EXPRESSION AND ACTIONS OF CONNECTIVE TISSUE GROWTH FACTOR DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Amy Wilson Rachfal ***** The Ohio State University 2003 Dissertation Committee: Approved by Professor David R. Brigstock, Advisor Professor K. Reed Clark _________________________________ Professor Nick Flavahan Advisor Molecular, Cellular Developmental Biology Program Professor Tom Sferra ABSTRACT Connective tissue growth factor (CTGF/CCN2) is an extracellular matrix-associated matricellular protein that regulates diverse cellular activities including adhesion, migration, mitogenesis, differentiation and survival. The broad biological properties of CTGF/CCN2 in many cell types likely reflect its ability to bind to a variety of cell surface molecules (integrins, low density lipoprotein receptor-related protein, heparin sulfate proteoglycans) as well as to other bioactive molecules. CTGF/CCN2 synthesis can be stimulated by a variety of molecules including transforming growth factor β1 (TGF-β1) which appears to play a major role in the transcriptional activation of the CTGF/CCN2 gene in matrigenic and fibrogenic pathways. This dissertation encompasses the relationship between CTGF/CCN2 and organ fibrosis, TGF-β1 action, gene regulation, pediatric desmoplastic tumors as well as its role in uterine function. Recombinant adeno-associated virus delivery of the CTGF/CCN2 gene was attempted to determine whether CTGF/CCN2 was directly linked to the progression of organ fibrosis. However, since little or no protein was generated in vivo using this system, it could not be deduced whether over-expression of CTGF/CCN2 alone could drive fibrosis. In vitro studies with fibroblasts and hepatic stellate cells (HSCs) demonstrated that TGF-β1, alcohol or CTGF/CCN2 itself could up-regulate CTGF/CCN2 ii transcription. Additionally, after heparin- or EDTA-dependent HSC adhesion to CTGF/CCN2, preliminary gene array experiments demonstrated a series of genes that were transcriptionally up-regulated including, collagen α-1-type I, fibronectin, GRO oncogene, secreted acidic cysteine rich glycoprotein, tenasin C and tissue inhibitor of matrix metalloproteinase. Further, RT-PCR analysis of similar experiments showed that hepatocyte growth factor, a potent mitogen that accelerates recovery from hepatic fibrosis, was down regulated. In addition, two pediatric fibrotic diseases, congenital hepatic fibrosis and desmoplastic small round cell tumor, were shown to produce elevated levels of CTGF/CCN2, consistent with its pro-fibrogenic properties. Finally, studies demonstrated that uterine CTGF/CCN2 gene transcription is regulated by maternal sex steroids and TGF-β1-dependent and –independent mechanisms. Further data indicated that the epithelium is a key source of CTGF/CCN2 in the mammalian uterus and that CTGF/CCN2 likely plays a role in regulating stromal cell function and placental neovascularization. iii Dedicated to my family and husband iv ACKNOWLEDGMENTS I wish to offer my heartfelt thanks to my advisor, David R. Brigstock, Ph.D. His encouragement, intellectual guidance and enthusiasm made this degree possible and for that, I am tremendously grateful and deeply honored. I am also thankful to my committee, K. Reed Clark, Ph.D., Nicholas Flavahan, Ph.D. and Thomas Sferra, M.D. for their support, scientific discussions and guidance. I also wish to thank the Viral Vector Core and Microarray Core staff for help with large-scale viral production, technical assistance and microarray hybridization. I am grateful to Essam Moussad, Ph.D. and Mona Rageh, Ph.D. for their work with porcine and murine uteri, Ruping Gao, Ph.D., M.D. for isolation of rat hepatic stellate cells, DeAnna Ball, Ph.D. and Sherri Kemper for generating recombinant CTGF/CCN2, Kris Backstrom, M.S. for assistance with viral delivery to the liver, Guliang Xia, Ph.D., M.D. for assistance with viral delivery to the muscle, as well as Mark Luquette, M.D. for pathology samples, scientific discussions and microscope assistance. This research was supported by grants from the USDA (grant 9803693), NIH (grant AA12817-02) and Children’s Research Institute. v VITA Born: November 6, 1972 Columbus, OH, USA Education 1998-2003 Ph.D., Molecular, Cellular and Developmental Biology (MCDB) The Ohio State University, Columbus, OH Principal Investigator: David R. Brigstock Ph.D. 1994-1997 M.S., Molecular Genetics, The Ohio State University, Columbus, OH 1990-1994 B.A., Ohio Wesleyan University, Delaware, OH Major: Zoology, Minor: Chemistry Employment History 2000 Preparing Future Faculty fellowship recipient Developed a mentoring relationship with Dr. Simon Lawrence at Otterbein College for the spring semester. Designed and taught multiple labs as well as prepared and gave a lecture to a molecular genetics class. 1998-2003 Graduate Research Assistant, David Brigstock, Ph.D. Children’s Hospital, Columbus, OH Project: Research toward Ph.D 1998 Graduate Teaching Assistant, The Ohio State University, Columbus, OH General Biology (101), Autumn quarter Professor: Dr. Michael Mangino 1997-1998 Research Associate, David Brigstock, Ph.D. Children’s Hospital, Columbus, OH Project: Recombinant production and signal transduction of CTGF vi 1994-1997 Graduate Research Assistant, Ellen Gottlieb, Ph.D. The Ohio State University, Columbus, OH Project: Research toward M.S. 1995 Graduate Teaching Assistant, The Ohio State University, Columbus, OH General Biology (113), spring quarter Professor: Dr. Johnson 1993 Research Assistant, The Ohio State University through Ohio Wesleyan University, funded by Howard Hughes Summer Research Fellowship Project: The detection of conserved microsattellite regions in the 21-OH region of domesticated species. 1993 Independent Study, Ohio Wesleyan University, Delaware, OH Project: Comparing ancient enterobacter from a mastodon gut to the modern day equivalent. PUBLICATIONS Published Papers Rachfal A. W., Brigstock, D. R., (2003) Connective tissue growth factor in hepatic fibrosis. Hepatology Research 26(1) 1-9 Xia, G., Rachfal, A. W., Besner, G. E., (2003) Upregulation of endogenous heparin- binding EGF-like growth factor (HB-EGF) expression after intestinal ischemia/reperfusion injury. J Invest Surg 16(2) 57-63 Ball, D. A., Rachfal, A. W., Kemper S. A., Brigstock D. R., (2003) The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion. J. Endocrinology 176 R1-R7 Park, H.O., Kang, P. J., Rachfal, A. W. (2002) Localization of the Rsr1/Bud1 GTPase involved in selection of proper growth site in yeast. J. Biol Chem, 277(30) 26721-4 Moussad, E.E.A., Rageh, M.A.E., Wilson, A.K., Geisert, R.D., Brigstock, D.R. (2002) Temporal and spatial expression of connective tissue growth factor (CTGF/CCN2) and transforming growth factor beta type 1 (TGF-β1) at the utero-placental interface during early pregnancy in the pig. Mol Pathol, 55 186-192 Rageh, M.A.E., Moussad E., Wilson, A.K., Brigstock, D.R. (2001) Steroidal regulation of connective tissue growth factor (CCN2; CTGF) synthesis in the mouse uterus. Mol Pathol 54(5) 338-346 vii Surveyor, G.A. Wilson, A.K., and Brigstock, D.R. (1998) Localization of connective tissue growth factor during the period of embryo implantation in the mouse. Biol Reprod 59 1207-1213 Published Abstracts Rachfal A.W., Clark, KR, Luquette, M. Brigstock DR. (2003) rAAV-mediated CTGF gene overexpression in skeletal muscle in vivo is associated with muscle fiber atrophy. Mol Pathol 56 P08. Wilson, A.K., Clarke, K.R. and Brigstock, D.R. (2001) Novel models of CTGF transgenesis in vivo: Recombinant adeno-associated viral (rAAV) mediated delivery of the CTGF gene. J Clin Pathol: Mol Pathol 54, P5. Wilson, A.K., Clark K.R., Sferra T.J. and Brigstock D.R. (2001) Gene transfer to hepatic stellate cells using recombinant adeno-associated virus. Hepatology 34 No.4 Pt 2 Abstract #1303. FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology viii TABLE OF CONTENTS P a g e Abstract. ii Dedication. iv Acknowledgments . v Vita . vi List of Tables. xiii List of Figures . xiv List of Abbreviations. xvi Chapters: 1. A general review of the connective tissue growth factor (CTGF/CCN2). 1 molecule 1.1 Discovery. 1 1.2 CCN gene family. 1 1.3 CCN family modular structure. 5 1.4 CTGF/CCN2 and heparin interactions. 8 1.5 CTGF/CCN2 and intracellular signaling. 8 1.6 The link between TGF-β and CTGF/CCN2. 10 1.7 CTGF/CCN2 action in normal processes. 12 1.7.1 CTGF/CCN2 in development. 13 1.7.2 CTGF/CCN2 in uterine biology. 13 1.7.3 CTGF/CCN2 and bone development. 15 1.7.4 CTGF/CCN2 in limb regeneration. 15 1.7.5 CTGF/CCN2 in wound healing. 16 1.7.6 CTGF/CCN2 in apoptosis and cell survival. 17 1.7.7 CTGF/CCN2 in angiogenesis. 18 ix 1.8 CTGF/CCN2 action in pathological disease processes. 20 1.8.1 CTGF/CCN2 and tumorigenesis. 20 1.8.2 CTGF/CCN2 in fibrotic skin disorders. 23 1.8.3 CTGF/CCN2 in atherosclerosis. 23 1.8.4 CTGF/CCN2 in organ fibrosis. 24 2. rAAV mediated CTGF/CCN2 gene delivery. 31 2.1 Introduction. 31 2.1.1 CTGF/CCN2 in muscle fibrosis. 31 2.1.2 CTGF/CCN2 in liver fibrosis. 32 2.1.3 In vivo CTGF/CCN2 delivery. 34 2.1.4 Viral delivery of CTGF/CCN2. 36 2.2 Materials and methods. 39 2.2.1 Materials. 39 2.2.2 Infection of HSC-T6 cells with rAAV-CMV-β-gal, rAAV-CMV-GUS, rAAV-EF1α-GUS. 41 2.2.3 Cloning strategy. 42 2.2.4 Verification of CTGF/CCN2 protein production. 43 2.2.5 Passage assay. 44 2.2.6 Verification of correct replication intermediates. 44 2.2.7 Selection of rAAV-CTGF/CCN2 producer cell lines. 45 2.2.8 Characterization of final producer cell lines. 47 2.2.9 rAAV-CMV-CTGF and rAAV-EF1α-CTGF infection of HSC-T6 cells. 48 2.2.10 rAAV-CTGF/CCN2 delivery to mouse muscles and liver.