of exon 1 were sufficient to drive basal versican expression in a non-tissue specific fashion. As part of this dissertation, the focus was on 632 and 118 by up- and down-stream of the transcription start site, respectively, however the remote areas of the versican promoter were not explored.  93  In order to identify possible transcription factors interacting with this promoter region, the transcription factor search programs TFSEARCH was used to search for the consensus binding sequences contained within the versican promoter regions 632 by upstream and 118 by downstream of the transcription start site. Figure III-1 is a diagram generated to demonstrate the location of the potential consensus binding sites of several transcription factors within the region mapped. In this chapter, I identified three potential enhancer (-632 to -584, -397 to -335, and 150 to -90) and two repressor regions (-584 to -397 and -335 to -150). My preliminary data also indicate that members of the C/EBP family differentially control constitutive and cAMPdependent transcription of versican in HeLa cells and RASMC (this chapter). Besides the transcription factors described in chapter IV and VI, in the last section of discussion of this chapter I discuss the identified transcription factors by computation search likely important for versican regulation and function in health and disease. C/EBPs mediate constitutive and cAMP-mediated regulation of versican gene transcription: differential role in HeLa epithelial cells and SMC — A 632-bp region upstream of  the transcription start site was identified as necessary for proximal promoter activity (Figure III4). Furthermore, multiple putative transcription factor binding sites within this region, including C/EBP and CRE, were found to be potentially important in optimal basal promoter activity (Figure III-1 and 111-2). Here, I show for the first time that factors which increase the cAMP level in cells [such as Forskolin and (Bu)2cAMP] cause an increase and decrease in human versican promoter activity in epithelial HeLa and SMC, respectively. These results suggest that cAMP-mediated versican transcription is cell type dependent. The results also suggest that region of -584 to -250 by from human versican promoter is likely responsible for cAMPmediated repression of human versican promoter activity in SMC. This region of the human  94  versican promoter includes two potential binding sites for C/EBP. Furthermore, my results show that cAMP is not capable of inhibiting luciferase activity from dVCN-90 region of the human versican promoter. This area also includes a CRE. It appears that the C/EBP binding sites are involved in cAMP-dependent repression of human versican promoter activity in SMC, but not the potential CRE. These two transcription factors are known to mediate cAMP-mediated regulation of target genes (17). Interestingly, co-transfection of C/EBP0 with VCN-632-Luc in HeLa and SMC was able to significantly enhance versican promoter function (Figure 111-7). Further, cAMP agonist enhanced C/EBP-mediated induction of versican promoter activity in HeLa cells (Figure 111-8), but cAMP converted the enhancement of versican promoter activity by C/EBP/3 to repression in SMC (Figure 111-8). These results suggest that C/EBP13 is a key factor in the constitutive and cAMP-dependent regulation of human versican transcription; however, the mode of action of this transcription factor on versican transcription seems to be cell type specific. An increased expression of the C/EB1 3 13 encoding gene after exposure to cAMP agonist has already been reported (24). But the cAMP responsiveness mediated by the C/EBP proteins could also occur via an increase in their phosphorylation by cAMP-dependent protein kinases. Indeed, Tae et al. (25) showed that in preadipocytes, a treatment which increases cAMP levels increases the phosphorylation and activity of C/EBP[3. This phosphorylation of C/EB1 3 13 could then stimulate its translocation into the nucleus (15) and activate the transcription. cAMP induced activity of C/EBP has been described for other genes, such as the phosphoenolpyruvate carboxykinase gene (26), where both C/EBPa and p can participate in the cAMP-inducible function. It is known that C/EBP proteins can interact with various factors to form heterodimers, in particular with CREB (27). An increased level of nuclear C/EBPP and an increased binding of this factor to its DNA site were observed under Forskolin treatment (28), but interaction(s) with  95  other protein(s) are not excluded. Therefore, mediators which are capable of inducing an increase in cAMP levels are potential activators of the versican transcription partly through an increased expression and/or activation of the C/EBP transcription factors. While there is compelling evidence that C/EBP contains both constitutive and cAMPinducible domains, they differ from CREB in that for the most part, the cAMP-inducible domains of C/EBP lack a PKA phosphorylation site [reviewed in (29)], with the exception being that C/EB13 13 can be phosphorylated in its bZIP domain which can in turn affect its DNA binding activity (29). Thus, the mechanism whereby these domains in C/EBP are 'activated' by cAMP is unclear. It is recently hypothesized that there may be a coactivator which is able to interact with the cAMP-inducible domains of C/EBP only after being phosphorylated itself by PKA. This scenario is suggestive of a model similar to that established for the activation mechanism of CREB, whereby CREB is phosphorylated by PKA which then allows recruitment of the coactivator CBP, however, the phosphorylation of the coactivator rather than the DNA-binding protein would regulate the interaction. Indeed, there is recent evidence that phosphorylation of a coactivator can selectively modulate which transcription factors it interacts with (30). Members of the C/EBP family of transcription factors have been shown to play important roles in regulating genes that control processes such as adipocyte differentiation and glucose homeostasis. Interestingly, it has recently been reported that versican and its partner hyaluronan play a role in the differentiation of 3T3-L1 cells into preadipocytes and mature adipocytes (31). Given the importance of C/EBP transcription factors in adipocyte differentiation and the regulatory role of this transcription factor on versican transcription, it opens the field for a more thorough study of their role in differentiation of several tissues including adipocyte and vascular SMC in normal and disease states.  96  CREB is involved in regulation of versican gene transcription in SMC — Numerous  intracellular signaling pathways are involved in transmitting information initiated by membrane receptor-mediated actions to the nucleus, where they interact with CREB to trigger processes that culminate in gene transcription. The effects of signaling pathways involving adenylate cyclase and cAMP (32), Ca 2+ (32) and mitogen activated protein kinase (MAPK) (33) on CREB and CREB-regulated gene transcription have been the focus of much research (34). The key steps involved in CREB-mediated gene transcription include dimerization, binding at response elements in DNA, and phosphorylation (14). CREB is one of several transcription factors that bind as dimers to the CRE, a specialized stretch of DNA that contains the consensus nucleotide sequence TGACGTCA. CRE sites are found within the regulatory (promoter or enhancer) region of numerous genes; if a promoter contains CRE, then it could be subject to regulation by CREB, depending on several tissue-specific factors including the conformation of the nearby chromatin. Traditionally, it has been thought that CREB dimers are bound to their CRE sites under basal conditions, but are inactive. According to this view, events at the cell membrane, which stimulate intracellular signaling cascades, cause the phosphorylation of both members of the CREB dimer and trigger its transcriptional activity (35). However, recent work raises the question of whether CREB is constitutively bound to its CRE sites in all cases or, instead, whether CREB phosphorylation sometimes initiates this interaction (36). These factors are likely to contribute to tissue-specific differences in CREB function. There is a CRE binding site at the proximal promoter region of the human versican gene (Figure III-1). I co-transfected the VCN-632-Luc reporter construct and CREB expression plasmid in HeLa cells and SMC in the presence or absence of cAMP inducer, (Bu)2cAMP (Figure 111-9). Our results demonstrated that CREB enhanced versican promoter activity through  97  cAMP-dependent and independent mechanisms in both cell line. In contrast to the finding that cAMP converted the enhancer function of C/EBPf to a repressor in SMC, cAMP did not inhibit CREB-mediated induction of versican transcription in these cell lines. Altogether, these results show that cAMP differently modulate CREB- and C/EBP-mediated regulation of the versican gene in SMC. CREB-mediated gene transcription can be directly altered experimentally by several molecular tools which have been the focuses of my ongoing investigations. As one example, engineered mutations of CREB have enabled the development of dominant-negative forms of the protein that act as CREB antagonists. CREBM1 — also known as mCREB — contains a point mutation at serine 133 (serine is replaced by alanine) that prevents both phosphorylation at this site and transactivation of genes by CREB (37). This mutation does not affect the ability of mCREB to dimerize with endogenous (wild-type) CREB or another mCREB molecule, nor does it affect binding at CRE sites. As such, transfer of mCREB into a cell can reduce or block CREB mediated gene transcription either by occluding CRE sites as mCREB homodimers or by heterodimerizing with wild-type CREB; such heterodimers can bind to available CRE sites, but cannot recruit CBP because only one subunit of the dimer can be phosphorylated at serine 133. KCREB is a distinct dominant-negative mutant: it contains a point mutation in the DNA-binding domain that prevents it from binding to CREs. It dimerizes with endogenous CREB, and the resulting heterodimers are unable to bind to DNA-binding sites (38). Conversely, CREB-VP16 is a constitutively active form of CREB; it can homodimerize, or heterodimerize with wild-type CREB, and activate transcription — via its VP 16 viral transcription-activation domain — even in the absence of serine 133 phosphorylation. Use of these various mutants and genetic deletion of CREB have increased our understanding of the molecular mechanisms of gene transcription, and have enabled us to gain unique insights into how CREB might ultimately affect complex  98  behavior in diverse species. In a series of ongoing experiments, alteration of the function of CREB, either through cAMP-mediated activation, overexpression of a dominant negative CREB, or gene-specific inhibition, will be used to identify direct functional regulation of versican transcription by this transcription factor. Specifically, I am co-transfecting the VCN-632-Luc with several combinations of CREB, mCREB, and KCREB in presence and absence of cAMP agonists to further identify the detailed mechanisms of CREB-mediated regulation of the versican gene transcription. Putative binding sites and potential involvement of transcription factors in the regulation of versican transcription — As I discussed in the method and result sections of this  chapter, I analyzed the -634/+118 by region of the human versican promoter using the computer search programs and database Matlnspector and ConSite (21, 22) to identify sequences in the versican gene which contain motifs for known transcription factor binding sites (Figures III-1 and 111-2). This search revealed the presence of two potential LEF/TCF binding sites at positions -546 by and -492 bp, respectively. Furthermore, a computational search suggested that this sequence contained two potential androgen response elements (ARE) at positions -392 and +83. These computational searches, in part, have been one of my motivations for further investigating these transcription factor complexes and upstream signal transduction pathways involved in the regulation of the versican gene (chapter IV and VI). Here, I also discuss the possible contribution of additional transcription factors with potential binding sites that were again suggested through a computer-assisted search of the human versican promoter region. I provide evidence from the literature to support their roles in versican transcription, but I did not perform any experiment to investigate their involvement in transcriptional regulation of the versican gene.  99  GATA — There are potential binding sites for GATA in the identified enhancer and repressor regions of the human versican promoter (Figure III-1). Members of GATA family of transcription factors are implicated in synergistic cooperation with other factors in the regulation of target genes. For example, synergistic cooperation between C/EBP13 and GATA-4 were found to influence the regulation of steroidogenic acute regulatory protein transcription (39). Further, functional interaction of CP2 with GATA-1 induced activation of erythroid promoters (40). There are potential binding sites for all three transcription factors, GATA, CP2, and C/EBP, in the regulatory region of the human versican gene. It is possible that these transcription factors synergistically or antagonistically enhance or repress the versican promoter activity in a cell type specific manner. Heat shock factors —There is also a potential binding site for heat shock factors (HSF) in 634 to -584 region of the versican promoter (potential enhancer region). Heat shock factors are the main transcriptional regulators of the stress-induced expression of heat shock protein genes. Four HSF (HSF1-4) exist in vertebrates (41). HSF1 is activated within minutes of an increase in temperature. The heat shock response enables the cell to cope with the deleterious effects of protein-damaging stresses, e.g. heat, heavy metals, and viral and bacterial infections. A characteristic of the heat shock response is down-regulation of gene transcription and protein synthesis in general, whereas an increase in the transcription of a specific subset of genes called the heat shock genes is induced (42). During heat shock, the heat shock proteins function as molecular chaperones that bind to and aid the folding of damaged proteins, thereby preventing protein aggregation under stressful conditions (43). Given the transcriptional regulation of versican in response to several different types of stress stimuli including mechanical associated stress during development (44), shear stress-mediated intimal growth (45), tensile stress (46),  100  mechanical stress (47), and oxidant injury (48), it will be of interest to elucidate whether HSF are involved in versican gene transcription. Interferon regulatory factors  —  Interferon (IFN)-a/(3 genes are induced through the  coordinate activation of transcriptional regulatory proteins including the interferon regulatory factors (IRFs) and NF-KB. One of the members of IRF, IRF-3, is expressed constitutively in a variety of tissues and maintained in a latent conformation in the cytoplasm. Ligands through Toll-like receptors (TLR) mediate phosphorylation of IRF-3, leading to dimerization, cytoplasmto-nuclear translocation, association with CBP/p300 coactivators, and stimulation of DNA binding and transcriptional activities through binding to IFN-stimulated responsive elements (4951). In addition to its involvement in the transcriptional induction of immediate-early IFN genes, IRF-3 also directly controls the expression of the CC chemokine RANTES in response to paramyxovirus infection (52) and induction of proinflammatory genes such as IL-12 (53). Tolllike receptors have been established to play an essential role in the activation of innate immunity by recognizing specific patterns of microbial components (54). Interestingly, it has recently been demonstrated that versican is upregulated in the ECM of vascular SMC during a TLR3-induced inflammatory response by the TLR3 ligand poly I:C (55). Endogenous or exogenous ligands can stimulate TLR-bearing cells in the vascular wall, such as macrophages, dendritic cells, EC, and SMC. Several lines of evidence, including the presence of a potential binding site for IRF-3 as one of the transcription factors downstream of TLR signaling in the human versican promoter (Figure III-1), the recent evidence that TLR3 mediates regulation of versican expression (55), and an abundant expression of versican in inflammatory disorders, suggest the possible involvement IRF-3 in regulation of versican transcription. Further experiments are required to support this hypothesis.  101  Egr-1, HIF- 1 a, and C/EBPs: the hypoxia related transcription factors — There are  potential binding sites for Egj -1, hypoxia inducible factor (HIF)-1a, and C/EBP in the human -  versican promoter region. Egr-1 is an immediate-early gene that encodes a nuclear phosphoprotein containing three zinc finger elements; these bind target GC-rich elements in the promoter of many different genes, thereby regulating transcription (56). Versican is one such gene that has an Egr-1 site in its promoter region. Other genes include the hypoxia-responsive genes of tissue factors, various growth factors, cytokines/chemokines, and adhesion receptors. Even VEGF, a classical HIF-1 a responsive gene, has hypoxia-responsive Eg-1 elements in its promoter region (57). Egr-1 is rapidly activated and expressed in hypoxia, both in vitro and in vivo, and is responsible for the expression of a diverse array of effector genes in  hypoxic/ischemic conditions (58, 59). Hypoxia-inducible factor 1 is another DNA binding protein comprised of a heterodimer between two basic helix-loop-helix transcription factors: HIF-la and the aryl hydrocarbon receptor nuclear translocator (ARNT) (60). When cells are exposed to oxygen concentrations considered to be normal (normoxia), newly synthesized HIF-1 a undergoes rapid degradation through a cascade of events and subsequently by proteasome degradation system (61). Under prevailing hypoxic conditions, HIF-1 a is stabilized, reflected by increased half-life, accumulates, and migrates to the nucleus, where the HIF-la-ARNT complex interacts with hypoxiaresponsive elements in transcriptional regulatory regions of multiple target genes such as erythropoietin (62), VEGF (63), inducible nitric-oxide synthase (NOS) (64), plasminogene activator inhibitor-1 (PAI-1) (65) and possibly versican. CCAAT /enhancer binding proteins comprise a family of transcription factors that each contains a highly conserved, basic C-terminal leucine zipper that mediates dimerization and  102  DNA binding. There are at least six known members of the C/EBP family that have been isolated and characterized so far, including C/EBP-a, 43, -y, -8, -E, and (66). I have also presented in this chapter that at least C/EBP-a and -13 induce versican promoter activity several fold in HeLa cells and SMC. There are several pieces of evidence that suggest the possible roles of Egr-1, HIF-la, and C/EBPs in regulation of versican transcription. Many of the hypoxic-induced genes, such as those encoding for glycolytic enzymes, growth factors, and vasoactive peptides, share the common feature of blunting tissue damage in situations of oxygen scarcity. Recent studies identified upregulation of versican in the acute (67) and chronic (68) myocardial ischemic injury, potentially important in early and late stages of response to ischemic injury. Further, it has been shown that versican protects cells from oxidative stress-induced apoptosis (48). It has also been identified that fetal lung fibroblasts, in response to elevated oxygen concentrations, downregulate versican synthesis (69). On the other hand, Ingemansson et al. (70) showed that hypoxia induce versican mRNA levels 20-fold compared to normoxic condition in human primary macrophages. Given the existence of the potential binding sites for the above transcription factors involved in hypoxic-induced gene expression in the human versican promoter and the role of versican in response to ischemic injuries; it is possible that these transcription factors are involved in versican transcription in response to ischemic injury and oxygen tension. MAZ and Sp 1 transcription factors — The MYC-associated zinc finger protein (MAZ) was identified as a transcription factor that binds to the promoter region of the c-myc gene (71). It can regulate the expression of numerous genes, such as c-myc (71), insulin I and II (72), CD4 (73), and NOS (74).  103  Spl was originally characterized as a transcription factor that recognized GC-rich sequences in the early promoter of Simian Virus 40 (75). Spl is considered to be a constitutively expressed transcription factor and has been implicated in the regulation of a wide variety of housekeeping genes, genes that are expressed in a tissue-specific manner, and genes involved in the regulation of growth (76). The sequences of the binding sites for Spl and MAZ, GGGCGG and GGGAGGG, respectively, are very similar and they are often found within the same gene [reviewed in (77)]. In fact, Spl and MAZ bind to the same cis-elements in the promoters of the genes for the receptor for serotonin lA (HTlAr), endothelial nitric-oxide synthase (eNOS), the receptor for parathyroid hormone (PTHr), MAZ and the major late promoter of adenovirus (AdMLP). Spl and MAZ activated the expression of the genes for HT1Ar and PTHr, as well as AdMLP. Both Spl and MAZ inhibited the expression of the gene for MAZ, while expression of the gene for eNOS was enhanced by Spl and repressed by MAZ. These observations suggest that both Spl and MAZ might have dual functions in the regulation of gene expression. There are potential binding sites for MAZ and SP1 in the human versican promoter. It will be of interest to identify whether these two transcription factors are involved in induction or repression of versican transcription. Activated protein-2 — Functional activated protein-2 (AP-2)-binding sites have been identified in the enhancer regions of viral genes such as simian virus 40 (SV40) and human Tcell leukemia virus type I, as well as several other cellular genes [reviewed in (78)]. AP-2 plays a pivotal role in regulating the expression of several genes whose products are involved in tumor growth and metastasis of melanoma [reviewed in (78)]. Interestingly enough, some genes regulated by AP-2 are also regulated by the CREB:ATF-1 transcription factors family (79). It is  104  suggested that the outcome of the metastatic phenotype in melanoma may well be dependent on the delicate balance between the expression of AP-2 and the CREB:ATF-1 transcription factors. Loss of AP-2 and over expression of CREB:ATF-1 in metastatic melanoma cells may work in concert to regulate several genes contributing to the malignant phenotype. Versican is one of the major PG highly expressed in malignant melanoma, and they have a role in the regulation of cell adhesion, migration, and differentiation (80). It has been shown that versican expression was negative in benign melanocytic nevi, weakly to strongly positive in dysplastic nevi, and intensely positive in primary malignant melanomas and metastatic melanomas (80). These results indicate that versican is involved in the progression of melanomas and may be a reliable marker for clinical diagnosis. There are several potential binding sites for AP-2 and CREB/ATF in the human versican promoter region. Given the potential roles of AP-2, CREB/ATF transcription factors, and versican in development and progression of melanoma, it is possible that AP-2 alone or in cooperation with CREB/ATF play a role in normal or aberrant regulation of versican transcription.  111.6. CONCLUSIONS I have characterized the versican promoter upstream from the transcription start site (+1) to — 632 by and down-stream to +118 by using transient transfection of luciferase reporter constructs in HeLa cells and SMC. In these regions, I have identified three potential enhancer regions (-632 to -584, -397 to -335, and -150 to -90), which are present in the two cell lines tested and do not appear to be cell line specific. These regions contain the transcriptional start site as well as transcription factor binding sites, indicating the presence of a basal expression promoter complex for the versican gene. I have also identified possible regions of suppression  105  within the versican promoter (-584 to -397 and -335 to -150) that do not appear to be cell line specific. These regions also contain potential binding sites for previously suppressing transcription factors and may therefore contain novel consensus binding sites that result in transcriptional repression. In ongoing investigations of our laboratory, we have been using band shift and supershift analysis involving point mutation oligonucleotides to determine the exact mechanisms of the enhancers and suppressor implicated in versican gene control. I have also demonstrated that C/EBP is required not only for the constitutive versican transcriptional activity but also in the cAMP-induced versican promoter activity in HeLa and SMC where it enhances and decreases versican transcription in epithelial and SMC, respectively. Furthermore, I demonstrated that CREB enhanced versican promoter activity through cAMPdependent and independent mechanisms. In an exciting ongoing investigation, I will pursue experiments to identify the requirement of putative CRE and C/EBP sites for versican transcription and to examine whether CREB and C/EBP are able to bind to versican regulatory regions in vitro and in vivo. Further, I will examine the role of CREB and C/EBP alteration, either through activation or depletion, on endogenous versican gene transcription. Specifically, I co-transfected the dominant negative and constitutively active CREB constructs with a versicanLuc reporter vector to show versican promoter activity is directly dependent on these transcription factors. Furthermore, I will investigate the influence of cAMP agonists as well as adenoviral-mediated expression of C/EB13 13 and CREB proteins on versican mRNA levels. I will also use band shift and supershift assays to investigate specific binding of these transcription factors to oligonucleotides corresponding to a potential C/EBP binding site and CRE in the human versican promoter. In addition to binding assays, I will directly assess the dependence of  106  versican promoter activity on C/EBP binding sites and CRE by using site-directed mutagenesis of the C/EBP site and CRE.  107  0)  0 El  H  C)  H C.)  4  E.:4  H H H H H  CD  H H H C.) H C.) H  8  8 E-I  8  C)  C.7  C.) C.)  C.)  H H  C.) C.) C.)  H H  0 < 0 1— E., 0  LI Z  0 N u_  U EH  H  C) H C.) C.) H  H H  NI  C.) 03 4 E.1 C.)  H  CD  CD  CD C.)  H < Ci 2 H CD H CD H H H H  H  0- E4  ES 8 -  L3 EI E-1 0 E-1  E-1  C.)  CD  w  CD  H  CD CD  H  CD CD  H  CD  H CD C.)  H C.) C.)  H H H H  E-1  CD CD  Fr4 CL. E_,  0  CD  wV  C.)  C.)  H  CD  0  H  ce 4 X U H  CD C.)  csi  C o Ei)  H^C.) C.) H  C.)^CD  CD  CD Li]  H  H  C.)  H  0 0< 1— CNI CO^C0^CO  LL  CO  n  H^u u^4 co H^u_ C)^I— E.., <^— 0  CD^03 E4 Lu CD^CC 0 H 0 0 C) CD^E, <^U  <  C.)^H 0^0 H^CJ H^H H^C.) C.)^H CD^CD H 0^C<D C)^EH  0^c_i C.)^CD C.)^C)  H^C.) H^EH  8^  E^H C.) H cl F, 0 0^C.) C.)^EH 0^H 0 i  . 0^C.) EL C)^0 Z CD^H H^CJ C)^C.) CD^C.) g^H 0^C.) 0^H 0_^EH  r, ....,^0 Q-. ■ esi^0  EH  CD <^0 C)^0  0^ cv 0^C) a ,,, U < C.) "^ d^U  0 0 .-J. c..)^0  4 < H H C.) C.)^CD CJ^F, H^CCD CD^H, CD^C.) C.)^CJ CD^0 CD^4  (NI  CO  CD^CD CD^EH CD^0 E-,^0 CD^CD CD^H CD CAD^ CD^< CD CD C.)^< C.)^E-,  CD  HCD CJ0  CD U CD^< CD^0 CD c\I^CD EH C1-^C.) < 0^CD 0^H  Cl^0 CD^H 0^H N^H g CD^H Z CD^C.) 0^CD  108  0  H  X  0-  7  CD H CD C.) CD  C.)  H  C.) C.)  CJ  CD C.)  CD 0 C.)  C.)  E., El El  4  <  H H  CD CD CD  H  5  4  < <0 0 CD  81  CO  c0  H H CCD C.) CD  H  CD C.)  CCD  H  H H  CD H CD  H H  CD C.)  CO CO  w  w  Figure III-1. Sequence map, potential and known transcription factor binding sites within the -632 to +118 by region of the human versican gene. The human versican promoter transcription start site at by +1 is marked by an arrow. Significant transcription factor binding sites are underlined and titled. TATA box site is indicated by red box. A search for potential consensus transcription factor binding sites in the versican promoter using Matlnspector program revealed the presence of several binding sites in proximal promoter region. Confirmed transcription factors are labeled with an asterisk (*).The abbreviations used are: IRF3, Interferon regulatory factor 3; NF1, Nuclear factor 1; LEF/TCF, lymphoid enhancer factor/T cell factor; XRE, Xenobiotic response element, HIFI, Hypoxia inducible factor; EGR1, Early growth response 1; SP 1, Stimulating protein 1; MAZ, Myc associated zinc finger protein; NF-KB, Nuclear factor kappa B; CREB/ATF, cAMP response element binding protein/Activating transcription factor; GATA3, GATA-binding factor 3; PXR/CAR, Halfsite of PXR (pregnane X receptor)/ CAR (constitutive androstane receptor); and PRE/ARE; Progesterone receptor element/Androgen response element.  109  A. HS F2 GATA-2 IRF3  EGR I HIF I MAZ CP2^C'EBP^SP I TCF'LEF^AP2^AP2^AP2  NF1 AP2a GATA-2 TCF/LEF  CREB/ATF CREB GATA3 PXR/CAR TATA-box^ARE/PRE  AP2 MAZ AML-1 a NF-KB  -632bp^-584^-527^-465^-397^-335^-250^-150^-90^+47^+118  B. 5'  Luc  VCN-632  EXON 1 Luc  dVCN-584 Luc  dVCN-527 Luc  dVCN-465 Luc  dVCN-397 Luc  dVCN-335 Luc  dVCN-250 Luc  dVCN-150 Luc  dVCN-90 Luc dVCN+47  Figure 111-2. Schematic representation of identified and potential transcription factor-binding sites within the versican 5' flanking sequence (A) and regions encompassed by construct generation (B). Constructs were designed with the 3' ends of constructs at the same location within the versican promoter gene and 5' end were extended to different lengths upstream. The potential transcription factor binding sites in the versican promoter constructs are shown. The abbreviations are as in Figure III-1. 110  Figure 111-3.  me O9  A  BglIl (36)  o cv^7r.^t---^Le-)^r---^t.n^ct^kr)^t--o en^oo^N^0•■^M^VI^,--■^ 71-^cd  f  r  9,^+^-9 z^z ^Z'^ 1^6 6 z^ (..)^ c..)^c..)^:3 6^(..)^u^(..)^ M  (-i  I.'  › › › ",,,^"c,^..c,^ › > › "p > -,,$^›^>^(... ,.,,^c, -,1^..,,^ ^t  OM two  is* W$ .6*  1018  506 460040,  396  ASPOift  344 298 220 201 154 134  75  111  Figure 111-3. Human versican reporter luciferase and serial deletion construct design. (A) The schema of reporter vector pGL3-basic (Promega) and construct assembly. This reporter vector lacks eukaryotic promoter and enhancer sequences, allowing maximum flexibility in cloning putative regulatory sequences and served as an ideal host for the PCR generated versican promoter regions in order to identify areas which will regulate transcriptional activity. A model of the pGL3-basic vector and the construct assembly are shown in this figure. (B) Restriction enzyme digestion of pGL3basic vector and versican luciferase constructs containing versican upstream promoter elements. Versican-Luc promoter serial deletion constructs and basic pGL-3-luciferase vector were digested with M/u/ and BglII. Restriction enzyme digested construct products and DNA size marker were loaded on a 2% agarose-1000 gel and separated by electrophoresis in lx Tris-AcetateEDTA (ETDA) buffer at 120 volts/cm for two hours. M/u/ and BglII restriction enzyme digested construct products appear with different lengths ranges from 752 to 50 by with pG13-basic construct origin at the top of the gel at 4818 by length. Lanes represent DNA fragments resulting from digestion of versican-Luc constructs and basic pGL3-luciferase with M/u/ and BglII and DNA size marker.  112  ^ ^  ^-  GO  I) C)  ^a,  kr-)  ^C..)  71cn --,-  0  2(..) e9^  crl cn 0  (...,1 a.)^ 0^ -0 c)) ct cct o^ 0 21 c :s. r - ..-, cd '70 1.) • .-' _ -0^ , ,,c71-,^'^ 4)',7,4 -0 40 4Z  --, c 0^0 0 Z •k-'^ 00 •-.0 0 cn 7,4 -r.z: Cr 0 00  C a 0 !DI° . _-)_, -H P 71cL^ a) v-) 1 -7^ 3^ ,-4 o 1:1, al 0 o 4 hz)^ r,^-cs i--.1  -,..1^'''...1^0^f —'^-4--^;■-■^  M  0 v3 '.. :i Z ).) Fi^ct o ce)^0 a) "-^,-, -0 0.--4 '^' Cl^• '' c - c(I ) `c cs 1) 0 4 , - .^bl : 1 f'^(1) ._, '..4^  0^-^ 1-1^(1) 1—■ c)-4^ '-'0^ a,^:-. LO^ bkl C1) ,+-(^L. • ,--■ 0 CA N^ ''' "---, Z^• 0^ Cl3 , +-1 0 ‘2 L-d --'‘ = ^1-. -(--(^C/) © ,,I, . C.) 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(..) > > >° > > > > > > > > -0 70 -0 -0 70^-0 70 70  ^cn  Cal  113^04  ct  J1  10^CO^(I) CO^CV  CO CD  O  O  CV 10^LO  .  X11A11DV 011-1 - Z£9 - NIDA pondwop a2 1.trtp ptoj  E 1st O  O  O  O  O  o^co^CO  C°.^(SD^'Sr^CV^C)^CO^CO^•sr^CV^C::). 0 0 0 0 0  -  -  ^E A11^11 0 V orrl Z£9 1\13A^c.> pondwop afuuto pioj^1-4z M  z  E  5  E  Figure 111-5. cAMP responsiveness of human wild-type versican promoter construct. HeLa cells (A and B) and RASMC (C and D) were transfected with 0.3 lig VCN-632-Luc plasmid, and parallel plates were treated with DMSO or two inducers of cAMP, Forskolin (10 and 20 ptM) and (Bu) 2 cAMP (1 and 2 mM), for 24 hours. The cells of all groups were harvested at the same time, and luciferase activity was measured. Each experiment was repeated at least in three separate independent experiments. Samples were performed in triplicate and luciferase activity was normalized to P-galactosidase activity. Shown are the mean relative luciferase/13-galactosidase values ± SEM presented as the fold increase compared with the basal activity of the specific promoter construct from one representative experiment. Dose-dependent activation of the human versican promoter was observed after 24 h of cAMP treatment by Forskolin in HeLa cells, however (Bu)2cAMP did not altered promoter activity in this cell line. In contrast both cAMP inducers, Forskolin and (Bu) 2 cAMP, reduced promoter activity about 75% compared to DMSA treated cells. *, P < 0.05 vs. untreated basal promoter activity.  115  A.  C/EBP  C/EBP^  CREB  -634bp^-584^-527^-465^-397^-335^-250^-150^-90^+47^+118  ■ Untreated 0 (Bu)2cAMP Treated]  J. 2000 t'  1800  a)  1600  )  Ct  (7D'  2  -  1400 1200  -  1000 800 -  n  600400 200 -  dVCN-584^dVCN-465^dVCN-250^dVCN-90 Figure 111-6. Regulatory regions in the human versican promoter mediate cAMPinduced inhibition of versican luciferase activity in RASMC. (A) Schematic  representation of potential C/EBP and CRE transcription factor-binding sites within the versican 5' flanking sequence. The transcription factor abbreviations are as in Figure III1. (B) RASMCs were transiently transfected using constructs containing deleted fragments of the human versican promoter as depicted in Figure 111-2. Cells were treated for 24 hours in the presence of 1 nM (Bu)2cAMP for 24 hours after transfection. Experiment was repeated at least three times. Samples were performed in triplicate and luciferase activity was normalized to 13-galactosidase activity. Shown are the mean luciferase/13-galactosidase values ± SEM. * values significantly different between nonstimulated and stimulated from those obtained with the same construct (P < 0.04).  116  A.  40 0  35-  0  30 sm 0¢25 c.)  -  20  tab  -  15  o^C_)  10 5-  0 VCN-632-Luc (0.3pg/m1) pCMV (0.3pg/m1) pCMV C/EBPf  B.  + 0.3 p.g/m1  16 14 .;>  ct^4-,  p< o^c.) C.)  a.)  (- 1  12 -  10 8  6  >.< -  4  o  ›-  2 0  VCN-632-Luc (0.3pg/m1) pMEX (0.3pg/m1) ^ pMEX C/EBPa  + 0.1 pg/m1^+ 0.3 pg/m1  Figure 111-7. C/EBP a and 13 isoforms significantly enhance luciferase activity of the versican promoter. HeLa cells were co-transfected with VCN-632-Luc and one of two C/EBP isoform expression vectors, (A) C/EB113 or (B) C/EBPa as indicated in the figure for 24 hours prior to harvesting and luciferase assay. Data are represented as fold difference compared with the cells only transfected with VCN-632-Luc vector and respective empty vectors. Data are presented as the mean ± SEM from a representative experiment of three independent experiments. Statistically significant difference is indicated by an asterisk (*, p < 0.05).  117  ■ Untreated ^ Forskolin Treated  A. 16 °^14 'D 12 • c3 +—■^  -  C.)  o u 10  HeLa cells  a.)^8  tv)  ■.o^6  Uz o U  o  4 2-  ■ Untreated ^ (Bu)2cAMP Treated  B.  • (1..)  5.0  -  4.5  -  4.0 -  .„ 3.5 a., (_,-? 3.0 o ^a.)^, 2.5 •  RASMC  ci  • ct rn 2.0 -  U 4  1.5  7:;^1.0 4, 0.5 0.0  VCN-632-Luc (0.3ng/m1) pCMV C/EBPII (0.6ug/m1) C/Edn (0.6ng/m1)  4  Figure 111-8. cAMP converts the enhancement of versican promoter activity by C/EBPI3 to repression in RASMC, but not in HeLa cells. The VCN-632-Luc vector was transfected into HeLa (A) or RASMC (B) in the presence of C/EB13 13, C/Edn or both pC/EBPI3 and C/Edn in the absence or presence of cAMP inducers as indicated in the figure. Cell lysates were prepared 24 h post-transfection. Data are represented as fold difference compared with the cells co-transfected with VCN-632-Luc vector and corresponding empty vectors without treatment by cAMP inducers. Data are presented as the mean ± SEM from a representative experiment of three independent experiments. Statistically significant difference is indicated by an asterisk (*, p < 0.05).  118  A.  ■ Untreated 0 With (Bu)2cAMP 2.0 1.8 1.6 -  ct  .__,^1.4 — 4-J  1.21.2 O U L) 1.0 U a.) 0.8 • M  a  0.6  HeLa cells  -  -0 U 0.4 0 0.2 0.0 ^  B.  Untreated ^ (Bu)2cAMP Treated 3.0  O ▪  2.5  •  < 2.0  c.)  T  O c..) U  RASMC  bo (■1 1.5 cn  773  Z  1.0  ""^0.5 0. 0  VCN-632-Luc (0.3pg/m1) pCMV (0.3pg/m1) CREB (0.3pg/mB  Figure 111-9. CREB enhances versican promoter activity through cAMP dependent and independent mechanisms in RASMC. RASMC were co-transfected with VCN632-Luc, CREB expression construct or corresponding empty vector as indicated in the figure for 24 hours. Cells were then treated with the cAMP agonist (Bu) 2 cAMP (1mM) for another 24 hours prior to harvesting and luciferase assay. Data are represented as fold difference compared with the cells co-transfected with VCN-632-Luc vector and corresponding empty vectors without treatment by cAMP inducers. Data are presented as the mean + SEM from a representative experiment of three independent experiments. Statistically significant difference is indicated by an asterisk (*, p < 0.05).  119  Table III-1. Oligonucleotides. Name sequence pGL3-Seq  Sequence^(5'^to^3'^with^restriction enzyme site underlined) CTA GCA AAA TAG GCT GTC CC  VCN-Up  TTC TGG ATC CGG GGA AAG GAG  VCN-Dw (anti-sense primer for the VCNUp as well as the following primers) dVCN-584  CCG AAG ATC TTG GTC CCA GCT TCT TAC GCG TGT CTC TTT CAG G  dVCN-527  TCT TAC GCG TAA GCT CTG TGG G  dVCN-465  TCT TAC GCG TTG CTC CCG AGA A  dVCN-397  TCT TAC GCG TCT TTC CCC TAA C  dVCN-335  TCT TAC GCG TAG GGC AGT GGT T  dVCN-250  TCT TAC GCG TTT GTC AGG AAG AA  dVCN-150  TCT TAC GCG TTG CAA GCC TGG A  dVCN-90  TCT TAC GCG TAA CCC TCC TCC T  dVCN+45  TCT TAC GCG TAA GAA CTC CAG G  * ACGCGT and AGATCT are restriction enzyme sites for M/u/ and BglII, respectively.  120  111.7. REFERENCES 1.  Lin, H., Wilson, J.E., Roberts, C.R., Horley, K.J., Winters, G.L., Costanzo, M.R. and McManus, B.M. 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I further examined the roles of PKB and GSK-3p, downstream effectors of PI3K, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active PKB constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3I3 activity by LiC1 augmented accumulation of13-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. P-catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for TCF, proteins which confer transcriptional activation off3-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and J3-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding  A version of this work has been published. Rahmani M, Read JT, Carthy JM, McDonald PC, Wong BW, Esfandiarei M, Si X, Luo Z, Luo H, Rennie PS, McManus BM. (2005) Regulation of the Versican Promoter by the P-catenin/TCF Complex in Vascular Smooth Muscle Cells, J Biol Chem. 280:13019-28.  129  assays, I directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 by relative to the transcription start site, markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of P-catenin and TCF-4 significantly stimulated basal versican promoter activity. My findings suggest that versican transcription is predominantly mediated by the GSK-3p pathway via the P-catenin/TCF transcription factor complex in SMC, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events.  IV.2. INTRODUCTION AND RATIONALE Findings from our laboratory and others indicate that the PG versican is one of several ECM molecules that accumulates in vascular lesions (1-3). Versican is generally considered to be pro-atherogenic because of its ability to trap cholesterol-rich lipoproteins (4, 5) in addition to its crucial role in regulation of cell adhesion, survival, proliferation, and migration, and ECM assembly, all fundamental processes involved in vascular disease (4-7). The complete versican gene structure has been elucidated in humans (8, 9) and the mouse (10). The human and murine genes prove to be remarkably conserved in genomic organization. Both versican genes extend for approximately 90 kb and contain 15 exons that align in an identical manner with the protein subdomains. Naso et al. (9) report that the human versican gene has one transcription start site. Meanwhile, sequence analysis reveals potential binding sites for several transcription factors in addition to the TATA box. Transient expression assays of reporter constructs driven by an 876 by (-632/+240 relative to the transcription start site) piece of the versican promoter in HeLa cells and IMR-90 embryonic lung fibroblasts have shown  130  significant expression. These results indicate that the human versican 5'-flanking sequence contains promoter and enhancer elements able to drive reporter gene expression in cells derived from epithelial or mesenchymal tissues (9). Various growth factors and mediators influence the expression of versican. Studies using arterial SMC have demonstrated that TGF-I31 and PDGF-AB increase versican mRNA levels, core protein synthesis and GAG chain length (11). Similarly, normal human gingival fibroblasts respond to treatment with either TGF-J3 or PDGF-BB by increasing versican mRNA levels (12, 13). In contrast, the pro-inflammatory cytokine interleukin-1-13 appears to decrease the synthesis of this PG in human gingival fibroblasts and arterial SMC (14). Data from recent investigations suggest that versican synthesis by mesanchymal cells can be regulated by physical stimuli, including cell density and mechanical strain (15). In vitro experiments using monkey arterial SMC have shown that PDGF-BB stimulates versican core protein expression; this signaling apparently occurs through a RTK-dependent, protein kinase C (PKC)-independent pathway (16). Angiotensin II-mediated stimulation of SMC versican expression is regulated by epidermal growth factor receptor (EGFR)-dependent tyrosine kinase pathways (17). The canonical Wnt—wingless signaling pathway regulates various biologic processes including early embryogenesis and neoplasia by increasing the stability and transcriptional activity of a key mediator, p-catenin (18-20). In the absence of Wnt ligand, GSK-3 promotes the phosphorylation off3-catenin at key serine/threonine residues, targeting it for degradation through the ubiquitin—ligase pathway (21). In response to Wnt, the GSK-3-binding protein inhibits GSK-3 activity (22). Some growth factors can regulate GSK-3 activity by mediating its phosphorylation at serine-9 phosphorylation independent of Wnt ligand. Although serine-9 phosphorylation of GSK-3 is associated with its inactivation, Wnt ligand doesn't necessarily  131  regulate this phosphorylation (23). GSK-3 inactivation leads to P-catenin stabilization and translocation into the nucleus, where it binds to TCF/LEF family proteins to form a transcription factor complex that activates target genes such as the matrix metalloproteinase (MMP)-7, fibronectin, VEGF, cyclin D1, and c-myc (24). A variety of mitogenic stimuli including Wnts, insulin, EGF and PDGF result in catalytic inactivation of GSK-3. The catalytic inactivation of GSK-3 induced by most polypeptide mitogens is reversible by treatment with serine/threonine-specific phosphates (25). The inactivation event has been demonstrated to be due to phosphorylation of serine-21 and -9 of GSK-3a and -3P, respectively (26-28). These residues are specific targets for several proteinserine kinases, including PKB, pp90rsk, and cAMP-dependent PKA (29, 30). The inactivating biochemical consequence of phosphorylation by all three enzymes is identical — what differs is the initiating signal. Thus, activation of PI3K pathway (usually via RTK activation) results in stimulation of PKB. Inactivation of GSK-3 in response to many mitogens can be inhibited by antagonists of PI3K such as LY294002 inhibitor (25). These mechanisms are all independent of Wnt-induced regulation of GSK-3. Most protein kinases are induced by cellular stimuli, whereas GSK-3 is shut down. In addition, the enzyme has a broad variety of target proteins, most of which are inactivated by phosphorylation of GSK-3. Thus, inhibition of this one enzyme will tend to induce the functions of a diverse array of targets including transcription factors and other regulatory molecules (31). Despite the importance of versican in vascular pathophysiology, the function and regulation of expression of this versatile molecule in vitro and in vivo is unknown. My results suggest that a signaling molecule activated by 3 phosphoinositides, namely PKB, plays a critical role in serum-stimulated versican transcription. Furthermore, I provide evidence that  132  phosphorylation and inhibition of GSK-3P by PKB and subsequent activation of the J3catenin/TCF complex is essential for transcription from the versican promoter.  IV.3. EXPERIMENTAL PROCEDURES IV.3.1.^Isolation and primary culture of rat aortic SMC — A rat aortic SMC culture was established by a modification of the enzymatic dispersion technique (32, 33). Briefly, four adult male Fisher rats (275-350g) were euthanized in accordance with ethical guidelines set out by the University of British Columbia Animal Care Committee. The thoracic aorta was removed and immediately washed in Molecular and Cellular Developmental Biology (MCDB) 131 medium (Sigma-Aldrich, Oakville, ON). Enzyme I (0.5 mg/ml collagenase II; Worthington Biochemical Corp, Freehold, NJ) was applied to the exposed media and the tissue was incubated for 20 minutes at 37 ° C to loosen the media from the underlying adventitia. Medial strips were removed with sterile forceps, taking care not to reach the adventitial layer, transferred to a 35 mm tissue culture dish containing 500 ial of Enzyme II (0.5 mg/ml collagenase II, 0.2 mg/ml elastase; Worthington Biochemical Corp, Freehold, NJ), and minced. Medial tissue from the aortas of all four rats was pooled, additional Enzyme II solution was added to the dish, and the tissue was incubated at 37 ° C for 2.5 hours with pipetting at regular intervals to disperse cells. Liberated cells were subsequently pelleted at 1000 rpm for 5 minutes, resuspended in 1 ml MCDB-131 containing 20% newborn calf serum (CS), and seeded into a 35 mm tissue culture dish. Cells were grown to confluence, released by trypsinization and subcultured at a density of 1.0x 10 4 cells/cm 2 in MCDB-131 supplemented with 5% CS. Cell Culture — Rat vascular SMC were maintained in MCDB-131 plus 5% CS as before in a controlled atmosphere of air-CO2 (5%) at 37 °C until confluence (6-7 days). Confluent SMC at  133  passages 4-8 were used for experiments. Human prostate cancer PC3 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5% FBS (fetal bovine serum; Gibco-BRL) at 37 °C in 5% CO2. A stable HeLa cell line expressing dominant negative mutant of PKB1 construct (DN-PKB), constitutively activated mutant of PKB1 (CA-PKB) and corresponding empty plasmids was generated and selected as previously described (34). IV.3.2.^Generation of promoter reporter, mutant, and deletion constructs — A 752-bp versican promoter (-634/+118) and a shorter fragment (-438/+118) corresponding to the versican promoter sequences (9) were generated by PCR from the human genomic DNA with appropriate sets of primers as described in chapter III of this dissertation. These inserts were cloned into a pGL3 basic vector (Promega) by standard molecular biology techniques and called VCN-632-Luc and dVCN-438-Luc. The putative TCF binding sites, -546 by TCCCTTTGATGG and -492 by TTCTTTGCTGAT contained in the VCN-632-Luc were mutated by site-directed mutagenesis using a Quick Change mutagenesis kit from Stratagene as described before (35). The mutated inserts were generated by PCR and then inserted into the promoterless luciferase vector pGL3-Basic. All constructs were verified by sequencing.  IV.3.3.^Plasmid constructs — The dominant negative mutant of PKB1 construct (DN-PKB) (Upstate Biotechnology), constitutively activated mutant of PKB1 (CA-PKB) (Upstate Biotechnology), and the empty vector control were used in transient and stable transfection of SMC and HeLa cells, respectively. The cDNAs for wild type PTEN (wt-PTEN) and its mutant (mut-PTEN) were kindly provided by J. Dixon (Department of Pharmacology, University of California, San Diego) and were subcloned into pXJ41-neo expression vector kindly provided by C. Pallen (Department of Pediatrics, University of British Columbia, Vancouver). A dominant stable f3-catenin construct was a kind gift of B. Gumbiner (Cellular  134  Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, USA). The expression vectors TCF-1E and TCF-4E harboring the wild-type human TCF-4 gene and chimeric TCF-1 construct lacking C terminal Binding Protein (CtBP) structural domain, respectively, were a gift from A. Hecht (Max-Planck-Institute of Immunobiology, Freiburg) (36). The myc-tagged dominant negative TCF-4 (ANTCF-4) was kindly provided by H. Clevers (Department of Immunology, University Medical Center Utrecht, The Netherlands) (37). Wild type GSK-30 was a kind gift from G. Cooper (Department of Pathology, Harvard Medical School, Boston, USA) (38). IV.3.4.^Transfection and luciferase activity assays — Starved SMC were  transiently transfected in six-well plates using up to 2 pig of plasmid DNA and FuGENE6 reagent (Roche Applied Science) according to the procedure recommended by the manufacturer. In brief, a ratio of 3:1 for FuGENE reagent (1.11):plasmid (pg) was incubated for 30 min at room temperature in incomplete medium before addition to 70-80% subconfluent cells in medium containing the mediator of interest or in complete medium for the period of time indicated in the legends of the figures. After the indicated incubation period, cells were lysed, and luciferase activities were measured with a kit from Promega according to the manufacturer's protocol. Protein concentrations were measured with a Bradford protein assay kit from Bio-Rad, and luciferase values were normalized to the obtained protein concentrations. In some transfection experiments, normalization was done by LacZ reporter (Promega), and p-galactosidase (P-gal) assays were performed according to the manufacturer's protocol. IV.3.5.^Immunoblotting — SMC were grown in six-well plates. Cells were lysed  with 200g1 of lysis buffer. For PKB, lysis buffer contained 50 mM HEPES (pH 7.6), 1 mM EDTA, 5 mM EGTA, 10 mM MgC12, 50 mM f3-glycerophosphate, 1 mM Na3VO4, 10 mM  135  NaF, 30 mM sodium pyrophosphate, 2 mM dithiothreitol, 1 mM AEBSF. For GSK3, lysis buffer contained 20 mM Tris (pH 7.5), 25 mM P-glycerophosphate, 100 mM NaC1, 1 mM Na3VO4, 2 mM EGTA, 2 µg/m1 leupeptin, 1 gg/m1 aprotinin, 1 mM AEBSF. Samples were subjected to SDS-PAGE, and transferred onto nitrocellulose membranes. The membranes were probed with anti-phospho-PKB (Ser-473) (Cell Signaling Technology) or anti-phospho-GSK3 (Ser-9) (Oncogene Research Product) antibodies. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies were used as secondary antibodies. The levels of wt-PTEN and mut-PTEN were determined 48 hours post-transfection of PC3 cells by Western blot analysis using antibody against PTEN (Santa Cruz Biotechnology). The bound secondary antibody was detected by the enhanced chemiluminescence (Amersham Pharmacia Biotech). IV.3.6.^RNA extraction and cDNA synthesis — Total RNA was isolated from treated and untreated SMC using RNeasy Mini Kit according to the manufacturer's protocol (Qiagen). All preparations were treated with RNase-free DNase (Qiagen) to remove genomic DNA. 0.5 to 1 gg of RNA was reverse transcribed in a total volume of 20 pil in the presence of 200 U SuperScript RNase H- Reverse Transcriptase (Invitrogen, Carlsbad, CA), 40 U RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), and 0.511g Oligo(dT) Primer (Invitrogen) according to the manufacturer's instructions. IV.3.7.^SYBR-green quantitative real-time reverse transcriptase-polymerase chain reaction — Quantitative two-step real-time RT-PCR was performed using a LightCycler (Roche Molecular Biochemicals) in order to assess versican mRNA expression in SMC. Beta actin was used as a housekeeping gene. Primer pairs were designed to flank an intron-containing sequence. PCR conditions used included 3 mM Mg2+, 0.3 i.tM forward and reverse primers, and 2 'IL of LightCycler FastStart DNA Master SYBR Green I Mix (Roche Molecular Biochemicals)  136  in a final volume of 20 pL. The samples were loaded in the LightCycler glass capillaries, closed, centrifuged, and placed in the LightCycler rotor. The cycling program consisted of 10 minutes of initial denaturation at 95°C, and 45 cycles at 95°C for 5 s, TA°C for 5 s, 72°C for 20 s, and single detection for 1 s with a single fluorescence acquisition (ramp rates 20°C/s). The analytical melting program was 95°C for 0 s and 65°C for 15 s, increasing to 95°C at a ramp rate of 0.2°C/s, with continuous fluorescence acquisition. Each sample was run in triplicate. A standard curve was included in each run. Standards were prepared by cloning the target sequence into plasmid DNA. The data were analyzed by using the second-derivative maximum of each amplification reaction and relating it to its respective standard curve. The results from the quantitative PCR were expressed as the ratio of the mean target gene measurements to the mean housekeeping gene value for a given sample (Target:Reference). IV.3.8.^Electrophoretic mobility shift assay — Individual oligonucleotides were 5'-  end y- 32 P deoxyadenine triphosphate labeled with T4 polynucleotide kinase. Labeled oligonucleotides (100 ng) were annealed to equimolar amounts of their complementary strands (unlabeled) by heating to 95 ° C for 5 min in Tris-EDTA supplemented with 50 mM NaC1 and slowly cooling to room temperature. Double-stranded oligonucleotide probes were purified on a 5% (w /v) polyacrylamide 0.5 X TBE non-denaturing gel, eluted in 500111 of elution buffer [0.6 M NI-140AC / 0.1 % (w /v) SDS / 1 mM EDTA] and ethanol precipitated prior to use in electromobility shift assays (EMSA). Nuclear extracts used in EMSA were isolated from SW480 human colorectal carcinoma cells, using a modified Dignam method (39). Final nuclear protein preparations were collected in buffer C [400 mM NaC1 / 20 mM HEPES (pH 7.4) / 25% glycerol / 1.5 mM MgC12 / 0.2 mM EDTA / 0.5 mM dithiothreitol (DTT)]. For band shift experiments, 20,000 cpm of labeled oligoduplex probes were added to 5 Ill of nuclear extracts in 40 ul of  137  DNA binding buffer [10% glycerol, 1mM EDTA, 1mM DTT, and 60 mM KC1]. To prevent nonspecific binding of nuclear proteins, poly dI-dC was added to a concentration of 100 ng/iil and the binding reaction was incubated at room temperature for 10 minutes prior to electrophoresis. For supershift experiments, 1 pg of goat anti-TCF-4 polyclonal antibody (0.4 pg/p.1, Santa Cruz Biotechnology, N-20) was added to the binding reaction and incubated for 10 minutes at room temperature prior to electrophoresis. For EMSA interference experiments 0.2 and 0.414 of goat anti-13-catenin polyclonal antibody (0.4 lig/111, Santa Cruz Biotechnology, H102). For both supershift and interference experiments antibody control reactions contained an equivalent mass of normal mouse IgG and antibody negative reactions were supplemented with an equal volume of PBS. Protein-DNA complexes were separated from unbound DNA using a 5 % (w / v) polyacrylamide, 0.5 X TBE non-denaturing gel, run at a constant voltage of 400 V for 1 hour. The oligonucleotides corresponding to the LEF/TCF site from versican promoter used for EMSA were: (5'-ACT TCC CTT TGA TGG GAC AG-3' and 5'-CTG TCC CAT CAA AGG GAA GT-3').  IV.4. RESULTS IV.4.1.^The 3 phosphoinositides-dependent signaling mediates versican transcription in vascular SMC— I examined the role of the PI3K-PKB pathway in induction of  versican transcription as a result of serum stimulation in vascular SMC. First, the levels of activated PKB were determined by Western blot analysis using phosphospecific antibody, after 30 min pre-incubation of quiescent SMC by different concentrations of LY294002, a pharmacological inhibitor of P13K, followed by serum stimulation. LY294002 inhibited activation of downstream PKB at all concentrations tested (Figure IV-1A). Next I examined  138  whether activation of PI3K and subsequent generation of 3 phosphoinositides is necessary for the stimulation of versican transcription by serum. I transfected SMC with wild type versican-Luc reporter construct, and 24 h after transfection, serum-starved, growth-arrested cells were pretreated with LY294002 for 30 min prior to stimulation with 5% CS for 24 h. versican promoter activation was significantly inhibited (Figure IV-1B). I next determined the role of the PI3K pathway in the induction of expression of versican mRNA by real time RT-PCR analysis. SMC were growth-arrested for 24 h pretreated with LY294002 for 30 min and stimulated with 5% serum for 24 h. The induction of versican mRNA expression by serum was significantly suppressed by LY294002 pre-treatment (Figure IV-1C). These results suggest that reduced production of 3 phosphoinositides inhibits versican transcription (Figure IV-1A and 21B). To examine further the role of 3 phosphoinositides, I investigated the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in control of versican expression. PTEN is a phosphatase that selectively dephosphorylates the 3 position of both phosphatidylinosito1-3,4,5trisphosphate and phosphatidylinositol-3,4-bisphosphate (40) antagonizing the diverse downstream signaling effector pathways activated by PI3K-derived phospholipids. To investigate the effects of this protein on versican expression levels, I over-expressed it in a cell line normally lacking PTEN, the PC3 prostate cancer cell line. In Figure IV-2A, the first lane shows the levels of endogenous PTEN in HeLa cells. The other 3 lanes show PTEN expression in PC3 cells transiently transfected with empty vector, wild type PTEN (wt-PTEN) expression vector and mutant PTEN (mut-PTEN) expression vector expressing a lack of functional PTEN. The results of the co-transfection of the versican-Luc plasmid with empty vector, wt-PTEN and mut-PTEN are shown in Figure IV-2B. Expression of wt-PTEN and mut-PTEN reduce and increase promoter function, respectively, which supports my previous findings regarding the role  139  of PI3K in versican promoter regulation. Taken together, these results are consistent with the notion that increases in 3 phosphoinositides levels are necessary for serum-induced increases in versican transcription. IV.4.2.^PKB is a downstream effector of PI3K in serum stimulation of versican transcription in SMC— PKB was suggested to be a major downstream mediator of the PI3K in gene regulation in SMC (41). To investigate whether it is also involved in the serum stimulation of versican transcription, I co-transfected expression vectors for either a DN-PKB plasmid that is incapable of being phosphorylated in response to different stimuli or a CA-PKB expression plasmid with versican-Luc into SMC. Empty vector was used as a control plasmid. As shown in Figure IV-3A and B, DN-PKB expression significantly decreased and CA-PKB increased versican promoter activity as compared to the corresponding empty vector control. To further study the contribution of PKB to versican regulation, I used HeLa cells stably transfected with DN-PKB, CA-PKB, or empty vector control plasmids. As expected, the DN-PKB mutant blocked PKB 1 phosphorylation and activation in transfected HeLa cells when compared to control cells as determined by measuring the phosphorylation of PKB1 and GSK-3 a/P, a PKBregulated downstream protein (Figure IV-3F). In contrast, HeLa cells stably transfected by CAPKB significantly augmented phosphorylation of PKB1 and GSK-3 a./13 as compared to control cells containing empty vector (Figure IV-3E). I transiently transfected a versican-Luc reporter construct into HeLa cells stably expressing CA-PKB, DN-PKB and corresponding empty vectors (Figure IV-3C-E). The results were similar to those observed in transient transfections; versican promoter activity was significantly increased and decreased in HeLa cells stably transfected with CA-PKB and DN-PKB, respectively. The effects of DN-PKB and CA-PKB expression on versican-Luc activity are similar to those seen when PI3K is inhibited by LY294002 treatment or  140  co-transfection of the mut-PTEN expression construct. Overall, these data suggest that PKB is a downstream mediator of PI3K in the serum regulation of versican transcription in SMC. IV.4.3.^Cytoplasmic accumulation of/J-catenin via GSK-3/J inhibition stimulates transcription of versican in SMC — I have used various approaches to show the role of GSK-313  in versican regulation (Figure IV-4A). To assess whether GSK3-0 is involved in up-regulation of versican expression via P-catenin, SMC were transfected with the versican-Luc promoter construct and treated with 30mM LiC1, an inhibitor of GSK3-(3. LiC1 is an inhibitor of GSK3 that induces accumulation of dephosphorylated13-catenin and increase the activity of synthetic TCFdependent reporter constructs. Addition of LiC1 resulted in a more than 2-fold increase in versican promoter activity (Figure IV-4A). Up-regulation of versican mRNA was also evidenced in SMC after treatment with LiC130 mM (Figure IV-4B). To confirm the involvement of GSK30 in versican activation, SMC were co-transfected with increasing amounts of GSK3-13 expression vector and this was shown to dose-dependently decrease versican promoter activity (Figure IV-4C). These findings are consistent with my results using another inhibitor of GSK3B, SB415286, which caused a similar increase in versican-Luc promoter activity and versican mRNA levels (data not shown). My results support the notion that GSK-30 regulates versican transcription in SMC via accumulation of f3-catenin. IV.4.4.^LEF/TCF DNA binding is involved in fi-catenin-induced versican transcription —13-catenin contains no DNA binding domain and therefore cannot regulate  versican promoter activity directly. The proximal versican promoter was searched for transcription factor binding sites that would confer 13-catenin transactivation. A search for consensus TCF binding sites in the versican promoter using MatInspector program (42) revealed the presence of two potential LEF/TCF binding sites, TCCCTTTGATGG and  141  TTCTTTGCTGAT sequences, at positions -546 by and -492 bp, respectively (Figure IV-5A). To determine the functional significance of consensus LEF/TCF binding sequences at nucleotides 546 to -534 and -492 to -480, I first employed 5' promoter deletion construct (Figure IV-5A). I co-transfected SMC with a versican-Luc deletion construct lacking the LEF/TCF binding sites (438del-versican-Luc) and P-catenin expression plasmids. Of the deletion reporter showed no f3catenin-induced versican promoter activation (Figure IV-5B). The functional significance of the consensus TCF/LEF binding motifs was assessed further by introducing point mutations in the sites within the versican-Luc promoter (Figure IV-5A). In SMC, basal activity of the -546mutversican-Luc construct was not significantly different from that of the wt-versican-Luc, but the 492mut-versican-Luc and -546/-492mut-versican-Luc showed significantly lower basal versican promoter activity (9-fold), indicating that the TCF binding site at position -492mu-versican-Luc plays a critical role in versican promoter function and potentially contributes to a significant extent to constitutive versican expression in these cells (Figure IV-5C). I observed similar effects of these mutations on versican promoter activity in the SW480 colon carcinoma cell line and HeLa cells (data not shown). Moreover the mutation of the LEF/TCF binding motif abolished transactivation of this construct by P-catenin in SMC (Figure IV-5D) as well as in other cell types (data not shown). These results indicate that induction of versican by p-catenin is controlled at the transcriptional level and depends on a LEF/TCF binding site. IV.4.5.^The versican promoter is activated by the /J-catenin-TCF complex — The  presence of possible LEF/TCF binding motifs in the versican promoter prompted us to examine the effects of13-catenin and TCF-4 expression on versican promoter activity. Co-transfection of versican-Luc with a f3-catenin expression vector significantly and dose-dependently increased  142  versican promoter activity (Figure IV-6A). These results demonstrate that the accumulation of cytosolic 13-catenin results in increased versican promoter activity. In humans, there are four known isoforms of TCF; LEF-1, TCF-1, TCF-3, and TCF-4, in which the most conserved portions among these transcription factors are the P-catenin binding domain and the HMG box, the later being facilitory for DNA binding. Some isoforms of TCF do not possess the P-catenin binding domain or contain domains which function as repressors such as CtBP binding structural domains and can therefore act in a dominant negative fashion. These isoforms are incapable of activating transcription and are thought to ordinarily function as tumour suppressors. These differences are thought to contribute to the bimodal behaviour of TCFs wherein they are known to be capable of both repression and activation. I co-transfected the versican-Luc reporter vector with the TCF-4 expression vector, containing the CtBP binding domain, in the absence or presence of a P-catenin expression plasmid. The results showed that TCF-4 inhibited versican promoter activity in the absence of P-catenin, but in the presence of Pcatenin was able to induce versican promoter activation (Figure IV-6B). In contrast, cotransfection of the versican-Luc reporter with a TCF-1 chimeric construct (TCF-1E) which does not contain a CtBP domain does not inhibit promoter activity and versican promoter activity significantly increased regardless of the co-transfection of 13-catenin (Figure IV-6C). These findings support a repressor role for CtBP in P-catenin-mediated versican promoter activation by TCFs. To assess whether versican transcriptional activation was TCF-4-dependent, I performed a co-transfection of the P-catenin expression vector with a TCF-4 dominant negative mutant (ANTCF-4) devoid of P-catenin binding domains. The \NTCF-4 mutant inhibited P-catenindriven transactivation (Figure IV-6D).  143  Taken together these data suggest that the P-catenin-TCF complex activates the versican promoter in SMC. My data also suggests the levels of different isoforms of TCF transcription factors containing different repressor or co-activator domains may determine the repression of TCF target genes, including versican in SMC. IV.4.6.^TCF-4 binds to the versican LEF/TCF site — In the next set of  experiments, I analyzed the binding of nuclear extract protein to the LEF/TCF site in the versican promoter by gel shift analysis (Figure IV-7). Oligonucleotides corresponding to the indicated DNA elements were labeled with P 32 and incubated with nuclear extract derived from the SW480 human colorectal cancer cell line. The versican TCF site element showed a strong double shift consistent with previously observed TCF-4 and TCF-4/13-catenin complexes and a supershift on addition of anti-TCF-4 antibodies (Figure IV-7A) (43, 44). Addition of anti-13-catenin antibodies to the shift experiment resulted in interference of the formation of the larger complex which is consistent with antibody binding preventing the TCF-443-catenin interaction (Figure IV-7B).  IV.5. DISCUSSION  In the present study, I found that PI3K and its downstream effector PKB mediate serum stimulation of versican transcription in SMC. Furthermore, I showed that cytoplasmic accumulation of 0-catenin via inhibition of GSK-313 stimulates transcription of versican. Finally, I demonstrated that a LEF/TCF binding site in the versican promoter is involved in p-catenininduced versican transcription and that at least one of the members of TCF family of transcription factors, TCF-4, binds to the LEF/TCF site in the human versican promoter. These observations may support a new concept for the pathophysiological role of PI3K/PKB/GSK-313 signaling through the 13-catenin/TCF transactivation complex, modulating the gene expression  144  patterns observed in atheromatous lesions and provisional matrix formation involved in normal and aberrant vascular injury and repair processes (1-7, 45). Numerous studies have demonstrated that after mechanical arterial injury, vessel walls follow a response-to-injury pattern of wound healing leading to restenosis secondary to the neointimal accumulation of SMC and ECM, with the ECM accounting for >90% of the neointimal volume (46, 47). The PG versican is one of several ECM molecules that accumulate in vascular lesions (1, 2). Vrsican is generally considered to be pro-atherogenic because of its ability to trap cholesterol-rich lipoproteins (4, 5) in addition to its role in regulation of fundamental events involved in vascular disease (4, 5, 7). The dynamic process of vascular injury and repair involves molecular signaling cascades that govern arterial SMC migration, differentiation, proliferation, and fate (48-50). The canonical Wnt—wingless signaling pathway regulates various biologic processes including early embryogenesis, neoplasia, and healing by increasing the stability and transcriptional activity of a key mediator, P-catenin (51-53). Upon its stabilization and accumulation (51-53), P-catenin translocates to the nucleus and activates the LEF/TCF family of transcription factors (53). Evidence suggests a potentially important role for  the 0-catenin/TCF signaling in vascular remodeling events (54-57). The present study suggests that upon activation of growth factor receptors and Wnt signaling, p-catenin increases and activates TCF target genes, including versican, which, in turn, retains atherogenic lipoproteins and contributes to lesion development. The production of versican also influences cellular functions and survival directly or indirectly. My finding may suggest one of the molecular mechanisms by which versican influences vascular injury and repair events. In vitro experiments using monkey arterial SMC show that PDGF-stimulated versican core protein expression apparently occurs through a RTK-dependent, protein kinase C (PKC)-  145  independent pathway (16). The concentration- and time-dependent increase in mRNA levels by AT1-receptor-expressing cells in response to Ang II inhibited by the AT1-receptor antagonist losartan, the EGFR inhibitor AG1478 and the MAP kinase inhibitor PD98059. The increase in versican gene expression was not inhibited by the PKC inhibitors chelerythrine and staurosporine, indicating that Ang II-mediated stimulation of SMC versican expression is regulated by RKT-dependent mechanisms (58). My data, for the first time, suggest that PI3K/PKB signaling is involved in the serum regulation of versican transcription in SMC. I established this regulatory sequence of events in a series of experiments. First, I showed that the pharmacological inhibitor of PI3K, LY294002, inhibited activation of downstream PKB at all concentrations tested and resulted in significant inhibition of versican promoter reporter activity and a reduced versican mRNA level in SMC. Second, consistent with previous results, expression of wt-PTEN and mutant-PTEN reduced and increased versican promoter function, respectively. PTEN antagonizes the diverse downstream signaling effectors pathways activated by PI3K-derived phospholipids, which supports my previous finding regarding the role of PI3K in the versican promoter regulation. PKB was suggested to be a major downstream effector of PI3K-related gene regulation in SMC. Finally, the results of my experiments demonstrate that versican promoter activity of versican-Luc was significantly increased and decreased in transient co-transfection with CA-PKB and DN-PKB constructs, respectively. Overall, these results suggest that reduced production of 3 phosphoinositides inhibit serum induced PKB signaling stimulation of versican transcription in SMC. GSK-3 inactivation leads to 13-catenin stabilization and translocation into the nucleus, where it binds to LEF/TCF family proteins to form a transcription factor complex that activates target genes such as MMP-7, fibronectin, VEGF, cyclin D1, and c-myc (24). Recent work  146  suggests a role for the Wnt/P-catenin signaling pathway in pathophysiological remodeling within the cardiovascular system (21, 22, 59, 60). There is also evidence that differential inhibition of GSK-3p in intimal tissue following vascular injury acts as a critical signal mediating SMC survival (21). Finally, Mao and colleagues (59) recently demonstrated altered expression of the Frizzled receptor family in SMC in response to vascular injury. There is a recent report that the versican gene was identified as a target gene of Wnt signaling using microarray technology to analyse human embryonic carcinoma cells stimulated with active Wnt protein (61). The promoters of nearly all the target genes identified include putative TCF binding sites including the versican promoter. Kishimoto et al. (62) also showed that co-culture of the cells expressing a secreted Wnt3a protein maintain the expression of green fluorescent protein (GFP) placed under the control of a fragment of the human versican promoter. In this study, I have observed that the versican gene expression is up regulated by p-  catenin. I show that endogenous versican mRNA is induced in SMC by LiC1 treatment, which inactivates GSK3 and therefore stabilizes wild type P-catenin. This regulation occurs at the transcriptional level, since the versican promoter responded to P-catenin in presence or absence of co-transfected TCF-4 expression plasmid whereas dominant negative TCF-4 expression inhibited these effects. My data demonstrate that a single consensus LEF/TCF binding sequence located -492 by from the transcriptional start site is critical for P-catenin responsiveness, thus identifying versican as a direct target of the P-catenin-TCF complex. I also show a potential repressor function of some TCF isoforms on versican transcriptional activity by co-transfecting a versican promoter reporter and a vector expressing a TCF-4 isoform containing a CtBP structural domain in the presence or absence of p-catenin expression plasmid. The results showed that the CtBP domain of TCF-4 mediated inhibition of versican promoter activation in the absence of p-  147  catenin, but in the presence ofp-catenin was able to induce versican promoter activity. In contrast, co-transfection of the versican promoter with a TCF chimeric construct which does not contain CtBP domains did not repress promoter activity (discussed in further details in chapter V of this dissertation). These findings support the hypothesis that certain TCF isoforms containing the CtBP structural domain can repress TCF target genes such as versican. Taken together these data suggest that (3-catenin-TCF complex activates the versican promoter in SMC. My data also suggest that the level of different isoforms of TCF transcription factors containing different repressor or co-activator domains determine the repression activity of TCF on target genes in SMC.  IV.6. CONCLUSIONS I suggest that the PI3K/PKB pathway plays a critical role in serum-stimulated versican transcription. Further, I provide evidence that phosphorylation and inhibition of GSK-3p by PKB and subsequent (3-catenin/TCF complex formation is essential for versican transcription (Figure IV-8). The roles of versican in cell growth, motility, adhesion, and angiogenesis strongly indicate that this PG of the ECM is an important downstream effector of the Wnt pathway during developmental and pathological processes. I also suggest that P-catenin through regulation of this versatile PG versican and other candidate target genes, augments the establishment of a provisional ECM, induces proliferation and survival of vascular cells, as well as modulating adhesive, migratory and angiogeneic processes that lead to normal or aberrant vascular injury and repair.  148  Figure IV-1. A. — o 10% Serum, 30 min  B.  0 10 20 40 LY294002 (uM) p-PKB  t-PKB  DMSO 10iM 20nM 40uM LY294002  149  Figure IV-1. PI3K is involved in versican promoter activity and transcription in smooth muscle cells (SMCs). A, Inhibition of PI3K signaling pathway inhibited PKB phosphorylation in SMCs. Confluent serum-starved (24 h) SMCs were pretreated with DMSO or various concentrations of LY294002 for 30 min and then stimulated with 10% newborn calf serum for 30 min. The levels of activated PKB were determined by Western blot analysis using phosphospecific antibody (anti-phospho-S473 PKB) (top panel). Blots were stripped and reprobed with anti-PKB antibody (bottom panel) to normalize for PKB protein. B, Inhibition of PI3K signaling reduces versican promoter reporter activity in SMCs. Fifty to 70% confluent SMCs were transfected with versican-luciferase reporter (2 ,ug). After 3 h, the DNA:liposomes mixture was removed and cells were kept in MCDB-131 medium containing 0.2% Bovine Serum Albumin (BSA). Twenty-four hours posttransfection, the cells were pretreated with DMSO or LY294002 (10, 20, and 40 gM) for 30 min and then stimulated with 10% newborn calf serum for 24 h. Cell extracts were obtained and luciferase activity was assayed. Transfection efficiency was monitored by cotransfecting the cells with 0.2 ,ug of RSV/I3-galactosidase construct. The values are representative of three independent experiments each performed in duplicate or triplicate and are shown relative to the luciferase activity of cells treated with DMSO (1). Values are mean±S.D. C, Inhibition of PI3K in SMCs reduces versican mRNA levels. Confluent serumstarved (24 h) SMCs were pretreated with DMSO or LY294002 (10, 20, and 40 p,M) for 30 min and then stimulated with 10% newborn calf serum for 24 h. Total RNA was isolated and the versican mRNA level was quantified by real-time RT-PCR as described under "Experimental Procedures". Expression was normalized with respect to 13-actin RNA levels in the same samples and presented relative to mRNA level from the cells treated with DMSO. Results shown are the mean±S.D. of two cDNA samples analyzed in duplicates representative of three independent experiments. Asterisk denotes a significant difference (p<0.05) between versican expression in cells treated with LY294002 and DMSO.  150  Figure IV-2. PC3 cells  A.  Control HeLa cells Mock ^wt-PTEN mut-PTEN PTEN  B.^2.5 * p<0.05 -= 2 a)  1.5  z —11^1  *  cc  a) IX  0.5  Mock wt-PTEN mut-PTEN Figure IV-2. PTEN inhibits versican promoter activity in PC3 cells. A. Transient overexpression of wt-PTEN, mut-PTEN, and corresponding empty plasmids in PC3 cells. PC3 cells were transiently co-transfected with the wt-versican-Luc plasmid and either empty vector or constructs expressing wt-PTEN or mut-PTEN as described under "Experimental Procedures". The levels of wt-PTEN and mut-PTEN were determined 48 hours posttransfection by Western blot analysis using an antibody against PTEN. B, Overexpression of wild-type PTEN blocks serum-mediated induction of versican promoter reporter in PC3 cells. Transient transfection of PC3 cells with the wt-versican promoter plasmid together with either empty vector or a construct expressing wt-PTEN or mut-PTEN. Data are expressed as fold induction with the induction of luciferase activity from versican-Luc relative to empty vector. Induction of reporter activity in the presence of wt-PTEN differed significantly from empty plasmid controls. Co-transfection of wt-versican-Luc plasmid with mut-PTEN lacking lipid phosphatases activity did not show significant reporter augmentation in response to serum. 151  Figure IV-3. 2.5  ^  * p<0.05  B.^1.4  *  Vector CA-PKB  Vector DN-PKB  2.5  D.  1.4  *  Vector^DN-PKB  Vector CA-PKB  10% serum  F. p-PKB  p-PKB  GSK3a  GSK3a  GSK3p  GSK313 152  Figure IV-3. Overexpression of dominant negative PKB (DN-PKB) and constitutively active PKB (CA-PKB) blocks and augments induction of versican promoter reporter, respectively. A, Transient overexpression of CA-PKB increases versican promoter reporter activity. SMCs were transiently co-transfected with the wt-versican-Luc plasmid and either empty vector or CA-PKB construct in serum free conditions as described under "Experimental Procedures". Data are expressed as fold induction relative to empty vector control transfections. B, Transient overexpression of DN-PKB decreases versican promoter reporter activity. SMCs were transiently co-transfected with the wt-versican-Luc plasmid and either empty vector or DN-PKB construct in the presence of 10% newborn calf serum as described under "Experimental Procedures". Data are expressed as fold induction relative to empty vector controls. C, Stable overexpression of CA-PKB in HeLa cells increases versican promoter reporter activity. Transient transfection of wt-versican-Luc into HeLa cells constitutively expressing CA-PKB or corresponding empty vector in serum free condition. Twenty-four hours post-transfection cells were harvested and luciferase reporter activity measured as described under "Experimental Procedures". Data are expressed as fold induction compared to cells stably transfected with empty vector. D, Stable overexpression of DN-PKB in HeLa cells decreases versican promoter reporter activity. Transient transfection of wt-versican-Luc promoter into HeLa cells constitutively expressing DN-PKB or corresponding empty vector in the presence 10% newborn calf serum. Twenty-four hours post-transfection cells were harvested and luciferase reporter activity measured as described under "Experimental Procedures". Data are expressed as fold induction compared to cells stably transfected with empty vector. E and F, Stable overexpression of CA-PKB and DNPKB in HeLa cells results in a sustained increase and decrease (respectively) in GSK-3/3/a phosphorylation (inactive form). Confluent HeLa cells constitutively expressing CA-PKB, DN-PKB or corresponding empty vector were harvested and cell lysatse prepared for assessment of levels of total and phosphorylated PKB and GSK-313/a. The levels of total and activated PKB were determined by Western blot analysis using the antibody detecting total (data not shown) and phosphorylated forms (anti-phospho-S473 PKB) (top panel). The levels of total and phosphorylated GSK-3 were analyzed by immunoblotting with antibodies detecting the total and phosphorylated forms of GSK-313/a (serine-9 and -21). The expression levels of total GSK-3f3/a did not significantly change in cells constitutively expressing CA-PKB and DN-PKB plasmids (data not shown).  153  Figure IV-4. p<0.05  4  A.  14.4 c.4  0.4  B.  3.5  3.5  3  itC^3  2.5  0.0  2  5^2  1.5  o  if.) 4  2 .5  •5  • .1 */1^  1  1  -C-44^0.5  0.5  0  0 ^ Vehicle LiC1 30 rnM  Vehicle^LiC1 30 inIVI  C.^1.2  o ^ Empty Vector  0.1ng 0.2ng 0.4ng GSK-311  154  Figure IV-4. GSK-3P inhibition stimulates transcription of versican in SMCs. A, Lithium chloride, a GSK-313 inhibitor, increased versican promoter luciferase activity. SMCs were transfected with wt-versican-Luc reporter (2 pg/well). After 3 h, the DNA:liposome mixture was removed and cells were kept in serum free medium containing 0.2% Bovine Serum Albumin (BSA) for 24 h. Twenty-four hours post-transfection the cells were incubated in presence or absence of LiC1 (30 mM) for 24 h. Cell extracts were obtained and luciferase activity was assayed. Transfection efficiency was monitored by cotransfecting the cells with 0.2 pg/well of RSV/13-galactosidase construct as described under "Experimental Procedures". The values are representative of three independent experiments each performed in duplicate or triplicate and are shown relative to the activity of luciferase of cells treated with vehicle. Values are mean±S.D. B, Lithium chloride increased versican mRNA levels. Confluent serum-starved (24 h) SMC were incubated in the presence or absence of LiCI (30mM) for 24 h. Total RNA was isolated and the versican mRNA level was quantified by real-time RT-PCR as described under "Experimental Procedures". Expression was normalized with respect to [3-actin RNA level in the same samples and presented relative to mRNA level from untreated cells. Results shown are the mean±S.D. of two cDNA samples analyzed in duplicates representative of two independent experiments. Asterisk denotes a significant difference (p<0.05) between versican expression in cells treated with LiC1 and those treated with vehicle. C, GSK-3/3 inhibited versican promoter activity. SMCs were co-transfected with the versican-Luc reporter and wild type GSK-313 construct or with the empty vector, incubated for 24 h in the presence of 5% serum, and assayed for luciferase activity. The values are representative of three independent experiments each performed in duplicate or triplicate and are shown relative to the luciferase activity in cells transfected with empty plasmid. Values are mean+S.D.  155  Figure IV-5. A.  -634  TCF/LE  TCF/LEF  F  wt-versican-Luc -546 ^534^-492^-480 TCCCTTTGATGG^TTCTTTGCTGAT  -546mut-versican-Luc  -492mut-versican-Luc  -546/-492mut-versican-Luc  TCCAgsTGATGG  ^  :•O ITCTTTGCTGAT  TCCCITTGATGG^T-TagsTGCTGAT  TCCAuTGATGG^TTIKKTGCTGAT -438  -438-del-versican-Luc  B.  Empty fl-catenin Empty P-catenin Vector^Vector wt-versican-Luc^-438mutversican-Luc  156  Figure IV-5.  C.  D. 1.8 1.6 1.4  * p<0.05 1.2 * p<0.05 1 0.8 0.6  *  •4  *  0.4  0:1  0.2  74 0.2  0 wt-versican -546mut ^-492mut -546/492mut -Luc  157  Versican -Luc  LiC1 fi-catenin 30m_111 0.8ng -492mut-versican-Luc  Figure IV-5. The TCF/LEF binding sequence at position -492 is crucial for activation of the versican promoter by the p-catenin-TCF complex. A, schematic representation of the sequence for TCF/LEF binding sites in wild type human versican promoter and mutant constructs. Two putative TCF/LEF binding sites are localized at position -546 by and -492 by  from the transcription start site. Mutations known to abolish TCF binding were introduced into the TCF/LEF recognition sites to make the mutant constructs -546mut-versican-Luc, -492mutversican-Luc, and -546/-492-mut-versican-Luc in the context of the wt-versican-Luc promoter. A deletion construct lacking the TCF/LEF binding sites, -438del-versican-Luc, was also generated as described under "Experimental Procedures". B, Mutation at position -492 of the versican promoter inhibits versican luciferase promoter activity. SMCs were transfected with wt-versican-Luc or mutant versican promoter luciferase vectors and harvested 24 h posttransfection. The values are representative of three independent experiments each performed in duplicate and are shown relative to the activity of luciferase at cells transfected with wt-versicanLuc construct. C, Mutation at position -492 of the versican promoter inhibited the response of luciferase promoter activity to Lia treatment and fl-catenin transactivation in SMCs. SMCs containing normal endogenous [3-catenin were transiently transfected with 0.5 gg of wt-versicanLuc and treated with 30 mM LiCl. The cells were co-transfected with 0.8 i.tg of p-catenin expression vector or equal amount of empty vector and harvested 24 h after transfection. Total amounts of plasmid DNA were kept constant by adding the empty pCDNA3.1 vector. All experiments were performed in duplicate and repeated at least three times. Mean + S.D. is presented. D, A deletion construct lacking the TCF/LEF binding sequences showed no /3-cateninmediated versican promoter transacativation. SMCs were transfected with wt-versican-Luc or 438del-versican-Luc reporter, along with D-catenin (0.8 p.g) or empty vector as indicated in the figure. Total amounts of plasmid DNA were kept constant by adding the empty pCDNA3.1 vector. All experiments were performed in duplicate and repeated at least three times. Mean ± S.D. is presented.  158  Figure IV-6. *^B. 2.5  A. p<0.05  Vector  Vector^TCF-4^TCF-4E + ji-catenin  0.8 pg 1.6 pg  P-catenin  C.  D.  1.4  1.2 7.  1  1 0.8  40L"  0.6  z 0.4 C4  0.2 0 Vector^ANTCF-4  P-catenin (0.5 pg) and  Vector TCF-1E TCF-1E + 13-catenin  wt-versican-Luc (0.5 ug) 159  Figure IV-6. The versican promoter is activated by p-catenin/TCF complex. A, /3-catenin induces wt-versican-Luc promoter activity in SMCs. SMCs containing normal endogenous 13catenin were transiently co-transfected with 0.514 of wt-versican-Luc, along with 0.8 and 1.6 gg of 13-catenin expression vector or equal amount of empty vector and harvested 24 h after transfection. Total amounts of plasmid DNA were kept constant by adding the corresponding empty vector. All experiments were performed in duplicate and repeated at least three times. Mean S.D. is presented. B, TCF-4E inhibits versican promoter activation in the absence of fi-catenin, but induces activation in the presence of fl-catenin. SMCs were co-transfected with the versican-Luc promoter construct and the TCF-4 isoform expression vector, TCF-4E, which contains the C terminal Binding Protein (CtBP) structural domain in the absence and presence of f3-catenin cotransfection. Cell lysates were harvested and analyzed for luciferase activity after 36 hours. C, TCF-1E induces a significant increase in promoter activity in the absence and presence of fi-catenin. SMCs were cotransfected with a versican-Luc promoter construct and chimeric TCF-1 expression vector, TCF-1E, which lacks CtBP structural domain in the absence and presence of f3-catenin co-transfection. Cell lysates were harvested and analyzed for luciferase activity after 36 hours. D, Dominant negative TCF-4 (ANTCF-4) expression vector decreases /3-catenin-induced versican promoter activity in SMCs. SMCs cells were transiently transfected with 0.5 pg of wt-versican-Luc, 0.5 ug of13-catenin expression vector and 0.5 lig of ANTCF-4 expression vector or corresponding empty vector. Total amounts of plasmid DNA were kept constant by adding the empty pCDNA3.1 vector. All experiments were performed in duplicate and repeated at least three times. Mean ± S.D. is presented.  160  Figure IV-7. >. 0 ^ .r...a.^c ._to  ^CI  cu + th^  ^+  o^uj  C^  0^Z  iC  ^C ^8  ui  c g• LUc  E^Z as •o EO r,^0 CO cn. vE^'Cr ^'42 0 a^vi 03  111111111011101101011100tit ^1111111111111111M111111111•111111111L  B.  A.  0.2pg 0.4 ug 0.2ug 0.44g  TCF-4/13-catenin  TCF-4 supershift TCF-4/J3-catenin  TCF-4  TCF-4  Free Probe  Figure IV-7. EMSA of a potential TCF-4 binding site in the versican promoter. A double stranded oligonucleotide corresponding to a potential TCF-4 binding site within the versican promoter was radiolabelled and incubated with nuclear extracts derived from the SW480 cell line. The indicated antibodies were added to the DNA binding reaction prior to electrophoresis. A) Anti-TCF-4 antibodies generated a supershifted complex while B) anti-P-catenin antibodies interfered with the formation of the higher molecular weight complex present in the control reactions.  161  Figure IV-8.  Wnts Growth Factors  Figure IV-8. Hypothetical model of versican promoter regulation via PI3K/PKB signaling and 11-catenin/TCF transcription factor complex. In the resting cells, under unstimulated conditions, cytoplasmic 13-catenin is associated with GSK-313 and other scaffolding proteins, and GSK-313 is active and phosphorylates 0-catenin in the cytoplasm and renders it susceptible to degradation via the ubiquitin-proteosome pathway. Activation of PI3K or Wnt signaling leads to phosphorylation of GSK-30, but induces inactivation of GSK, destabilizes the destruction complex, and I3-catenin accumulates in the cytoplasm and subsequently translocates to the nucleus. 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Genes Dev, 14, 1181-5.  168  CHAPTER V- ISOFORMS OF LEF/TCF TRANSCRIPTION FACTORS DIFFERENTIALLY CONTROL TRANCRIPTION OF WNT TARGET GENES  *  V.1. SUMMARY  Different TCF family members activate or repress target genes in a cell-context and promoter-specific manner. I investigated whether TCF members differentially regulate Wnt target genes via variations in their C-terminal structure. HEK293 cells were co-transfected with different combination of expression vectors of Wnt target genes (either a TCF reporter or endogenous Wnt response gene human VCN-632-Luc promoter), several natural and chimeric TCF isoforms with long and short C-terminal tails, non-phosphorylatable 0-catenin, and p300. Furthermore, inhibitors of p300 histone acetyltransferase, curcumin, or HDAC inhibitor, TSA, were used to identify p300 HAT- or HDAC-mediated regulation of Wnt target genes. I showed that TCF isoforms including an E-tail domain strongly enhanced 13-catenin/TCF- and 13-catenin/TCF-p300-mediated transcriptional activity of TCF reporter and the Wnt target gene, versican promoter. Furthermore, curcumin significantly inhibited the activity of Wnt target genes as a consequence of both 0catenin- and f3-catenin/p300-induced Wnt signaling irrespective of TCF isoforms. My results revealed that HDAC likely inhibits Wnt-responsive genes in the absence of activation by Wnt signaling. I also demonstrated that nuclear 13-catenin or activation of Wnt signaling is required for the induction of Wnt-response gene by TSA. In contrast, TSA, in the absence of Wnt signaling activation, is not capable of inducing p300-mediated Wnt targets, TCF reporter or versican gene promoter. Furthermore, TSA did not induce promoter activity in a TCF isoform-dependent manner. In conclusion, my results suggest that variations in C-terminal regions of TCF family members determine their repressor or enhancer properties on Wnt target genes. TCF with short or * A manuscript based on the results of this chapter is in preparation.  169  incomplete C-termini repressed promoter activity and those TCF with long C-terminal structures were required for [3-catenin- and 13-catenin/p300-mediated Wnt target gene promoter induction. Furthermore, I showed that curcumin is a strong inhibitor of the [3-catenin/TCF-p300 mediated gene expression. In addition, the repressor functions of HDAC on Wnt-responsive genes are more likely dependent on their interaction with TCF partners or chromatin mediated modifications of TCF binding site than variations in TCF structures or E-tail domain.  V.2. INTRODUCTION AND RATIONALE  In the absence of Wnt ligand, GSK-313 promotes the phosphorylation of13-catenin, targeting it for degradation through the ubiquitin—ligase pathway (1). Canonical Wnt signaling can inhibit the activity of the so-called P-catenin destruction complex, a complex of proteins including APC, Axin, and GSK-313 that otherwise phosphorylates 13-catenin, thus preventing its destruction by the ubiquitin-proteasome pathway. These and other mechanisms allow Wnt ligands to both stabilize 13-catenin and promote its entry into the nucleus where it recruits transactivators to HMG box DNA binding proteins of the LEF/TCF family (2). In the nucleus, the transcription factor family of LEF/TCF proteins transmits Wnt signals by binding to 0-catenin and recruiting it to target genes for activation. The LEF/TCF family of transcription factors in mammals is comprised of four different proteins [reviewed in (3, 4)]. The founding family members, human and mouse LEF-1 and TCF-1, were first discovered in differentiating T and B lymphocytes (5-7). Two other mammalian LEF/TCF proteins (TCF-3 and TCF-4) were later identified in other tissues and their expression patterns differ (8). LEF/TCF proteins are expressed in many tissues during embryogenesis in overlapping, but distinct patterns  170  (8, 9). Extensive analyses of transcripts from TCF genes have revealed the presence of up to at least 17 exons which undergo extensive alternative splicing [reviewed in (3)]. These variations mainly affect the amino acid sequences located downstream of the HMG domain. Roughly, long (E-), medium- and short (B-) isoforms can be distinguished which differ with respect to the length of the C-terminal domains. TCF proteins are composed of an N-terminal domain of approximately 300 amino acids, an 80-amino acid HMG domain, an adjacent nuclear localization signal, and a C-terminal tail which is highly variable in length. The binding domain for 0-catenin is a short amino acid sequence motif close to the very N terminus. Several domains located at the very N terminus and within the central part of the N terminus contribute to context-dependent transcriptional activation by TCF proteins. The C-terminal tail regions of certain TCF isoforms (TCF-1E and TCF-4E) harbor an additional promoter-specific activation domain. Transcriptional repression mediated by TCF depends mainly on a region within the N-terminal half of the proteins. TCF-3 and TCF-4 variants with long C-terminal tails contain additional repressing elements composed of two short peptide motifs at their C-termini, so called CtBP. The inclusion or omission of certain amino acid motifs, such as the CRARF domain specifically present in the TCF-1E and TCF-4E isoforms or the CtBP-binding motifs found only in the TCF-3E- and TCF4E isoforms confer distinct functional properties to these variants. Two important activities have recently been ascribed to the 'E' tail of TCFs. One motif that is present in the E-tail of some TCF is recognized by the transcription co-repressor CtBP (10). A second motif in the E-tail but juxtaposed to the HMG DNA binding domain of TCF has been found to comprise a13-catenindependent transcription activation domain, a so called CRARF motif, which is highly conserved among all TCF orthologs (11, 12). TCF-1 does not have the CtBP binding elements in its E-tail, but it carries the potent CRARF motif for transcription activation. TCF-3 has CtBP binding  171  elements but is missing key residues of the CRARF motif; TCF-4 carries both CtBP binding elements and the CRARF activation domain. Finally, the LEF-1 locus cannot generate LEF-1 isoforms with an E-tail because the alternative exon needed in the splicing pattern is not present in the gene (13). While it thus remains largely unclear to what extent TCF proteins themselves are targets for acetylation in vertebrates, TCF nonetheless utilize acetylases and deacetylases to execute their regulatory functions in mammalian cells. A novel activation domain present specifically in the "E" isoforms of TCF-1 and TCF-4 has been shown to interact with p300 in vitro and in cellular extracts (11). It is likely that this interaction is important for the context-dependent activation of certain promoters by TCF-1 and TCF-4. The CBP and the related p300 are transcriptional co-activators which can modulate transcription processes by acetylating histones and nonhistone transcription factors (14). The functional consequences of these interactions appear to be vary between species (15-17) and perhaps also between TCF isoforms. The mechanism underlying p300 activity in this context is unkown but it could involve acetylation of TCF. In addition to this stimulatory interaction between TCF-4 and the p300 acetylase, TCF can also interact with a deacetylase to repress transcription. LEF-1 has been found in a complex with HDAC1 (18). The interaction may occur either directly or indirectly through Grg/TLE repressors which are known to bind HDAC1. Inhibition of HDAC1 by an inhibitor of HDAC TSA enhances Wnt/13-catenin target gene expression and alters the acetylation state near Wnt/13-catenin regulated promoters (18). This indicates that association of LEF-1 and HDAC1 contributes to the transcriptional repression of target genes in the absence of a Wnt stimulus through a chromatinbased mechanism.  172  Thus, I hypothesize that variation in structure and expression of different LEF/TCF isoforms and their capacity to interact with different co-activators and co-repressors results in differential regulation of Wnt-response genes. By transient transfections of a TCF luciferase reporter and a Wnt-regulated gene (19), VCN-632-Luc construct, with various TCF isoforms and TCF chimeric constructs in context of Wnt signaling activation, I have been able to show that TCF with short or incomplete C-termini repressed promoter activity while those with long C-terminal structures were required for 13-catenin- and 13-catenin/p300-mediated Wnt target gene promoter induction. Furthermore, I showed that curcumin is a strong inhibitor of the f3catenin/TCF-p300 mediated gene expression. In addition, the repressor functions of HDAC on Wnt-responsive genes are more likely dependent on their interaction with TCF partners or chromatin mediated modifications of TCF binding site than variations in TCF structures or E-tail domain.  V.3. EXPERIMENTAL PROCEDURES V.3.1. Cell culture and reagents — HEK293 cells were obtained from the American Type Culture Collection (Rockville, MD). HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum. For conditioned medium, control L cells and Wnt3a-secreting L cells obtained from the American Type Culture Collection (Rockville, MD) were cultured in DMEM containing 10% FBS as described (20). When cells were grown to 70-80% confluence, culture medium was changed to fresh medium and maintained for 24 h. The conditioned media were collected, stored at 4 °C, and used within 2 days. Alternatively, conditioned medium was stored at -80 °C; then thawed once, before refrigeration and use within  173  2 days. Curcumin was purchased from Acros Organics (Geel, Belgium). Histone deacetylase inhibitor TSA was purchased from Sigma. V.3.2. Plasmid constructs — The reporter plasmid used for transfections was the VCN632-Luc plasmid which contains the firefly luciferase gene under the transcriptional control of the -632 to +118 by fragment of the human versican promoter (19). 13-catenin/TCF luciferase reporter pTOPflashx8-Luc construct, which has eight tandem TCF binding sites, was used for assaying TCF transcriptional activity (a gift of R.T. Moon University of Washington, Seattle). TCF-4E, TCF-4AC, LEF-1 and LEF-1T4C were kindly provided by A. Hecht (Max-PlanckInstitute of Immunobiology, Freiburg). TCF-1E, TCF-1B and TCF-3E were supplied by M. Waterman (University of California, Irvine). A dominant stable 0-catenin construct was a kind gift of B. Gumbiner (Memorial Sloan-Kettering Cancer Center, New York). A p300 construct was a kind gift of D. Livingston (Dana-Farber Cancer Institute, Boston) via S. Grossman (University of Massachusetts Medical School, MA). V.3.3. Transfection, and luciferase activity assays — For the transfection studies cells were transiently transfected with the corresponding plasmid DNA as indicated in the legend to figures and in the text using FuGENE6 reagent (Roche Applied Science) according to the procedure recommended by the manufacturer. In brief, a ratio of 3:1 for FuGENE reagent (0):plasmid (pig) was incubated for 30 minutes at room temperature in incomplete medium before addition to 70-80% subconfluent cells in serum-free DMEM containing 0.2% BSA for the period of time indicated in the figure legends. After the indicated incubation period, cells were lysed, and luciferase activities were measured with a kit from Promega according to the manufacturer's protocol. Protein concentrations were measured with a Bradford protein assay kit (Bio-Rad), and luciferase values were normalized to the obtained protein concentrations. In some  174  transfection experiments, normalization was done with the LacZ reporter (Promega), and 0-gal assays were performed according to the manufacturer's protocol.  V.4. RESULTS LEF/TCF are sequence-specific DNA binding transcription factors that function in the Wnt signaling pathway by recruiting 13-catenin to Wnt target genes. 13-catenin has potent transcription activation domains at the N- and C-termini, but it has no intrinsic ability to bind to DNA, thus, it relies on interactions with DNA-binding factors to regulate gene expression. In vertebrate system, there are four family members: TCF-1, LEF-1, TCF-3 and TCF-4 (21-23). LEF/TCF are produced as a group of isoforms through alternative splicing and promoter usage, and Figure V-1 presents their basic domain structure and diverse isoforms. The N-terminal part contains a short amino acid stretch that mediates the interaction with 13-catenin and related proteins. The adjacent sections harbors binding sites for transcriptional coregulators, and has additional modulating effects on transcativation (24-28). This part abuts the HMG domain which is responsible for sequence-specific DNA binding of TCF (29). The C-terminal parts of TCF which are highly variable in length, provide contacts for coactivator proteins and again alter the transctivation properties of TCF (10-12, 30). The inclusion or omission of certain amino acid motifs, such as the CRARF domain specifically present in the TCF-1E and TCF-4E isoforms or the CtBP-binding motifs found only in the TCF-3E and TCF-4E isoforms confer distinct functional properties to these variants. Figure V-1 also shows the natural and chimeric LEF/TCF isoforms employed in this study.  175  V.4.1. /3-catenin and p300 are different in their ability to regulate synthetic TCF reporter (pTOPFLASHx8-Luc) and endogenous Wnt-responsive versican promoter gene — To see whether Wnt activation and p300 HAT cooperate to stimulate the activity of synthetic TCF luciferase reporter, HEK293 cell line was co-transfected with the pTOPFLASHx8-Luc and p300 expression plasmids as indicated in the Figure V-2A and described in the legend to figure. In order to activate Wnt signaling following overnight transfection, conditioned medium from negative control cells (without Wnt-3a) or medium containing Wnt-3a in the presence or absence of curcumin, a specific inhibitor of p300, were added over the 293 cells as indicated in the Figure V-2A. 293 cells were chosen because they express TCF factors as interaction partners for  0-  catenin (31) and their endogenous p300 is largely neutralized due to the presence of the adenovirus ElA oncoprotein (32). When cells were simultaneously co-transfected with TCF reporter and p300 and subsequently treated with Wnt-3a, reporter gene activity level was similar to the activity level of the cells treated only with Wnt-3a (Figure V-2A). In addition, expression of p300 had no effect on TCF reporter activity in the absence of Wnt-3a (Figure V-2A). Furthermore, curcumin significantly inhibited TCF reporter activity of cells treated both with Wnt-3a alone or simultaneous with Wnt-3a and co-transfection with p300 expression plasmid. These observations demonstrate that Wnt-3a is necessary and sufficient to induce a synthetic TCF reporter. In contrast, p300 neither induces TCF reporter activity nor enhances Wnt-3a mediated TCF reporter function. Furthermore, curcumin significantly inhibited Wnt-3a-mediated activity of TCF reporter. To test whether P-catenin cooperates with p300 in stimulating the endogenous Wntresponse genes, 293 cells were transfected with combinations of expression vectors for P-catenin, p300, and endogenous Wnt-responsive versican promoter gene (19) as indicated in Figure V-2B.  176  Following overnight transfection, medium was replaced by medium without or with curcumin as indicated in the figure. Without co-transfection of p300, 0-catenin stimulated versican promoter only 6-fold in 293 cells (Figure V-2B). However, in contrast to TCF reporter where, without cotransfection of 13-catenin, p300 alone did not enhance luciferase activity (Figure V-2A), expression of p300 alone increased versican promoter activity about 18-fold. Furthermore curcumin significantly inhibited both basal and13-catenin/p300-induced activities of Wnt responsive versican promoter gene. These observations suggest that (1) there are differences between synthetic TCF reporter and Wnt-target promoter gene in response to (3-catenin and p300, (2) activation of Wnt signaling by Wnt-3a is necessary and sufficient for significant stimulation of synthetic TCF reporter activity, (3) p300 alone or combination of p300 and Wnt-3a does not stimulate luciferase activity of TCF reporter, (4) the stimulatory function of 0-catenin and p300 on endogenous Wnt-response gene is promoter-specific, (5) 13-catenin and p300 govern an independent stimulatory function on transcription of the Wnt-response gene versican promoter, and (6) curcumin, an specific inhibitor of p300 HAT, significantly inhibited both basal and 13-catenin/p300-induced promoter activity of synthetic TCF reporter and Wnt-responsive versican gene promoter. V.4.2. TCF isoforms with various C-terminal lengths differ in their ability to support Wnt//3-catenin/p300-mediated activation of the Wnt-target promoters — The CBP and the related p300 are transcriptional co-activators which can modulate transcription processes by acetylating histones and nonhistone transcription factors (14). It appears that p300 and CBP are among the interaction partners of TCF proteins, but binding of TCF to p300/CBP may be mediated by multiple TCF domains. Moreover, the functional consequences of these interactions appear to vary between species (15-17) and, perhaps, also between TCF isoforms. To test  177  whether downstream components of the Wnt/f3-catenin signal transduction pathway with distinct structural domains contribute to the differential regulation of Wnt target genes, I asked whether different TCF isoforms were equally capable of supporting activation of Wnt-regulated gene promoters by 13-catenin and p300. First, 293 cell line was co-transfected with the pTOPFLASHx8-Luc, TCF isoforms, and p300 expression plasmids as indicated in Figure V-3. Following overnight transfection, DNA mixtures were removed and medium replaced by medium containing Wnt-3a without or with curcumin as indicated in the Figure V-3. TCF isoforms containing long C-terminal structures, TCF-4E, TCF-1E, and chimeric construct LEF1T4C showed the highest 13-catenin-mediated luciferase activity compared to TCF isoforms lacking C-terminal structures (TCF-40C, TCF-1B, and LEF-1). However, TCF-3E, which includes an imperfect C-terminal domain, showed the lowest 13-catenin-mediated luciferase activity. Furthermore, co-transfection of p300 and TCF isoforms containing C-terminal structure showed higher levels of luciferase induction compared to TCF isoforms with short C-terminal tails. However, LEF-1, which lacks a C-terminal structure, contain a context dependent activation domain, showed a prominent response to p300. In addition, curcumin inhibited Wnt3a/TCF/p300-mediated luciferase activities to similar extent for all TCF isoforms. In previous studies, I found specific binding of human TCF-4 and 13-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter (chapter IV) (19). In addition to binding assays, I directly showed the dependence of versican promoter activity on TCF binding sites. Thus, I tested whether various TCF and chimeric constructs with different C-terminal lengths differentially regulate versican promoter and further identifies the influence off3-catenin and p300 on specific TCF isoforms mediating Wnt target gene versican promoter activities. 293 cells were co-transfected with a human VCN-632-Luc promoter  178  containing TCF binding sites, natural and chimeric TCF vectors, and a p-catenin expression plasmid in the presence and absence of curcumin as indicated in Figure V-4. In contrast to pTOPFLASHx8-Luc reporter construct, VCN-632-Luc reporter construct showed similar luciferase induction in response to P-catenin for all TCF isoforms, but LEF-1 showed the highest luciferase induction. Furthermore, p300 expression plasmid significantly induced I3-cateninmediated versican promoter activity when co-transfected with TCF with long C-terminal structures (TCF-4E, TCF-1E, and LEF-1T4C) compared to those with short or imperfect Cterminal structures (TCF-40C, TCF-1B, TCF-3E and LEF-1). As mentioned above, curcumin significantly inhibited Wnt-3a/TCF mediated luciferase activity of a synthetic TCF reporter and this effect was not dependent on TCF isoforms. However, curcumin inhibited 13-catenin/TCFinduced versican promoter activity to about 50% for all isoforms of TCF (Figure V-4). On the other hand, similar to its inhibitory function observed on synthetic TCF reporter the inhibition of curcumin on 0-catenin/TCF/p300-induced versican promoter luciferase activity was not dependent on TCF isoforms. Altogether, these results suggest that p300 utilizes the E-tail domain of TCF isoforms to optimally induce luciferase activity of a synthetic TCF reporter and a Wnt responsive versican promoter gene. Furthermore, the effect of curcumin on Wnt-response genes such as versican differs in its inhibitory action observed on synthetic TCF reporter construct. These finding might suggest that curcumin has differential effects on Wnt-response gene promoters based on the promoter structure and associate chromatin neighborhood. V.4.3. Histone deacetylase inhibitor TSA potentiates the induction of Wnt-response genes in a Wnt-dependent but not p300-independent manner — Several authors linked the acetylation of the 13-catenin with its transcriptional activation (33, 34). Therefore, I investigated  179  the effects of inhibition of HDAC using an inhibitor of HDAC, TSA, on f3-catenin- and p300induced Wnt-response target gene activity. To see whether activation of Wnt signaling by Wnt3a, p300, and HDAC inhibitor TSA cooperates to stimulate the activity of synthetic TCF luciferase reporter, 293 cell line was transfected with the pTOPFLASHx8-Luc with or without p300 expression plasmid as indicated in Figure V-5A. Following overnight transfection, DNA mixtures were removed and medium replaced by serum-free medium or medium containing Wnt-3a with or without 300 nM TSA as indicated in the Figure V-5A. TSA significantly induced pTOPFLASHx8-Luc reporter activity when cells treated with Wnt-3a compared to cells treated with serum free medium. In contrast, TSA in the absence of Wnt-3a stimulation did not induced reporter activity of the cells transfected with p300 HAT expression plasmid. Furthermore, TSA treatment of the cells either transfected with p300 plasmid or stimulated by Wnt-3a did not enhance luciferase reporter activity beyond the luciferase activity resulted from cells treated only with Wnt-3a. My results demonstrate that TSA-induced activation of TCF luciferase reporter requires the activation of Wnt signaling by Wnt-3a. Furthermore, these results revealed that in the presence or absence of Wnt-3a stimulation, TSA does not modulate p300-induced activity of TCF reporter luciferase. To test whether TSA cooperates with f3-catenin and p300 in stimulating the Wntresponsive versican promoter, 293 cells were co-transfected with combinations of expression vectors for13-catenin and p300 and the VCN-632-Luc, as indicated in the Figure V-5B. Following overnight transfection, DNA mixtures were removed and replaced by medium without or with 300 nM TSA as indicated in Figure V-5B. Interestingly, similar to results from the TCF reporter plasmid (described above), TSA stimulation in the absence of Wnt signaling activation (co-transfection with 0-catenin) did not significantly altered versican luciferase activity. In  180  contrast, TSA significantly enhanced versican promoter activity in the presence of cotransfection with 13-catenin expression plasmid. Furthermore, TSA did not alter p300-mediated luciferase activity. However, in contrast to the TCF reporter, TSA significantly enhanced  0-  catenin/p300-induced versican luciferase activity compared to cells were not treated with TSA. Altogether these results suggest that TSA requires activation of Wnt signaling to induce TCF reporter and the endogenous Wnt-response versican promoter. In contrast, TSA in the absence of Wnt signaling activation is not capable of enhancing p300-mediated induction of either Wnt targets, the TCF reporter and the versican gene promoter. However, TSA differentially induced TCF reporter and Wnt-responsive versican promoter in presence of cotransfection with p300 and Wnt signaling activation. These results suggest that TSA has differential effects on synthetic TCF reporter and endogenous Wnt-response gene promoters emphasizing the roles of promoter-specific structures and associate chromatin neighborhood in response to external stimuli. V.4.4 HDAC inhibitor, TSA, induces Wnt-responsive versican gene in a TCF isoform-independent manner — LEF/TCF transcription factors are known to act in complex with HDAC as transcriptional inhibitors (18, 30). In order to better appreciate the functional importance of the interaction among HDACs, TCF isoforms, and p300 in the context of activated Wnt signaling, I investigated the effects of inhibition of HDACs, TSA, on synthetic TCF reporter and Wnt-target versican promoter. To investigate the interaction among HDAC inhibitor TSA, TCF isoforms, P-catenin and p300 on an endogenous Wnt target versican promoter, 293 cells were transfected with combinations of expression vectors for the TCF isoforms, 0-catenin, p300, and the VCN-632Luc as indicated in Figure V-6. Following overnight transfection, DNA mixtures were removed  181  and replaced by medium without or with 300 nM TSA. The results revealed that TSA alone induced (3-catenin/TCF mediated versican luciferase activity to a similar extent for all TCF isoforms. Furthermore, TSA did not modulate (3-catenin/TCF/p300 mediated versican promoter activity. Altogether these results suggest the notion that TCF isoforms with distinct structural domains does not reveal differential effects on TSA-induced versican promoter activity in context of activation of Wnt signaling. Furthermore, TSA does not modulate [3-catenin/p300 mediated regulation of versican promoter function specific to any of TCF isoforms used in these studies.  V.5. DISCUSSION Structural and functional diversity of TCF/LEF transcription factors — The culprits of the canonical Wnt signaling activities are heterodimeric transcription factor complexes formed by a transactivating component, 13-catenin and TCF family of proteins. Often, TCF are simply perceived as carriers for 13-catenin, but actually they carry out more complex functions in gene regulation. T-cell factor family members act in a cell-context and promoter-specific manner in which they fulfill partially redundant but also specific tasks (11, 12, 35, 36). In addition, they are part of both positive and negative regulatory feedback loops which dampen and enhance Wnt signals (25, 37, 38). LEF/TCF transcription factors mediate signaling from Wnt proteins by recruiting 13catenin as a transcriptional co-activator (39-41). However, studies of Drosophila, Xenopus, C. elegans, and more recently in mammals have indicated that TCF factors may also be transcriptional repressors (42-46). At a subset of Wnt-regulated promoters, the CRARF-domain specifically present at the C termini of TCF-1E and TCF-4E additionally functions as obligatory  182  but context-dependent transcriptional activation domain (Figure V-1) (11, 12). In mammals, the CRARF domain allows selective activation of the 13-catenin/TCF-responsive LEF-1 promoter by TCF-1E and the Wnt-responsive Cdxl promoter by TCF-4E (11, 12). A further contribution to promoter activation can be made by amino acid sequences encode by an alternatively spliced exon upstream of the HMG domain (26, 28). The N-terminal half of LEF-1 harbors yet another transactivation domain, which functions specifically in the context of the TCRa enhancer but not at Wnt-inducible promoters (47, 48). A similar scenario is probably true for the repression domains in TCF which were mapped to a region upstream of the HMG domain and to two short sequence motifs at the C termini of the TCF-3E and TCF-4E isoforms (Figure V-1). While the former interacts with Grg/TLE corepressors (24, 25, 27), the latter interacts with CtBP (10, 30). Which activation or repression domains are used is probably determined by the exact promoter architecture and the molecular framework provided by the various transcription factors and their cofactors which assemble into higher order structures at given regulatory elements. Interaction of/3-catenin and p300 in regulating Wnt target genes — In vertebrates, (catenin acts as transcriptional activator, which is needed to overcome target gene repression by Grg/TLE proteins and to permit promoter activation as the final consequence of Wnt signaling. However, the molecular mechanisms of transcriptional activation by f3-catenin are only poorly understood. By interacting with TCF factors 0-catenin may alter the promoter architecture and displace Grg/TLE or CtBP corepressors (10, 25, 43, 49-51). In addition, there is a strict correlation between the ability of fl-catenin to function in Wnt signaling and its ability to transactivate (52, 53), suggesting that 0-catenin facilitates additional steps during promoter activation. In support of this, 0-catenin possesses multiple transactivating elements at its N- and C-termini, which can operate independently from TCF, and 13-catenin can bind to the TATA box  183  binding protein (TBP) and to RuvBL1/Pontin52, which also interacts with TBP and RNA polymerase II (48, 52, 54-56). However, these interactions are not be sufficient to explain entirely how 13-catenin functions as a transcriptional activator (56, 57). It has been demonstrated that acetyltransferases p300 and CBP potentiate 0-catenin-mediated activation of the Wnt target genes (17). Further, the C-terminus off3-catenin interacts directly with p300 (17). In functional tests, however, several short fragments of the P-catenin C-terminus that do not appear to interact with p300 behave as potent transactivating elements (48, 52, 56). These transactivators also function in yeast cells (56), which possess neither p300 nor CBP. Additional, as yet unidentified, coactivators must act via these regions offi-catenin to enable p300-independent transcriptional stimulation of different promoters. In vertebrates, p300 and CBP are essential coactivators off3catenin which are necessary for the activation of Wnt targets (16, 17, 58-60). The ability to interact with and recruit different coactivators may be a necessity to enable 13-catenin to function in distinct promoter settings and to facilitate discrete steps during the transcription initiation process. Here I have provided further evidence for p300 as an important coactivator of 13-catenin. (i) Co-expression of p300 stimulates 0-catenin-dependent and -independent activation of specific human Wnt-target versican promoter in HEK293 cells (Figure V-2B). It has been reported that activation of the cyclin D1 promoter, a Wnt target gene, by 13-catenin is refractory to p300 stimulation (17). These observations suggest that 13-catenin and its co-activator p300 function in a promoter-specific manner. (ii) I also showed that curcumin, which is a known inhibitor of p300, diminishes activation of f3-catenin-regulated promoters. It has been demonstrated that curcumin and its derivative are excellent inhibitors of (3-catenin/TCF signaling in several cancer cell lines and the reduced 13-catenin/TCF transcriptional activity are due to decreased nuclear  184  p-  catenin and TCF-4, diminished association of f3-catenin with TCF-4 and to the reduced binding of TCF-4 to the consensus DNA (61). However my data showed that curcumin inhibits Wnt target gene promoter activity both in presence and absence of (3-catenin/p300 (Figure V-2A and 2B). These observations suggest that curcumin might have transcriptional inhibitory functions both through 13-catenin/p300 dependent and independent mechanisms. (iii) It has been reported that P-catenin and p300 synergize to stimulate a synthetic TCF reporter gene construct (17), whereas my results showed that p300 alone and in combination with one of the potent Wnt ligands, Wnt-3a, was not able to increase 13-catenin-mediated activity of a synthetic TCF reporter (Figure V-2A). However, Wnt-3a-mediated induction of the TCF reporter was inhibited by curcumin (Figure V-2A). These observations provide good evidence that f3-catenin, at least in part, utilizes p300 as coactivator to perform its function in Wnt signaling. As well curcumin inhibits basal versican promoter activity in addition to 13-catenin- and 0-catenin/p300-mediated induction of Wnt target genes. TCF isoforms differentially regulate 13-catenin/p300-mediated activation of the Wntresponse promoters — Recent reports revealed functional differences between TCF family members and between isoforms derived from the same TCF gene. For example, LEF-1 appears to mainly act as a 0-catenin-dependent transcriptional activator, whereas the repressor activities of TCF-3 prevail over its activating function (62-64). It has been reported that LEF-1 and TCF-4 have different transactivation capacities, although both factors contain the activating N-terminal exon. Additionally, whereas both LEF-1 and TCF-4 can act as activators, they appear to have distinct spectra of target genes, and a novel, promoter-specific CRARF transcriptional activation domain was identified in the TCF-4E variant (11, 12).  185  Two important activities have recently been ascribed to the 'E' tail of TCFs. One motif that is present in the E-tail of some TCF is recognized by the transcription co-repressor CtBP (10). Second motifs at region of the E-tail juxtaposed to the HMG DNA binding domain of TCF has been found to comprise a P-catenin-dependent transcription activation domain, so called CRARF motif, is highly conserved among all TCF orthologs (11, 12). CRARF, which is present specifically in the "E" isoforms of TCF-1 and TCF-4, has been shown to interact with p300 in vitro and in cellular extracts (11). It is likely that this interaction is important for the contextdependent activation of certain promoters by TCF-1 and TCF-4. The mechanism underlying p300 activity in this context is unkown but it could involve acetylation of TCFs. This is suggested by the observation that in TCF-4 each of the two binding sites for the transcriptional repressor CtBP is flanked by a lysine residue (65). In the case of E1A, which also binds both p300 and CtBP, a similar configuration near the CtBP-binding site is reversibly acetylated by p300/CBP and this acetylation modulates the interaction between E1A and CtBP. By extrapolation, acetylation of the C terminus of certain TCF splice variants might be required for promoter activation by weakening the interaction with the CtBP repressor proteins. This raises the possibility that regions outside of the HMG domain contribute to target gene recognition by TCF proteins. Although LEF-1 and TCF-4E recognized the same DNA sequences, their affinities for single or multimerized recognition motifs varied considerably. Pukrop et al. (28) also observed 37-fold differences in affinity for single TCF binding elements between LEF-1 and other TCF family members. These differences are likely to be of importance because TCF proteins are not only mediators of Wnt signaling but also contribute to Wnt/13-catenin-independent gene regulation, for example at the TCR enhancer or at the HIV-1 promoter (6, 66). How can inappropriate  186  activation of these regulatory elements by 13-catenin-TCF complexes be prevented? Perhaps significantly, both the TCR enhancer and the HIV-1 promoter contain single TCF binding motifs (6, 66), whereas the known Wnt target genes typically contain multiple TCF recognition elements. Under competitive conditions, when TCF levels are low, or when only a particular TCF family member is expressed in a cell, the differential recognition and occupancy of single versus multiple binding sites could be one way to distinguish non Wnt target genes from Wnt target genes. The CBP and the related p300 are bimodal transcriptional coactivators which can modulate transcription processes by acetylating histones and nonhistone transcription factors or by bridging transcriptional activators to components of the basal transcription machinery (14). In D. melanogaster, dCBP has been reported to be a negative regulator of Wnt signaling, whereas in vertebrates, p300 and CBP are essential coactivators of f3-catenin which are necessary for the activation of Wnt targets (16, 17, 58-60). p300 was shown to interact with a region containing the CRARF domain at the C terminus of the TCF-4E isoform but acetylation of TCF-4E was not investigated (11). Whether other TCF family members also interact with CBP or p300 and which domains in TCF are capable of interacting with p300/CBP is not yet fully investigated. Nonetheless, it appears that p300 and CBP are among the interaction partners of TCF proteins, but binding of TCF to p300/CBP may be mediated by multiple TCF domains. I investigated the role of the interaction of TCF isoforms withr3-catenin/p300 complex on both the synthetic TCF reporter and Wnt responsive versican promoter (Figure V-3 and 32). My results demonstrated that TCF isoforms containing long C-terminal structures, TCF-4E, TCF-1E, and chimeric construct LEF-1T4C conveyed the highest luciferase activity of TCF reporter in the context of both 13-catenin- and ii-catenin/p300 complex compared to TCF isoforms lacking Cterminal structures (TCF-4AC, TCF-1B, and LEF-1) (Figure V-3). However, TCF-3E which  187  includes an imperfect C-terminal domain (lacks CRARF domain) showed the lowest P-cateninmediated luciferase activity of TCF reporter (Figure V-3). These results further emphasize on the importance of complete E-tail domain of TCF isoforms on optimal Wnt mediated regulation of target genes. However, LEF-1, regardless of lacking C-terminal structure but containing a context dependent activation domain, showed both P-catenin- and a P-catenin/p300-mediated response (Figure V-3). These results suggest that both P-catenin and p300 utilize the E-tail domain of TCF isoforms to optimally induce luciferase activity of a synthetic TCF reporter. Furthermore, specific structures, such as the context dependent activation domain in LEF-1, are also important for 0catenin and P-catenin/p300 mediated responses of Wnt target genes. In contrast to the TCF reporter construct, the VCN-632-Luc reporter construct showed similar luciferase induction in response to P-catenin for all TCF isoforms, but LEF-1 showed the highest basal luciferase induction (Figure V-4). However, expression of p300 significantly induced P-catenin-mediated versican promoter activity similar to the luciferase activity observed using the TCF reporter, when co-transfected with TCF with long C-terminal structures (TCF-4E, TCF-1E, and LEF-1T4C) compared to those with short or imperfect C-terminal structures (TCF-4AC, TCF1B, TCF-3E and LEF-1). These results suggest the importance of promoter-specific sequences in P-catenin/TCF/p300 mediated transcriptional activation. Furthermore, my results reveal that the transcriptional activity observed by tandem recognition motifs in TCF reporters do not exactly mirror the activities of an endogenous Wnt target gene, at least in the case of the versican promoter. Although differences in DNA binding may contribute to the differential activation of the versican promoter by TCF isoforms, I believe that the C-terminal activation domain in TCF-4 and TCF-1 isoforms performs additional functions. TCF-4AC, TCF-1B and TCF-3 did not activate the  188  TCF reporter, however they were able to activate Wnt responsive versican promoter, similar to those of TCF isoforms with complete E-tail domains (Figure V-3 and V-4). Thus, an influence on promoter occupation or topology is likely to be one of the mechanisms for differential activation of the TCF reporter and versican promoter by TCF with and without E-tail domains. An additional mode of action could be that the E-tail domain mediates protein-protein interactions. Indeed, it interacts with p300 in vitro, and p300 co-immunoprecipitated together with TCF-4 from cellular lysates (11). Although p300 may not be the only factor that interacts with the E-tail domain, the match between the ability to form a complex with p300 and the ability to support activation of the TCF reporter and versican promoter strongly suggests that the interaction between TCF-4E and p300 is of physiological relevance. Preliminary results indicate that TCF isoforms which include the E-tail domain can cooperate with f3-catenin and p300 to activate the versican promoter. Temporal and tissue-specific inducibility of the versican gene by Wnt signaling thus may in part be restricted through the availability of an appropriate TCF isoform. More likely, mechanisms that govern tissue-specific Wnt target gene regulation act at the level of individual regulatory elements and their associated transcription factors. The inhibitory mechanism of curcumin against /i-catenin/TCF signaling — Several histone deacetylase inhibitors have been reported and used as antineoplastic drugs [reviewed in (67, 68)]. However, very few inhibitors of HAT are known so far. Two specific HAT inhibitors, Lys-CoA for p300 and H3-CoA-20 for PCAF (69), have been synthesized and tested. Although Lys-CoA has been extensively employed for in vitro transcription studies, cells are not permeable to it (70). Recently, two natural compounds with HAT inhibitor activity, anacardic acid, from cashew nut shell liquid and garcinol from Garcinia indica, were isolated which are nonspecific  189  inhibitors of p300/CBP and PCAF but are capable of easily permeating the cells in culture (71, 72). In a recent study, Balasubramanyam et al. (73) reported that curcumin (diferuloylmethane), a major curcumanoid in the spice turmeric, is a specific inhibitor of the p300/CBP HAT activity but not of p300/CBP-associated factor, in vitro and in vivo. Furthermore, curcumin could also inhibit the p300-mediated acetylation of p53 in vivo. It specifically represses the p300/CBP HAT activitydependent transcriptional activation from chromatin as well. Here, I present three important findings relevant to the roles and mechanisms of curcumin mediated inhibition of Wnt/13-catenin signaling pathway. First, curcumin is a strong inhibitor of the [3-catenin/TCF signaling. Second, the inhibitory effects of curcumin on Wnt signaling are not specific to TCF isoforms. Third, the extent of the curcumin mediated inhibition of Wnt signaling differs between presence and absence of cotransfection with p300. Bordonaro et al. (74) claimed that curcumin does not inhibit the 0-catenin/TCF transcriptional activity however, Jaiswal et al. (75) suggested that curcumin downregulates13-catenin's transcriptional activity in HCT116 intestine cancer cells. Park et al. (61) tested several cancer cell lines, namely gastric, intestine and colon cancer with curcumin. In all cell lines tested, 13-catenin's transcriptional activity was suppressed by inhibitors dependent on the concentration. My data also demonstrate that curcumin significantly inhibited Wnt-3a/TCF mediated luciferase activity of a synthetic TCF reporter and Wnt target versican promoter and this effect was not dependent on TCF isoforms (Figure V-2 to V-4). However, the inhibitory function of curcumin on the TCF reporter was stronger than its inhibition on versican promoter activity. These finding might suggest that curcumin has differential effects on Wnt-response gene promoters based on the promoter structure and associate chromatin neighborhood. My results strengthen the conclusion that curcumin is effective in inhibiting 13-catenin/TCF signaling, in agreement with the findings of  190  Jaiswal et al. (75) and Park et al. (61). It has been suggested that the inhibitory mechanism of curcumin is related to the reduced amount of P-catenin and TCF-4 products in the nucleus (61). Jaiswal et al. (75) reported that curcumin suppressed the13-catenin/TCF activity but they only investigated the consequence of diminished p-cateniniTCF transcriptional activity induced by curcumin, not how curcumin conferred an inhibitory effect on cancer cells. In order to ascertain the relevance to APC—Axin—GSK-3P complex, I tested HEK293 transiently transfected with a constitutively active mutant of P-catenin gene. Curcumin suppressed the P-catenin/TCF signaling and this indicates that the mechanism of suppression is not related to the P-catenin degrading machinery, but to P-catenin itself or to the downstream components of f3-catenin in this signaling (Figure V-2). Furthermore, Park et al. (61) used EMSA and showed that the binding of TCF complexes to DNA is severely impaired by curcumin. In addition, coimmunoprecipitation of 0catenin and TCF-4 using TCF-4 antibody shows that the association of P-catenin with TCF-4 is blocked by curcumin (61). From these data, I conclude that the reduced binding to DNA and the reduced association of f3-catenin with TCF-4 caused by curcumin leads to the inhibition ofPcatenin/TCF signaling. To investigate the cause of the suppressed binding to the TCF-binding element and the decreased association of (3-catenin with TCF-4, Park et al. (61) determined the amount of P-catenin and TCF-4 in treated cells. P-catenin is distributed widely in a cell. It is located at the membrane with a cell—cell adhesion function and is also located in the nucleus functioning as a transcriptional activator. These investigators showed that the amount of Pcatenin protein in the cytosol and membrane fraction was not altered; however, the amount ofPcatenin in the nucleus is decreased markedly by curcumin. This finding is very important for 13catenin must be in the nucleus to associate with TCF-4 to upregulate the P-catenin/TCF signaling.  191  Furthermore, I showed that curcumin differentially inhibits transcription of Wnt target genes in the absence and presence of transfected p300. Curcumin strongly inhibited luciferase activity of TCF reporter in cells co-transfected with different TCF isoforms followed by Wnt-3a treatment, however in the presence of p300 co-transfection, curcumin only neutralized the effect of p300-mediated induction (Figure V-3). A similar scenario was true for Wnt-target versican promoter (Figure V-4). These results suggest the possibility that p300 mediates modifications in the chromatin neighboring, TCF sequence binding sites, or TCF proteins to elicit the differential response of Wnt target genes to curcumin in presence of p300 HAT. In fact, it has been recently demonstrated that curcumin promoted proteasome-dependent degradation of p300 and blocked histone hyperacetylation in both PC3-M prostate cancer cells and peripheral blood lymphocytes (76). In this regard, I assume that the inhibitory activity of curcumin on Wnt/13-catenin signaling occurs via several avenues including the reduction of nuclear 13-catenin/TCF proteins and inhibition of p300 HAT through its direct degradation or blockage of its HAT activity. Activation of Wnt signaling is required for HDAC inhibitor-mediated induction of Wnt target genes — Inhibitors of HDAC (HDACis) are promising agents for cancer [reviewed in (67, 68)]. These agents preferentially induce growth arrest, differentiation, and apoptosis in malignant, but not normal, cells (67, 68). Several HDACis are currently in clinical trials, and, recently, the U.S. Food and Drug Administration gave approval for the HDACi vorinostat (SAHA) to be used in the treatment of cutaneous T-cell lymphoma. Thus, knowledge of how these agents express their antineoplastic properties is important. The major activity of HDACis is believed to involve inhibition of HDAC, resulting in modified chromatin assembly and altered  192  gene expression (67, 68); however, an increasing body of evidence suggests that non-histone proteins are essential mediators of HDACi function (77). It has been established that HDACis such as sodium butyrate and TSA modulate Wnt transcriptional activity in human colorectal carcinoma cells (74, 78). The constitutive activation of canonical Wnt signaling due to mutations in APC (79) and 13-catenin (80) is believed to promote cell proliferation and tumorigenesis in the colon. However, several other research groups have reported that relatively high levels of Wnt signaling result in apoptosis (81, 82). Based upon these results (81, 82), it is hypothesized that the relative levels of Wnt signaling determine whether cells proliferate or commit to undergo apoptosis. These observations and the findings that Wnt signaling is modulated by HDACis suggest that the reason HDACis induce reversible growth arrest or apoptosis in different cell types is at least partially determined by the levels of induced Wnt signaling. It has been recently reported that HDACis influenced the physiology of cells that do not carry Wnt activating mutations to a lesser extent; however, in cells with a deregulated Wnt pathway, HDACis induce higher levels of Wnt which lead to apoptosis. Bordonaro et al. (83) recently reported that the HDACi, sodium butyrate, enhanced Wnt signaling in colorectal carcinoma cells and apoptosis and a mechanism involved in this effect is an increase in S er37/Thr-41-dephosphorylated 13-catenin initiated at the ligand level. The findings suggest that nonhistone targets of HDACis likely mediate the effects of these agents on Wnt signaling and apoptosis. My results that TSA was able to enhance Wnt target gene promoter activity only in presence of Wnt signaling activation strongly supports the notion that at least in part 0-catenin or other Wnt signaling components are required for HDACis-mediated induction of Wnt target genes (Figure V-5A and V-5B). In contrast, TSA is not capable of inducing p300-mediated Wnt targets,  193  TCF reporter and versican gene promoter, in absence of Wnt signaling activation (Figure V-5A and V-5B). This finding also further emphasizes the critical role of Wnt signaling, likely via augmenting the amount of available nuclear 0-catenin in the modulation of this signaling by HDACi, TSA. TCF can also interact with deacetylases to repress transcription. LEF-1 has been found in a complex with HDAX1 (18). The interaction may occur either directly or indirectly through Grg/TLE repressors which are known to bind HDAC 1. Inhibition of HDAC 1 by TSA enhances Wnt/13-catenin target gene expression and alters the acetylation state near Wnt/f3-catenin regulated promoters (18). This indicates that the association of LEF-1 with HDAC1 contributes to the transcriptional repression of target genes in the absence of a Wnt stimulus through a chromatinbased mechanism. However, chromatin may not be the only target of LEF-1-associated HDAC1. In a recent report, HDAC 1 overexpression counteracted LEF-1-dependent nuclear accumulation and retention of f3-catenin (84). In addition, it has been recently demonstrated that 13-catenin interacts with HDAC 1 in a LEF-1-dependent fashion and that the enzymatic activity off3-catenin bound HDAC1 is reduced compared to that of unbound HDAC1 (18). It has been proposed that transcriptional activation by LEF-1-13-catenin involves the attenuation of HDAC activity and dissociation of HDAC1 from LEF-1 by f3-catenin. Thus, evidences suggest that TCF might use deacetylases to execute their inhibitory functions in mammalian cells. The novel domain present specifically in the "E" isoforms of TCF-3 and TCF-4 has been shown to interact with a repressor, CtBP. In fact, an interaction between the C-terminus of the TCF-4 protein and the CtBP1 transcriptional co-repressor has been recently shown (30). These investigators demonstrated that CtBP 1 represses the transcriptional activity of a f3-catenin/TCF-dependent synthetic promoter as well as the endogenous Wnt target, Axin2/Conductin, in a HDAC dependent manner (30). My  194  results demonstrated that in context of activation of Wnt signaling and presence of different TCF isoforms TSA does not reveal differential effects on versican promoter activity.  V.6. CONCLUSIONS I investigated whether TCF members differentially regulate Wnt target genes via variations in their C-terminal structure that enable interactions with different co-regulators and their post-translational modifications. Here, I showed that TCF isoforms which include the E-tail domain strongly enhance 13-catenin/TCF- and 13-catenin/TCF-p300-mediated transcriptional activity of TCF reporter and Wnt target versican promoter. Furthermore, curcumin, a specific inhibitor of p300 HAT, significantly inhibited both p-catenin- and 0-catenin/p300-induced Wnt signaling mediated activity of Wnt target genes irrespective of TCF isoforms. I also demonstrated that nuclear 0-catenin or activation of Wnt signaling is required for the induction of Wnt-response gene by HDAC inhibitor, TSA; however HDAC inhibitor did not induce promoter activity in a TCF isoform-dependent manner. These data might support the notion that the repressor functions of HDAC on Wnt-responsive genes are more likely dependent on their interaction with TCF partners or chromatin mediated modifications of TCF binding site than variations in TCF structures or E-tail domain. The inclusion or omission of certain amino acid motifs, such as CRARF domain specifically present in the TCF-1E and TCF-4E isoforms or the CtBP-binding motifs found only in the TCF-3E and TCF-4E isoforms confer distinct functional properties to these variants. In the context of Wnt signaling activation, all TCF that carry an E-tail (TCF-4E, TCF-1E, TCF-3E and chimeric LEF-1T4C) significantly induced Wnt target gene promoter activities. TCF with E-tails all contain the CRARF structural domain except TCF-3E, which lacks key residues of this  195  activation domain. Importantly, among TCF that carry an E-tail, TCF-3E demonstrated the least activation of the promoter in presence of13-catenin. These data support the idea that E-tail domain of TCF, likely through a complete CRARF structural motif, is important for 13-cateninJTCFmediated transcriptional activity of Wnt-responsive genes. My findings also suggest that variations in C-terminal tails of TCF might be mainly responsible for the bimodal functions of TCF on Wntresponsive genes. CtBP binds to a characteristic amino acid sequence motif `PLDLS' and closely related variants thereof. Two copies of this motif are present in TCF-3 (PLSLT and PLSLV) and TCF4 (PLSLS and PLSLV). Functional assays including my data indicated that TCF containing only CtBP motifs (TCF-3E) represses target genes even in presence of exogenous 13-catenin. In contrast, TCF-4E containing both CtBP and CRARF motifs enhances transcriptional activation of Wnt target genes in presence of Wnt signaling activation. The results that I report in this study support the intriguing possibility that not all LEF/TCF are functionally equivalent and that different isoforms may preferentially regulate different subsets of Wnt target genes. Such a possibility is consistent with genetic data from knockout and transgenic mice experiments in which the removal of the LEF-1 gene or TCF-1 gene generated partially non-redundant phenotypes in tissues where their expression patterns overlapped or where overexpression of dominant negative forms of LEF/TCF produced non-identical phenotypes (35, 63, 85). Whether a portion of these non-redundant activities is a result of the expression of E-tail isoforms and whether such specialized functions play a role in several disease conditions in which Wnt/f3-catenin signaling activity play critical roles are unknowns that are important to address if we are to understand the role that Wnt signals both in normal tissue function and in promoting diseases. Based on functional differences among TCF family members and TCF isoforms, the enormous structural diversity generated by alternative splicing, the largely  196  unexplored relevance of post-translational modifications and the still growing number of interaction partners, one can expect that more and more sophisticated scenarios of TCF function in gene regulation will emerge.  197  TCF-4E  I  TCF-4AC TCF-1E TCF-1B  •  TCF-3E LEF-1 LEF-1 T4C  13-catenin binding domain  I  l  i  HMG Box  CRARF domain  CtBP binding domain  NLS  Figure V-1. Transcriptional regulatory domains of LEF/TCF isoforms and schematic representation of natural and chimeric TCF isofroms employed in this study. TCF proteins are composed of an N-terminal domain of approximately 300 amino acids, an 80amino acid HMG domain (green bar) mediates sequence-specific DNA binding to the consensus recognition motif, an adjacent nuclear localization signal (NLS; black box), and a C-terminal tail which is highly variable in length. The binding domain for 13-catenin is a short amino acid sequence motif close to the very N terminus (blue. TCF-4E carries both CtBP binding elements and the CRARF activation domain, but TCF-4AC lacks E-tails including two of CtBP and CRARF domains. TCF-1E and TCF-1B are identical except for the Cterminal tail in which TCF-1B includes truncated C-termini. TCF-1E does not have the CtBP binding elements in its E-tail, but it carries the potent CRARF motif for transcription activation. Although TCF-3E carries an E-tail, the amino acid sequence is significantly divergent from TCF-1 and TCF-4. TCF-3E has CtBP binding elements but is missing key residues of the CRARF motif. Finally, the LEF-1 locus cannot generate LEF-1 isoforms with an E-tail because the alternative exon needed in the splicing pattern is not present in the gene. The chimeric LEF-1 T4C construct is the same as wild type LEF-1 but includes an additional E-tail from TCF-4E added to its C-terminus.  198  Figure V-2.  A.  25.0  20.0  E0 00' (—)  15.0  ci)  &19 ,4 C-) 7:3  a  10.0  O  f=1-'  5.0  0.0  Wnt-3a p300 Curcumin  B.  20.0 18.0 16.0  a)^14.0 ct  9^12.0  10.0 L.)c^ r) ,^8.0 C—)^6.0 4.0 2.0 0.0  13-catenin p300 — Curcumin  199  Figure V-2. 3-catenin and p300 differ in their ability to regulate pTOPFLASHx8-Luc reporter and endogenous Wnt-responsive versican promoter. A, Condition medium from fibroblast cells constitutively expressing Wnt-3a induce the activation of the TCF reporter in 293 cells and curcmin, specific inhibitor of p300 HAT, inhibits this induction. The HEK293 cell line was cotransfected with the TCF reporter construct (pTOPFLASHx8-Luc; containing 8 wild-type TCF-binding sites) and a p300 expression plasmid as indicated on the x-axis. Following overnight transfection, DNA mixtures were removed and condition medium from negative control cells (without Wnt-3a) or medium containing Wnt-3a were added over the 293 cells in the presence or absence of curcumin as indicated in the figure. Cultures were further grown for 24 h, then the cells were harvested together and processed to assay the reporter gene activities. All experiments were performed in triplicate and repeated at least two times. Average values and their standard errors from a representative experiment of three independent replicates are represented as fold change of the luciferase activity compared to basal luciferase activities. (B) 293 cells were transfected with combinations of expression vectors for 13-catenin and p300 and VCN-632-Luc as indicated. Following overnight transfection, DNA mixtures were removed and replaced by medium without or with curcumin as indicated in the figure. Cultures were further grown for 24 h, then the cells were harvested together and processed to assay the reporter gene activities as described above. * P < 0.05  200  0 0 0 -  0 0 0 •71  0 0 0 CV  0 0 0 0 -  1-1  C  0 0 0 0 0 0 0 0 0 0 0 00 00 CO^C \  /CTIApoy oni[ PHSVIAdOiLd  201  I O O  co  ^  I O O CO  ^  E L  O C O ^  •• •• • ••••▪ ••  ":, CD k nU )  7 7 1-)  cd '' o :. $ =^i.) ,...)^0 - A0 O ,i1 2 to _.- 7::i --- -  : : :,^  'CI a) b I ) V, Cll^cu  i■.I c'/ 0 t—,4 v.  • ,-.^• ^. °^;--,  f  cs) O2 ^c. 7c^ - cc1  ,:z O 9 -I Ti co % c4 S. O 0 . ti Cr' l 77j C6 0 p '-' c9  .-I.., •.c/) .1.M  ;--1  az^-0 0 a) at aj a) 0 1 (1  v. 1■•••■  `,--, --,' o .,-,^•,L, ' '''' = 7: '7: •7 c/D -I ^7Zi 7r) = C) . "" (1) 0 ••.*^C/) m t= 0 ;...,^,:. (14 cy c/D ,zt-' '-. 4-0 c....^ .... a.) I) 73 "" P 77) CI)^<—, ci ^'-,-, czt H r-r Ail 0^ i.: P. 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'-' • .--4 .r. ^,C1  0 ° bl H ;--. 0) ;....^;-.^to 0.4 0 ,z 4-^C-' .. q.1.1  o -,-, c E^cu e. ■ 0^'- .-CD 4 1-) 0n  0 I) .. > 4.81^;-■^c  ct s.I) cn a)  cn 0  ct^9.? a> 0^5 7:i ... .+.0 • 7)^,,, cf) -4,-,  i-■ 0  al a>  -  ,  ''=^:,-, a)^"-4-= . 1-, a> )a(1)^ cl--■^ cn^.... -4..1^  4-  cz;  0 a) 7-1 X, ti) ,..^  o^ bl) to  ,-.^,.....,^ U . ,--, ct (A-.^ ..^ ns ^tZ 0^-^ :a> c'.5 c.) -,-) ,_^ct  =  C <14  -  o bi) .,c.> ,-, ^a  ;.-.^a> rn rm.— 4) 0 -4.p ^t  .  l...) ■-•^. '-'^CD^;-■  •  0 7,  m Vat a) a> 7! 0 c.) ..-,_.0  co>^a, -T.-■^  .E, 0  .... ct -,-,^ c>) ctt c_) --: c>)^v., L)^Ct Ct cL) ' (i) Z LI-I WO Cn -tC4  a.4 czt ..-.^ ^,-1 ;..,^ s., -t:i^-ci a)  ..1^  ,-4^a)^Cip a> . -.4 .^g) ;--, CI 0 C/)^0  --,^P,  C3 C.) 0 1■,^ 0 =^C.) ,4 ;-4 1.4^CI)  s-, ^- 5 ,7 C1,4^C.) too 0 (}-I ® • m. m m CD mCt C.3 ^ •r:'' V) C-A 50 115  -  71  E 6 U, -_F_, -, = ,---. .^b1) e*-- • 5 0^.-Ci  4..1 l■  CD  . a) _ 2c'z I-T-1 c4^C» co) •,1 C) -..ci I.)  E-1 ct rn sm. P ct  0^,-.0 I..> 1 ^cz3 'CS ci,) C.)^00 > = • '''^0^> ■^ 0 / z tD1)^0^I)^e-, .--.^S.^=qj I) 4.4^00^u5.-, C.)  Figure V-5.  ^ No TSA ■ TSA 300nM  * 8000 • r•••1  •  7000  c.) 6000 oc) 4.4 C/) •-1 4-1  a,  5000 4000 3000  =1-1 2000  Wnt-3a p300  B. 50000  ^ No TSA ■ TSA 300nM -  45000 40000 ▪ C.)  -  35000 30000  -  25000 M  20000 15000  ▪  10000 5000 0 13-cat p300  203  Figure V-5. Histone deacetylase inhibitor ,TSA, potentiates the effect of Wnt- but not p300-mediated induction of Wnt-response genes. A, TSA potentiates Wnt-3a-induced pTOPFLASHx8-Luc reporter activity. The 293 cell line was transfected with the pTOPFLASHx8-Luc with or without p300 expression plasmid as indicated on the x-axis. Following overnight transfection, DNA mixtures were removed and medium replaced by serum-free medium or medium containing Wnt-3a with or without 300 nM TSA as indicated in the figure. Cultures were further grown for 24 h, then the cells were harvested and processed to assay the reporter gene activities. B, TSA requires 13-catenin-mediated induction of Wnt signaling to enhance Wnt-responsive versican promoter. 293 cells were cotransfected with combinations of expression vectors for 13-catenin and p300 and the VCN-632Luc as indicated in the figure. Following overnight transfection, DNA mixtures were removed and replaced by medium without or with 300 nM TSA as indicated. Cultures were further grown for 24 h, then the cells were harvested together and processed to assay the reporter gene activities. * P < 0.05  204  ^---■  00—  0^0^CD 0^CD^0 CD^0^CD CD^CD^CD CID^,1-^CN  AT/A-93V 0111 – Z£9 -- NIDA  205  •  •^ •• • •  a  75 P -s--^ -t+-4 '"" (-) 0 ct^a) czt A 0 b.!) ,..^I) con • '--'  •■■ Q,  .--■  r,.....•^ ) -7:^2 r.  ^\•  ao^ a) ;., 1-)^<-^ I^ a) - .- !DO 1^ • -0 ;., -45 crl 0 a) I) -i--. 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(2002) Apoptosis in neural crest cells by functional loss of APC tumor suppressor gene. Proc Natl Acad Sci USA, 99, 297-302.  212  83.  Bordonaro, M., Lazarova, D.L. and Sartorelli, A.C. (2007) The activation of beta-catenin by Wnt signaling mediates the effects of histone deacetylase inhibitors. Exp Cell Res, 313, 1652-66.  84.  Henderson, B.R., Galea, M., Schuechner, S. and Leung, L. (2002) Lymphoid enhancer factor-1 blocks adenomatous polyposis coli-mediated nuclear export and degradation of beta-catenin. Regulation by histone deacetylase 1. J Biol Chem, 277, 24258-64.  85.^Okamura, R.M., Sigvardsson, M., Galceran, J., Verbeek, S., Clevers, H. and Grosschedl, R. (1998) Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1. Immunity, 8, 11-20.  213  CHAPTER VI - ANDROGEN RECEPTOR REGULATION OF THE VERSICAN GENE THROUGH AN ANDROGEN RESPONSE ELEMENT IN THE PROXIMAL PROMOTER *  VI.1. SUMMARY Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and DHT stimulate expression of a versican promoter driven luciferase reporter vector (VCN-632-Luc). Further, both basal and androgen stimulated VCN-632-Luc activities were significantly diminished in LNCaP cells when AR gene expression was knocked down using a short interference RNA. Methylation-protection footprinting analysis revealed an AR protected element between positions +75 and +102 of the proximal versican promoter which strongly resembled a consensus steroid receptor element. Electromobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated VCN-632-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce VCN-632-Luc activity in androgen positive prostate stromal fibroblasts. Interestingly, over expression of P-catenin and TCF-4 in the absence  * A version of this work has been published. Read JT*, Rahmani M*, Boroomand S, Allahverdian S, McManus BM, Rennie PS, (2007) Androgen Receptor Regulation of the Versican Gene through an Androgen Response Element in the Proximal Promoter. J Biol Chem. 282:31954-63. (*Authors contributed equally to manuscript)  214  and presence of DHT augmented versican promoter activity 10 and 30 fold in fibroblasts. In conclusion, we demonstrate that AR trans-activates versican expression which may augment tumor—stromal interactions and contribute to prostate cancer progression.  VI.2. INTRODUCTION AND RATIONALE Stromal tissue mediates the induced growth and development of embryonic epithelium into differentiated prostate (1). Prostatic stroma is also necessary in the maintenance of adult prostatic secretory epithelium (2, 3). In addition, stromal-epithelial interactions play a significant role in steroid-influenced prostate carcinogenesis, and in the progression to the hormoneinsensitive phenotype (4). Tumor cells must remodel the matrix to facilitate communication and escape control by the microenvironment. Remodeling can also include interactions with "alternative" ECM, leading to cellular proliferation, structural disruption, and circumvention of apoptosis (5). Current findings obtained through a variety of approaches increasingly point to the contribution of stromal components to oncogenic signals that mediate both phenotypic and genomic changes in epithelial cells (6-8). Versican is one of the main components of ECM, which provides a loose and hydrated matrix during key events in development and disease (9, 10). Versican also contributes to the development of a number of pathologic processes including atherosclerotic vascular diseases (11, 12), cancer (13), central nervous system injury and neurite outgrowth (14). Several reports have shown that versican plays a role in cell adhesion (15), migration (16), proliferation (17), differentiation (18), angiogenesis (19), and resistance to oxidative stressinduced apoptosis (20); all of which are important events in tumor initiation and/or progression. Versican is highly expressed in many malignancies, including prostate cancers (21). Recent  215  studies demonstrated that prostate cells from tumor and benign prostatic hyperplasia (BPH) tissues induce host stromal cells to accumulate versican levels via a paracrine mechanism (22, 23). Further, versican has been shown to be an important modulator of tumor cell attachment to the interstitial stromal matrix of the prostate; a factor likely important in cancer cell motility and local invasion of the prostatic stroma (24). As well it has been suggested that versican may be a useful marker of disease progression in patients with early-stage prostate cancer (21). Genes that are preferentially expressed in human prostate tissues are often regulated by androgens at the transcriptional level. The androgen testosterone, and the more potent form DHT, bind to the AR. Upon ligand activation the AR is phosphorylated and forms a homodimer that is transported to the nucleus where it activates transcription by binding to androgen-response elements (ARE) in promoter and enhancer regions of target genes (25). Androgen withdrawal is the most effective form of systemic therapy for men with advanced prostate cancer, producing symptomatic and/or objective responses in >80% of patients (26). Unfortunately, androgenindependent (AI) progression is inevitable, and the development of hormone-refractory disease and death occurs within 2 to 3 years in most men (26). AI progression is a multifactorial process by which cells acquire the ability to both survive in the absence of androgens and proliferate using non-androgenic stimuli for mitogenesis, and involves variable combinations of several processes including adaptive up-regulation of antiapoptotic genes, ligand-independent activation of the androgen receptor, and alternative signaling pathways. The elucidation of the mechanisms that mediate AI progression, including key cytoprotective molecules, is an important step towards identifying new targets for therapy. Several lines of evidence indicate that 13-catenin is important in the progression of prostate cancer. Cre-mediated excision of the 13-catenin (exon3) regulatory domain leads to  216  prostatic hyperplasia and transdifferentiation in mice at 18 wk of age but without metastatic behavior (27). In a similar model, stabilized p-catenin appears to be important for the initiation of prostatic neoplastic lesions which are the precursor to invasive carcinoma (28). Also, gain-offunction, truncated forms of f3-catenin occurring in metastatic prostate and breast specimens have been shown to preferentially locate to the nucleus, possibly serving as an additional "pool" ofilcatenin to promote cell proliferation during the AI phenotype (29). AR and 13-catenin interact by direct binding and complexing, as ascertained by yeast two-hybrid analysis (30), GST pull downs (31), coimmunoprecipitations (32), and transcriptional reporter assays (30, 32, 33). AR/I3catenin interactions are ligand sensitive, such that the complexes increase in the presence of androgen (33) and decrease in the presence of the pure AR antagonist, bicalutamide (30, 33). To understand the role of the AR in prostate cancer development and progression, it is important first to determine the AR signaling cascades and the genes that are regulated by AR. Despite the importance of versican in prostate cancer, the function and regulation of expression of this versatile molecule in vitro and in vivo is unknown. Our results, for the first time, indicate that versican is transcriptionally regulated by androgens in the AR positive human prostate epithelial and stromal cancer cells. Further, we show a strong and specific binding of recombinant AR to the DNA sequences located in the +75 region of the proximal human versican promoter. Using transient transfection of synthetic reporter and endogenous gene reporter constructs in prostate stromal fibroblasts, we demonstrated that [3-catenin is required for AR-mediated transcription in both ligand-dependent and ligand-independent manner in prostate stromal fibroblast cells. These data identify a novel role for p-catenin in nuclear hormone receptor-mediated transcription in prostate stromal cells.  217  VI.3. EXPERIMENTAL PROCEDURES VL3.1.^Tissue culture — HeLa cells, modified HeLa-FLAG-AR overexpressing cells (a gift of M. Carey, University of California at Los Angeles, Los Angeles, USA), and human prostate stromal fibroblast cell line WPMY-1 (Invitrogen) were maintained in DMEM medium (Invitrogen) while LNCaP cell line (human prostate cancer cells) were maintained in RPMI (Invitrogen). Both types of media were supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 100 units/ml penicillin/streptomycin. For androgen withdrawal experiments, media was instead supplemented with 10% charcoal-stripped serum (CSS) and 100 units/ml penicillin/streptomycin for at least 24 hours prior to harvesting of cells. VL3.2.^Oligonucleotides — Oligonucleotides were synthesized by Nucleic Acids Protein Services (University of British Columbia). Oligonucleotide numberings for steroid response element (SRE)-2 are relative to the transcription start site of the human versican gene. The sequences of the oligonucleotides are: SRE-2 forward: CGAGAACATTAGGTGTTGT and SRE-2 reverse: ACAACACCTAATGTTCTCG VL3.3.^Electrophoretic mobility shift assay — Individual SRE-2 oligonucleotides were subjected to EMSA as previously described (34). Nuclear extracts used in EMSA were isolated from HeLa and HeLa-FLAG-AR cells using a modified Dignam method [17]. For EMSA experiments, 10,000 cpm of labeled oligoduplex probes were added to 18 pg of nuclear extracts. For supershift experiments 2 gg of rabbit anti-AR polyclonal antibody (214/11.1, Santa Cruz Biotechnology), 2 pg of rabbit anti-AR monoclonal antibody (2 pg/til, Santa Cruz Biotechnology) or rabbit anti-FLAG polyclonal antibody (2 pg/111, Santa Cruz Biotechnology) were added to the binding reaction and incubated for 30 minutes on ice prior to electrophoresis.  218  VL 3.4.^Probe generation for DMS in vitro footprinting — The 764 by proximal  versican promoter was excised from the pGL-3 backbone by digestion with M/u/ and Bg1II. The DNA region of interest was radiolabelled by Klenow enzyme labelling using a32 P - deoxyadenine and deoxycytidine triphosphate. The labeled promoter fragment was then digested with PflMI to give single end labeled fragments of 426 and 338 by (referred to as Versicanl and Versican2) respectively. These fragments were separated from each other using a 5% polyacrylamide, 0.5 X TBE non-denaturing gel. Probes were then cut out of the gel, eluted in 500 IA elution buffer and ethanol precipitated to generate single-end labeled probes for DMS footprinting. DMS in vitro footprinting: methylation protection analysis —  Footprinting reactions were carried out on the Versicanl or Versican2 probes using 5-10 lig (-3 lig/i.t1) of His-AR-DBD as described previously (34). VL3.6.^Western blotting — Protein samples were boiled in Laemmli sample buffer  and resolved by SDS-PAGE electrophoresis. Proteins were then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in TBST (0.05% Tween 20 in PBS) for lh and then incubated for 1-2 h at room temperature with primary AR antibodies (Santa Cruz Biotechnology). Detection was achieved with anti-mouse horseradish peroxidase (Santa Cruz Biotechnology) and ECL Western blotting detection agents (Amersham Biosciences, Inc., RPN 2106). VL3. 7.^Plasmid construction — The reporter plasmid used for transfections was  the VCN-632-Luc plasmid which contains the firefly luciferase gene under the transcriptional control of the -632 to +118 fragment of the human versican promoter (35). The pRL-TK (Promega) renilla luciferase reporter plasmid was used as an internal transfection control. A dominant stable f3-catenin construct was a kind gift of B. Gumbiner (Memorial Sloan-Kettering  219  Cancer Center, New York, USA). The expression vector harboring the wild-type human TCF-4 gene was a gift from A. Hecht (Max-Planck-Institute of Immunobiology, Freiburg, Germany) (36). 13-catenin/TCF luciferase reporter constructs pTOPflashx8, which has eight tandem TCF binding sites, was used for assaying TCF transcriptional activity (a gift of R.T. Moon University of Washington, Seattle, USA). VL3.8.^Short hairpin RNA against AR — We recently reported the generation and cloning of short hairpin RNA (shRNA) against AR (37). In brief, the pSHAG-1-tet plasmid (gift from A. Mui, University of British Columbia, Vancouver, Canada) used to clone the shRNA was modified from pSHAG-1 (gift from G. Hannon, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA). The following sequence was found to have the greatest attenuating effect on AR expression 5'-GAAAGCACTGCTACTCTTCAGCATTATTCCA-3' (3,539-3,569). A scrambled sequence (5'-CCGTACCTACACGCAGCGCTGACAACAGTTT-3') was used as a negative control. Equal molar amounts of both forward and reverse complimentary oligonucleotides were annealed, kinase treated, and cloned into pSHAG- 1-tet plasmid. VL3.9.^Transfections and luciferase assay — HeLa and HeLa-FLAG-AR or LNCaP cells were plated in 6-well plates (3.0 x 10 5 cells per well) in DMEM or RPMI, respectively with 5% FBS for 2 days or until reaching 50-60% confluency. Each well was transfected with 2.0 lig of VCN-632-Luc reporter plasmid and 0.2 pg of pRL-TK (Promega) for purposes of normalization. Plasmid DNA was mixed with Lipofectin reagent at a ratio of 2 .tg DNA / 3 pil Lipofectin (Invitrogen) in serum free DMEM and incubated for 30 min at room temperature. Cells were incubated with the transfection mix for 16 hours following which transfection medium was replaced by DMEM supplemented with 5% charcoal-stripped serum (CSS; HyClone, VWR, West Chester, PA) and the appropriate concentration of steroid. LNCaP  220  cells were plated in 6-well plates (3.0 x 10 5 cells per well) in RPMI with 5% FBS for 2 days or until reaching 50-60% confluency. Each well was transfected with 1.0 pig of AR shRNA vector or the scramble control, 1.0 lag of VCN-632-Luc reporter plasmid and 0.2 pg of pRL-TK (Promega) for purposes of normalization. Plasmid DNA was mixed with of Lipofectin reagent at a ratio of 2 pg DNA / 3 ill Lipofectin (Invitrogen) in serum free RPMI and incubated for 30 min at room temperature. Cells were incubated with the transfection mix for 16 hours following which transfection medium was replaced by RPMI with 5% CSS with 10 nM R1881 and cells were collected after 24 h of incubation using passive cell lysis buffer (Promega). Luciferase activities were measured using a commercial kit from Promega according to the manufacturer's protocol. Luciferase activities were measured as luminescent units/min and normalized to renilla control values. All transfection experiments were carried out in replicates of three or six and repeated at least 2 times. Database analyses — The versican proximal promoter was searched against the Matlnspector (http://wl,vw. gsf de/bi odv/matin spector.html) (38) and ConSite (http://forkhead.cgr.ki.se egi-biniconsite) (39) databases to detect potential SRE sites. ,  VI.3.11.^Immunohistochemical analysis — Using anti-versican antibody (L1350; United States Biological) at a working dilution of 1/50 immunohistochemical staining was conducted on the Vantana autostainer model Discover XT TM (Vantana Medical System, Tuscan, Arizona) with enzyme labeled biotin streptavidin system and solvent resistant DAB Map kit. For antigen retrieval slides were incubated with EDTA, pH8 in 95° C for 8 minutes then slides were sealed with coverslips using a xylene based mounting media, Cytoseal (Stephen Scientific). Negative control slides were processed in an identical fashion to that above, with the substitution of normal rabbit IgG for the primary antibody.  221  VI.3.12.^Statistical analysis — Significant differences between experimental groups were determined using the Student's t test. Results for cell culture data are expressed as means ± SEM. Calculated P values were 2-sided, and those < 0.05 considered statistically significant.  VI.4. RESULTS VI.4.1.^Versican proximal promoter luciferase reporter vector responds to steroid stimulation — The VCN-632-Luc vector, in which expression of the luciferase gene is under the control of the versican proximal promoter (-634 to +118 relative to the transcriptional start site), was used in transient transfection assays to determine the effects of steroid stimulation on versican promoter activity (35). VCN-632-Luc transfected into LNCap prostate cancer cells responded to the addition of a potent synthetic androgen (10nM R1881) with —3 fold induction of luciferase expression while exposure of the cells to l OnM dexamethasone, a glucocorticoid receptor agonist, provoked a —2 fold increase in VCN-632-Luc activity (Figure VI-1A). Parallel transfections of VCN-632-Luc into HeLa-FLAG-AR and wild type HeLa cells were used to assess the dose response of the reporter to the natural androgen DHT. VCN-632-Luc responded with increasing activity across a range of DHT concentrations from 0 nM to 10 nM in HeLaFLAG-AR cells while failing to respond in wild type HeLa cells lacking AR (Figure VI-1B). Taken together these results demonstrate androgen-mediated regulation of the versican promoter. VI.4.2.^Database searches reveal two potential SRE candidates within the versican proximal promoter — To determine whether known nuclear receptor transcription factors are able to bind to the proximal versican promoter, the —634 to +118 by of the human versican promoter sequence (Figure VI-2A) was submitted to the MatInspector (38) and ConSite (39) databases to identify potential protein binding sites. Results suggested that the submitted  222  sequence contained two potential SRE (Figure VI-2B). SRE-1 (-392 to —373bp) showed most sequence similarity to a glucocorticoid response element (GRE) while SRE-2 (+83 to +102bp) most resembled a progesterone response element, although there is considerable overlap in the SRE family of recognition sequences and both sites showed sequence similarity to the consensus ARE. While the SRE-2 is located within exon 1 of the versican sequence, functional ARE within the transcribed regions of other genes have previously been reported (40, 41). VI.4.3.^Methylation protection reveals the interaction of the AR DNA-binding domain to SRE-2 of the versican promoter — Methylation protection (MeP) analyses were  conducted using single-end 32P-labeled fragments of the versican promoter to look for guanineprotein DNA contacts on incubation with bacterially produced recombinant His-tagged AR DNA binding domain (His-AR-DBD). The versican probes were incubated with His-AR-DBD or with the equivalent His extract from bacteria containing a control plasmid. In this assay, dimethylsulfate is used to methylate unprotected guanines at position N7 of the DNA double helix while piperidine is used to cut these methylated guanines. Following gel extraction and chemical cleavage, equal amounts of labeled DNA are then separated on a urea sequencing gel. When bound to proteins, DNA conformation may change causing certain bases to become more exposed and prone to methylation, resulting in hypersensitive sites or bases may be sterically protected from chemical modification resulting in protection. No protection or hypermethylation of the versican promoter region corresponding to the SRE-1 was observed (data not shown). In contrast, obvious protection (guanine +84) and hypermethylations (guanine +92) were detected in the versican UTR exon 1 region corresponding to the previously identified SRE-2 (Figure VI3). This data positively demonstrates the binding of the AR DNA-binding domain to SRE-2.  223  VL4.4.^AR binds to versican SRE sites in vitro — To further explore the interaction of the AR with the SRE-2 protected region of the versican promoter, a radiolabeled, double-stranded probe corresponding to the SRE-2 region was synthesized for EMSA studies. Figure VI-4 shows the results of incubating the SRE-2 probe with nuclear extracts from HeLa and HeLa-FLAG-AR cells treated with 10 nM R1881. The SRE-2 probe yielded a number of protein-DNA complexes. Three non-specific bands (Figure VI-4) of varying intensity and molecular weight were detected with both cell extracts, while a higher molecular weight species was present only on incubation with HeLa-FLAG-AR extract. Addition of Anti-AR (C-19 polyclonal, Sigma: N-441 monoclonal, Sigma) or Anti-FLAG (F-1804, Sigma) antibodies to the EMSA generated a further, higher molecular weight supershift in the presence of HeLa-FLAGAR extracts. Taken together the shift and supershift data demonstrate the binding of a full-length mammalian androgen receptor to the SRE-2 DNA sequence. VI.4.5.^Mutation of SRE-2 interferes with androgen stimulation of the versican promoter activity — In order to further verify the direct regulation of the versican promoter by AR, site-directed mutagenesis was used to alter the SRE-2 site within the versican proximal promoter in the VCN-632-Luc plasmid. SRE-2 site was mutated to exchange the "AA" present in the upstream half-site to "TT" (Figure VI-5A). Following sequence verification, HeLa-FLAGAR cells were transfected with either wild-type VCN-632-Luc or the VCN-632-Luc-SRE-2mt plasmid and the pRL-tk normalisation control reporter. Cells were treated for 24 hours posttransfection with 10 nM R1881 or vehicle in 5% serum DMEM. Luciferase activities were corrected for transfection efficiency and normalized to the steroid negative (Figure VI-5B). The mutation of SRE-2 completely abrogated R1881 stimulation when compared to the wild type  224  promoter (p<0.001), demonstrating the importance of the SRE-2 DNA element to androgen mediated regulation of the versican promoter in an in vivo system. VL4.6.^Knock-down of AR expression reduces ligand-dependent and ligandindependent versican promoter activity — Four shRNA constructs targeting AR were designed to evaluate the requirement for AR in versican promoter activation. To determine which of the four AR shRNA was most effective, each was screened for inhibition of activation of the androgenresponsive ARR3-tk luciferase reporter in transient transfection assays. When compared with empty vector (pSHAG-1), shRNA 4 was found to be the most potent construct, inhibiting ARinduced luciferase expression by 97% (37). To examine the role of AR in ligand-dependent and ligand-independent versican promoter activation, we next tested the effects of this shRNA on the activity of the versican promoter. VCN-632-Luc reporter activation by R1881 was compared in cells transfected with control scramble or AR shRNA 4 constructs in LNCaP human prostate cancer cells. The AR shRNA could effectively inhibit ligand-independent and ligand-dependent activation of versican promoter by 40% and 50%, respectively (Figure VI-5C). Because significant AR knock-down could not completely eliminate promoter activation, versican promoter activity seems to involve both AR-dependent and AR-independent mechanisms. VL4. 7.^Cross-talk between AR and fl-catenin/TCF signaling is essential for optimal transactivation of Wnt- and AR-responsive promoters in prostate fibroblast cells — Using transient transfection assays in the AR positive LNCaP cells we demonstrated that androgens stimulate expression of the VCN-632-Luc promoter reporter gene (Figure VI-1A). Recent studies demonstrated that prostate cells from tumor and BPH tissues induce host stromal cells to accumulate versican levels via a paracrine mechanism (22, 23). In order to show the transcriptional activity of the versican gene in prostate stromal fibroblasts, the primary source of  225  versican in prostatic tissues, we used an established AR positive prostate stromal fibroblast cell line WPMY-1 (42). First, we examined whether the AR agonist R1881 could induce versican promoter function in prostate fibroblasts. In contrast to LNCaP cell and HeLa cells engineered to express AR, R1881 was did not increase VCN-632-Luc activity in prostatic fibroblasts (Figure VI-6A). We have recently reported that the f3-catenin/TCF signaling pathway plays a critical role in versican transcription (35). Additionally, recent reports suggest that AR and ii-catenin interact by direct binding and that their interaction is ligand sensitive (33). We showed that cotransfection of VCN-632-Luc with 0-catenin and TCF-4 expression vectors into prostatic fibroblasts significantly increased versican promoter activity (—j 10 fold) (Figure VI-6A). Interestingly R1881 augmented VCN-632-Luc promoter activity in these fibroblasts over that of 13-catenin/TCF-4. Altogether these results suggest that 13-catenin is essential for AR liganddependent and ligand-independent regulation of versican gene transcription in prostate stromal fibroblasts (Figure VI-6A). To determine whether the interaction between AR and P-catenin/TCF signaling could modulate AR- and 0-catenin-dependent transcriptional activities of AR- and 13-catenin/TCFresponsive promoters, prostate stromal fibroblast cells were also transfected with PSA-Luc, ARR3-tk-Luc, or pTOPflashx8-Luc with or without P-catenin and TCF-4 expression vectors in presence or absence of AR ligand, DHT. The luciferase reporter plasmid VCN-632-Luc is both a AR- and TCF-responsive promoter; PSA-Luc and ARR3-tk-Luc reporter plasmids are ARresponsive promoters and pTOPflashx8-Luc is responsive to activation of wnt signaling. As mentioned above, transfection of endogenous reporter gene responsive to AR and 13-catenin signaling, VCN-632-Luc, with f3-catenin and TCF-4 expression plasmids mediated —10- and 30fold induction of luciferase activity compared to transfection of VCN-632-Luc reporter construct  226  alone in the absence and presence of AR ligand DHT, respectively (Figure VI-6A). In contrast, co-transfection of 13-catenin and TCF-4 expression plasmids with PSA-Luc (AR-responsive gene) mediated only 4- and 8-fold promoter induction, respectively (Figure VI-6B). These findings support the notion that AR and 13-catenin/TCF signaling require both a TCF binding site and ARE in the promoter for the optimal transcriptional activation by f3-catenin/TCF and AR signaling as evident with the higher response of VCN-632-Luc compared to PSA-Luc. Because 13-catenin/TCF complex modulated the transcriptional activity of endogenous Wnt- and ARresponsive promoters in absence and presence of AR ligands, we investigated the ability of the 13catenin/TCF complex to modulate reporter plasmids with either synthetic ARE or wnt responsive elements in prostate fibroblast cells. Co-transfectedr3-catenin and TCF-4 expression plasmids with ARR3-tk-Luc or pTOPflashx8-Luc augmented both AR and wnt-responsive promoters —15and 30-fold, in absence or presence of 1 nM DHT, respectively (Figure VI-6C and VI-6D). These findings emphasize that in prostate stromal fibroblast cells 13-catenin/TCF signaling is required for the optimal transactivation AR- and wnt-responsive genes. VI.4.8.^Versican immunolocalizes to the stroma of prostate gland and increases in human prostate cancer — Figure VI-7A and VI-7B illustrate the comparison of immunostaining of stromal versican in BPH and prostate cancer. Weak to moderate staining intensity is seen in BPH tissues (Figure VI-7A) and strong stromal staining was observed in prostate cancer tissues (Figure VI-7B). Prominent immunostaining for versican was observed in stroma associated with malignant areas of sectioned prostate tissue, whereas negligible deposits of versican were identified in stroma surrounding nonmalignant glands (Figure VI-7B).  227  VI.5. DISCUSSION In the present study, we have found that androgens dose-dependently enhance human versican promoter activity in LNCaP AR positive epithelial human prostate cancer cells. Furthermore, we have shown that inhibition of AR expression levels using shRNA diminishes both basal and androgen stimulated versican transcriptional activities in these cells. We also demonstrated that an SRE binding site located in the first exon of the human versican gene is involved in androgen-induced versican transcription and that at least one of the members of steroid hormone family of transcription factors, AR, binds to this SRE site in the human versican promoter. Finally, we showed that 13-catenin is required for both ligand-dependent and ligandindependent AR-mediated trans-activation in prostate stromal fibroblasts. Carcinoma cells, like normal epithelial cells, live in a complex microenvironment that includes the ECM, diffusible growth factors and cytokines, and a variety of non-epithelial cell types. Recent studies have provided evidence that stromal cells and their products can cause the transformation of adjacent cells through transient signaling that leads to the disruption of homeostatic regulation, including control of tissue architecture, adhesion, cell death, and proliferation (5, 6). Given the importance of the versican in cellular events involved in initiation and progression of tumors (15-17, 19, 20, 43); our observations may support a new concept for the role of steroid hormone signaling through the AR transactivation complex, modulating stromal gene expression patterns and versican-rich provisional matrix formation involved in BPH (23) and aberrant prostate cancer development and progression (21, 22, 24). Growing evidence suggests the existence of intriguing similarities between the process of wound healing and that of prostate tumorogenesis. In human prostate cancers, reactive stroma is characterized by an increase in myofibroblasts, a corresponding amplification in ECM protein  228  production, and an increase in local vascular density (44), properties similar to those seen in granulation tissue. The PG versican is one of several ECM molecules that accumulate in BPH (23) and prostate cancer (21, 22, 24). Versican is generally considered to be important in tumor initiation and/or progression because of its important roles in cell adhesion (15), migration (16), proliferation (17), differentiation (45), angiogenesis (19) and resistance to oxidative stressinduced apoptosis (20); all of which are important events in tumor formation and progression. The dynamic processes of normal prostate development and progression of prostate cancer are dependent on androgen acting through the AR (25). Androgens, testosterone and DHT, bind to the AR promoting dimerization and association with coregulators. AR then translocates to the nucleus and binds to ARE in the promoter regions of target genes, thereby modulating transcriptional activities. The present study suggests that versican is a positively regulated target gene of the androgen receptor. The production of versican also influences cellular functions and survival directly or indirectly. Androgens themselves have long been known to promote survival of prostatic epithelium and they appear to increase the cell's ability to withstand apoptotic stimuli (46). Our finding may suggest one of the molecular mechanisms by which versican influences prostate tissue behavior via stromal-epithelial interactions leading to either normal maintenance or BPH and possibly tumor progression. Recent data suggests that the gonadotropin hormones, LH and FSH, regulate versican mRNA and protein expression in the rodent ovary (47, 48). Given the role of gonadotropins in versican mRNA and protein expression (47, 48), it supports the notion that gonadotropinmediated regulation of the versican expression is via either signaling transduction pathways i.e. PI3K/PKB/Akt or androgens. I have recently reported that the PI3K/PKB/GSK-313 pathway plays a critical role in versican transcription (35). Therefore, it might be concluding that one of  229  the mechanisms through which gonadotropins induce versican transcription is mediated by PI3K/PKB/Akt. Furthermore, it has also been reported that in cultured granulosa cells, testosterone significantly enhanced versican mRNA and protein expression (47). In this study, for the first time, our results indicate that versican is transcriptionally regulated by androgens. We established this regulatory sequence of events in a series of experiments. First, we showed that R1881, a potent synthetic AR agonist, significantly enhanced human VCN-632-Luc activity in AR positive LNCaP human prostate cancer cells and in cervical HeLa cells engineered to express AR. Furthermore, consistent with this result, the native androgen, DHT, dosedependently increased VCN-632-Luc activity in HeLa-positive AR cells while failing to induce the same activation in wild type AR-negative HeLa cells. Finally, knock-down of endogenous AR levels in AR-positive LNCaP human prostate cancer cells reduced both the basal and androgen stimulated versican promoter driven transcription. Overall, these results suggest that AR, at least in part, is required for androgen-mediated stimulation of versican transcription in AR positive human prostate cancer cells. The location, sequence, and number of ARE associated with a given androgen target gene varies, although androgen-responsive regions typically contain multiple nonconsensus ARE (5'TGTTCT-3') (49). By analyzing the published sequence of the versican gene promoter (50), we noticed that there are two potential ARE-like motifs located in the promoter of the human versican gene (versican SRE-1 —392-AGAACTagcTGCACG-373 and versican SRE-2 (+83AGAACAttaGGTGTT+102). We have characterized the specific binding of recombinant AR to the SRE-2 site by EMSA and the guanine nucleotides involved in AR/DNA interactions were localized by footprinting. Moreover, transient transfection analysis of wild type and SRE-2 mutant VCN-632-Luc constructs showed that the mutation eliminated the hormonally induced  230  response, suggesting the functional im