Platelet-derived growth factor receptor alpha signaling pathways in development and liver disease Brian J. Hayes A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2014 Reading Committee: Jean Campbell, Chair Daniel Bowen-Pope William Mahoney Jr. Program Authorized to Offer Degree: Pathology ©Copyright 2014 Brian J. Hayes University of Washington Abstract Platelet-derived growth factor receptor alpha signaling pathways in development and liver disease Brian J. Hayes Chair of the Supervisory Committee: Jean S. Campbell, Assistant Professor Department of Pathology Platelet derived growth factor receptor alpha (PDGFR α) signaling is critical for development and disease. Insufficient PDGFR α signal transduction results in developmental anomolies, while excessive signaling results in fibrosis and carcinogenesis. PDGFR α signal transduction is regulated at multiple levels, two of which are ligand binding and Pdgfr α expression levels. In my thesis work, I describe a Pdgfc mutant mouse, Pdgfc tm1Lex , in which only growth factor coding exons are deleted. Viability of Pdgfc tm1Lex mice, which express only the complement components C1r/C1s, sea urchin EGF, bone morphogenetic protein 1 (CUB) domain of PDGF- C, depends on the presence of both Pdgfr α alleles. Alternative splicing in Pdgfc tm1Lex mice gives rise to a truncated transcript that contains the entire coding region of the CUB domain of PDGF- C, but lacks the majority of the exons encoding the growth factor domain (GFD). Contrary to Pdgfc knockout (KO) mice, Pdgfc tm1Lex mice are viable, suggesting that the CUB domain of PDGF-C contributes to PDGFR α signal transduction. The CUB domain of PDGF-C appears to maintain PDGFR α signal transduction above the minimum level necessary for development. At the other end of the spectrum, increased PDGFR α signal transduction can contribute to disease. PDGFR α levels are elevated in human liver disease, and after acute and chronic liver injury in mice. These correlative studies indicate that PDGFR α may be important for the liver’s response to injury. Activated hepatic stellate cells (HSCs) produce collagen resulting in fibrosis, which can progress to liver cirrhosis. Cirrhosis is the 12 th leading cause of death in the United States, and increases the risk of developing liver cancer. Utilizing a chemical model of fibrosis, chronic carbon tetrachloride (CCl 4) exposure, I found that mice with decreased WT/nGFP expression of Pdgfr α (Pdgfr α ) develop less fibrosis, than wild type mice following CCl 4 exposure. These results indicate that elevated Pdgfr α expression is associated with chronic liver injury, and suggests excessive PDGFR α signal transduction is detrimental. PDGFR α signal transduction is controlled at the level of ligand binding and gene expression, and needs to be maintained at a balance that allows for proper development, but does not result in fibrotic disease. Therapies targeted to maintain the balance of PDGFR α–specific signaling pathways may provide both sufficient PDGFR α for wound healing and therapeutic benefit for patients with chronic liver disease. TABLE OF CONTENTS Page Table of contents......……...………………………………………………………....… i List of Abbreviations .……...………………………………………………………...... ii List of Figures ..……………………...………………………………………………… v List of Tables ..............…………………………………………………………...…… vii Chapter 1: Introduction 1.1 Summary……………………………………………………….…….....1 1.2 Liver disease……………………………………………………..….….1 1.3 Liver cell involvement in disease…………………………………..…2 1.4 Mouse models of chronic liver disease..…………………………....3 1.5 Platelet-derived growth factor ligands and receptors.…….…….…5 1.6 Hypothesis………………………………………………………..….…9 Chapter 2: Function of the CUB domain in PDGF-C knockout mice 2.1. Introduction ..…………………………………………………….. 13 2.2. Materials and Methods………………………………………….. 14 2.3. Results ..……………….…………………………………………. 18 2.4. Discussion ..…………………………………………………...…. 21 Chapter 3: Platelet derived growth factor receptor alpha contributes to liver fibrosis 3.1. Introduction ..…………………………………………………….. 30 3.2. Materials and Methods………………………………………….. 32 3.3. Results ..……………….…………………………………………..35 3.4. Discussion ..…………………………………………………...…. 39 Chapter 4: Conclusions and Future Directions 4.1. Major Findings ...……………………………………………..….58 i 4.2. Future Directions …………………………………………..……59 4.2. Discussion……… …………………………………………….…61 References ...……………………………………………………………………...…. 67 ii Abbreviations BrdU 5-Bromo-2-deoxyuridine BDL bile duct ligation BHK baby hamster kidney cells CCl 4 carbon tetrachloride CRBP-1 cellular retinol binding protein 1 CTGF connective tissue growth factor CUB C1r/C1s sea urchin EGF bone morphogenetic protein 1 ECM exracellular matrix EGF epidermal growth factor ET-1 endothelin 1 FGF fibroblast growth factor GFAP glial fibrillary acidic protein GFD growth factor domain GFP green fluorescent protein Grb2 growth factor receptor-bound protein 2 HCC hepatocellular carcinoma HSCs hepatic stellate cells IF immunofluorescence IHC immunohistochemistry KCs Kupffer cells LSECs liver sinusoidal endothelial cells NAFLD non-alcoholic fatty liver disease NPCs non-parenchymal cells OPC oligodendrocyte progenitor cell Ph Patch mutant mouse iii Abbreviations PDGF platelet derived growth factor PDGFR α platelet derived growth factor receptor alpha PDGFR β platelet derived growth factor receptor beta PI3K phosphoinositol 3-kinase PLC γ phospholipase gamma 1 RasGAP Ras GTPase activating protein TAA thioacetamide TGF β1 transforming growth factor beta WT wild type iv List of Figures Figure Number Page 1.1 Carbon tetrachloride mouse model of liver fibrosis..…………………………………….11 1.2 PDGF-C transgenic mouse model of liver fibrosis .……………………………………..11 1.3 PDGF-C transgenic mouse Kaplan-Meyer survival curve……………………………...12 1.4 PDGFR induced intracellular kinase phosphorylation…………………………………..13 2.1 Design of targeting constructs to inactivate PDGF-C…………………………………...25 2.2 Recombination at the Pdgfc locus in the Pdgfc tm1Lex mouse……………………………26 2.3 Alternative mRNA transcribed from the Pdgfc locus in Pdgfc tm1Lex mice……………...27 2.4 Sequence of cDNA from Pdgfc tm1Lex mice………………………………………………..28 3.1 Perisinusoidal PDGFR α expression is localized to fibrotic or cirrhotic areas in tumor specimens by IHC……………………………………………………………………………….44 3.2 PDGF receptors are expressed in hepatic stellate cell lines…………………………..45 3.3 PDGFR mRNA expression increases in response to acute CCl 4 exposure……….…46 WT/nGFP 3.4 PDGFR α positive cells form fibrotic bands after chronic CCl 4 injection in Pdgfr α mice………………………………………………………………………………………………47 3.5 PDGFR α-positive cells co-localize with PDGFR β-positive cells in chronic CCl 4 injured liver……………………………………………………………………………………………….48 WT/nGFP 3.6 Compared to C57BL/6 mice, chronically CCl 4 injured Pdgfr α mice have reduced transcription of fibrotic genes………………………………………………………………….49 3.7 Chronically injured Pdgfr αWT/nGFP mice have less collagen deposition than C57BL/6 mice …………………………………………………………………………………………………...50 3.8 Expression of PDGFRs in human liver tumor and non-tumor tissue………………...51 3.9 C57BL/6 HSCs express PDGFR α…………..…………………………………………..52 4.1. Amino acid sequence of wild type PDGFC and PDGF-Cmut………………….…….69 4.2. Hypothetical model of a monomer of wild type PDGFC and PDGF-Cmut…….…...69 v List of Figures Figure Number Page 4.3. Hypothetical model of a dimer of wild type PDGF-CC and PDGF-Cmut…………..70 4.4 PDGFR α Activity has a narrow range compatible with developmental viability……71 4.5 PDGFR α as the limiting PDGF pathway component in the liver environment……..72 vi List of Tables Table Number Page 2.1 Heterozygous Pdgfc tm1Lex breeding pairs in two backgrounds produce offspring with normal Mendelian distribution…………………………………………………………………………..29 2.2 Heterozygous Pdgfctm1Lex breeding pairs with one copy of Pdgfr α do not produce Pdgfctm1Lex homozygous and hemizygous Pdgfr α offspring.………….…………………30 3.1 Antibodies used for immunohistochemistry and immunofluorescence………………53 3.2 Human and mouse hepatocyte and stellate cells used in this study…………………54 3.3 Oligonucleotide primers used for real time analysis……………………………………55 3.4 Summary of PDGFR α and PDGFR β immunoreactivity in human liver specimens…56 3.5 Immunoblot detection of PDGFR expression in macroscopically dissected human tumors and surrounding liver…………………………………………………………………..57 3.6 PDGF stimulates proliferation in stellate cell lines, but not primary hepatocytes or hepatoma cell lines……………………………………………………………………………..58 vii Chapter 1 Background and Introduction Summary This chapter provides background information on 1) the public health burden of liver disease, 2) the functions of parenchymal and non-parenchymal liver cells, 3) mouse models of liver disease, and 4) signaling pathways and molecular components involved in the response to models of hepatotoxic injury and the development of liver fibrosis. A brief synopsis of my thesis project is presented at the end of this chapter. Liver disease Chronic liver injury leads to fibrosis, which can progress to cirrhosis and hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer, and develops in cirrhotic liver in 90% of cases (1). The incidence and mortality from liver cancer are both increasing at a rate of around 3% a year (2); five year survival from time of diagnosis is only 16% (2, 3). Underlying cirrhosis in HCC prevents resection, thus the limited therapeutic options for these patients are in part due to the fact