Regulation of hepatic stellate cell phenotype and cytoglobin expression by extracellular matrix proteins By Louise Catherine Stone A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences The University of Birmingham October 2014 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract All chronic liver diseases can induce fibrosis and lead to liver cirrhosis. Within liver disease, hepatic stellate cells (HSC) are accepted as the major effectors of fibrogenesis and changes in the extracellular matrix (ECM). Cytoglobin (CYGB), a hexacoordinated globin, is upregulated in liver disease, and expression has been reported to be specific to HSCs in the liver, though this is disputed. Data presented in Chapter 3 of this thesis confirm upregulation of Cygb in murine models of liver disease and diseased human liver tissue. Chapter 4 shows how ECM can effect HSC morphology, behaviour and phenotype with collagen I, an important component of the hepatic scar conferring an activated HSC phenotype, and laminin, a basal protein in a normal liver, inducing a more quiescent phenotype in HSC cell lines HSC-T6 and LX-2. Chapter 5 demonstrates the novel observation of collagen I-induced downregulation, and laminin-induced upregulation, of Cygb in HSC-T6s. Chapter 6 explores the role of cell signalling through membrane receptors and regulation of Cygb expression, identifying phosphorylated focal adhesion kinase as a mechanism of signal transduction through integrin activation. These findings suggest that Cygb expression is modulated by ‘outside-in signalling’ and this is important in the activation status of HSCs. Acknowledgements I would like to thank Nik Hodges for supporting and guiding me throughout this process. I would also like to thank Mark Graham and Kevin Chipman for their advice, and Chris Weston for his assistance, with many aspects of this project. I would also like to express my gratidute to the BBSRC and Astra Zeneca for providing funding for the research and also to attend conferences, which has allowed me to develop my skills. The advice and support of the many members of the 4th floor has been invaluable, and special thanks must go to Nadine Taylor, Leda Mirbahai, Lorna Thorne, Bob Harris, Nicola Cumley, Rachael Kershaw, Chibuzor Uchea, Fiona McRonald, Tim Williams and Shrikant Jondhale. My family have been of great help over the last four years, my parents, Julie and Nigel Stone my sister Helen Stone and my fiancé Marc Lloyd providing love, support and a shoulder to cry on, as well as pride in my achievements. My Ph.D. would not have been possible without these people, and I thank them for helping me through it. Table of Contents Chapter 1 Introduction ..................................................................................................................... 1 1.1 Cytoglobin ........................................................................................................................ 2 1.1.1 Discovery ....................................................................................................................... 2 1.1.2 The globin family ........................................................................................................... 3 1.1.2.1 Evolutionary history of CYGB ..................................................................................... 5 1.1.2.2 CYGB in comparison with other globins .................................................................... 5 1.1.3 The structure of CYGB ................................................................................................... 6 1.1.4 Genetics of Cygb ......................................................................................................... 12 1.1.4.1 Regulation of Cygb expression ................................................................................ 13 1.1.5 Localisation of CYGB ................................................................................................... 15 1.1.6 Putative functions of CYGB ......................................................................................... 17 1.1.6.1CYGB and O2 .............................................................................................................. 17 1.1.6.2 CYGB, reactive oxygen species and reactive nitrogen species ................................ 19 1.1.6.3 CYGB in hypoxia ....................................................................................................... 21 1.1.6.4 Enzymatic activity of CYGB ...................................................................................... 23 1.1.7 CYGB and collagen I synthesis .................................................................................... 23 1.1.8 CYGB in disease ........................................................................................................... 25 1.1.8.1 CYGB in cancer ......................................................................................................... 25 i 1.1.8.2 CYGB in fibrosis ........................................................................................................ 27 1.2 Hepatic Stellate Cells ..................................................................................................... 29 1.2.1 HSC function................................................................................................................ 30 1.2.1.1 HSCs in a normal liver .............................................................................................. 30 1.2.1.2 Function on liver injury ............................................................................................ 31 1.2.1.3 Senescence of HSCs ................................................................................................. 33 1.2.1.4 Transcription factors involved in the regulation of HSC activation and quiescence in the context of Cygb regulatory elements ..................................................... 37 1.2.2 Liver fibrosis ................................................................................................................ 41 1.2.3 Changes in ECM .......................................................................................................... 42 1.2.4 The reversibility of liver fibrosis .................................................................................. 43 1.3 Liver disease ................................................................................................................... 44 1.3.1 Overview of liver disease ............................................................................................ 45 1.3.2 Types of liver disease .................................................................................................. 45 1.4 Hypothesis, Aims and Objectives ................................................................................... 51 1.4.1 Hypothesis .................................................................................................................. 51 1.4.2 Aims and Objectives .................................................................................................... 51 Chapter 2 Materials and Methods .................................................................................................. 52 2.1 Chemicals and consumables .......................................................................................... 53 ii 2.2 Cell lines ......................................................................................................................... 53 2.2.1 HSC-T6 cell line ............................................................................................................ 53 2.2.2 LX-2 cell line ................................................................................................................ 54 2.2.3 Primary rat HSCs ......................................................................................................... 55 2.3 Continual Cell Culture .................................................................................................... 55 2.3.1 Cell Cryopreservation .................................................................................................. 56 2.4 Culture on different ECM proteins ................................................................................ 56 2.4.1 Collagen coating procedure ........................................................................................ 57 2.4.2 Laminin coating procedure ......................................................................................... 58 2.4.3 Mixed matrix effects on HSC-T6 cells ......................................................................... 58 2.5 Cell imaging using Light Microscopy .............................................................................. 58 2.6 Retinoic Acid uptake on different culture surfaces ....................................................... 59 2.7 Ribonucleic acid Isolation .............................................................................................