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Transcriptional Regulation of the Human Cytochrome P450 2J2 Gene by Activator Protein-1

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Transcriptional Regulation of the Human Cytochrome P450 2J2 Gene by Activator Protein-1 Transcriptional Regulation of the Human Cytochrome P450 2J2 Gene by Activator Protein-1 by Nicole Yvonne Marden A thesis submitted for the degree of Doctor of Philosophy School of Medical Sciences Faculty of Medicine The University of New South Wales January 2006 Originality Statement Originality Statement I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged. Nicole Yvonne Marden ii Acknowledgements Acknowledgements Firstly, I would like to express my sincere thanks to my supervisor, Professor Michael Murray, for his constant guidance, support and encouragement throughout my PhD. In particular, I must thank Michael for the advice he has given me in terms of the design and analysis of my experimental studies, and for the generous amount of time he has devoted to reviewing this thesis. I would like to extend a special thank you to Dr Gloria Quee and Mrs Eva Fiala-Beer of the Murray Lab for all their help in teaching me experimental techniques, and for their continuous support and advice throughout my PhD. Your friendship and encouragement has been wonderful and has made the long hours in the lab a more pleasurable experience. I must also thank all of the other members of the Murray Lab, past and present, for their friendship and encouragement throughout my studies. I would like to thank Mr Stuart Purvis-Smith from the Molecular and Cytogenetics Unit at the Prince of Wales Hospital for generously providing me with access to a hypoxic incubator to undertake my hypoxic cell culture studies. I would also like to acknowledge Dr Kazuhiko Imakawa and Dr Michael Karin for generously providing various expression plasmids, and Dr Qing-Yu Zhang for generously providing the anti-(rat-CYP2J4) antibody. I would like to thank the members of staff at the Department of Physiology and Pharmacology for their help and interest in my project. A special thanks must go to Ms Rebekah Smith, Ms Sindy Kueh and Dr Kylie Mansfield for their friendship and support. To my fantastic friends Mandy and Beck: thank you for our weekly get-togethers over muffins and coffee. Your friendship has been priceless, and you have helped me to keep things in perspective and keep me smiling. I must also thank you both for your help with proof-reading this thesis. Finally, I would like to thank my wonderful family for their constant love, support and encouragement. Thank you to my Dad for giving me a love of science and learning in general, thank you to Lou for his amazing support and generosity, thank you to my Maxie and thank you to Mark and Nan for their love and encouragement. Most importantly, I would like to thank my wonderful mother, Marlena, and my amazing iii Acknowledgements partner, Christian, to whom I dedicate this thesis. You have taught me never to give up, and your unconditional love, patience and encouragement has allowed me to achieve my goals and dreams. iv Table of Contents Table of Contents Originality Statement ii Acknowledgements iii Table of Contents v List of Figures xi List of Tables xiv Abbreviations xv List of Publications and Abstracts xx Abstract xxi Chapter 1 Introduction 1 1.1 Cytochromes P450 1 1.1.1 Fundamental Aspects 1 1.1.2 Nomenclature of CYP Enzymes 1 1.1.3 Biochemistry 2 1.2 Metabolism of Xenobiotics by CYPs 4 1.2.1 CYP1 Family 6 1.2.2 CYP2 Family 7 1.2.3 CYP3 Family 11 1.2.4 CYP4 Family 12 1.3 Metabolism of Endogenous Substances by CYPs 12 1.3.1 CYPs Involved in Steroid Synthesis and Metabolism 14 1.3.2 CYPs Involved in Cholesterol Metabolism and Bile Acid Synthesis 15 1.3.3 CYPs Involved in Vitamin A and Vitamin D Metabolism 17 1.3.4 CYPs Involved in the Metabolism of Arachidonic Acid 19 1.3.4.1 Arachidonic Acid and the Arachidonic Acid Metabolic Cascade 19 1.3.4.2 The Third Pathway in the AA Cascade: Metabolism of AA by CYPs 21 v Table of Contents 1.4 The Z/Z-1 Hydroxylase Pathway of AA Metabolism 22 1.4.1 Biological Activities of 20-HETE 23 1.4.2 Vascular Effects of 20-HETE 24 1.4.2.1 Vasoconstriction and the Regulation of Vascular Tone 24 1.4.2.2 Role for 20-HETE in Vasodilation Pathways 26 1.4.2.3 20-HETE as a Possible Oxygen Sensor 26 1.4.3 Effect of 20-HETE on Ion Transport within the Kidney 27 1.4.4 Mitogenic Actions of 20-HETE 27 1.4.5 Role of 20-HETE in the Pathogenesis of Hypertension 28 1.5 The Epoxygenase Pathway of AA Metabolism 30 1.5.1 Biological Activities of EETs 32 1.5.2 Vascular Effects of EETs 32 1.5.2.1 Vasodilatory Effects of EETs within the Vascular System 32 1.5.2.2 EETs Proposed to be the Endothelium-derived Hyperpolarising Factor (EDHF) 34 1.5.2.3 Role of EETs in Reactive Hyperemia 35 1.5.3 Non-vasodilatory Effects of EETs within the Vascular System 36 1.5.3.1 Anti-Inflammatory Effects of EETs 36 1.5.3.2 Anti-migratory Effects of EETs 38 1.5.3.3 Fibrinolytic Effects of EETs 38 1.5.3.4 Mitogenic Properties of EETs 39 1.5.3.5 Effects of EETs on Platelets 40 1.5.3.6 Role of EETs in Protection Against Hypoxia-reoxygenation Injury in Endothelial Cells 40 1.5.4 Non-vascular Effects of EETs 41 1.5.4.1 Effects of EETs on Cardiomyocyte Function and Recovery After Cardiac Ischaemia 42 1.5.4.2 Effect of EETs within the Kidney and Potential Role in Hypertension 43 1.5.4.3 Anti-apoptotic Effects of EETs 45 1.5.4.4 Effects of EETs on the Release of Peptide Hormones 45 1.5.4.5 Effects of EETs in the Lung 46 1.5.4.6 Effects of EETs in the Liver 47 1.5.5 Factors Affecting the Level of EETs within the Body 48 1.6 Cytochrome P450 2J2 (CYP2J2) 49 1.6.1 CYP2J2 Gene and Protein Structure 49 1.6.2 Catalytic Activity of CYP2J2 52 vi Table of Contents 1.6.3 Tissue Distribution of CYP2J2 53 1.7 Biological Significance of CYP2J2 54 1.7.1 Potential Role of CYP2J2 in the Heart and Vasculature 55 1.7.2 Potential Role of CYP2J2 in Other Tissues 59 1.8 Regulation of CYP Gene Expression 62 1.8.1 Regulation of CYP Genes by Liver-enriched Transcription Factors 64 1.8.2 Receptor-mediated Regulation and Induction of CYP Gene Expression 65 1.8.2.1 CYP1A Induction by the Ah Receptor 66 1.8.2.2 Nuclear Receptors Involved in CYP Gene Expression 66 1.8.2.3 CAR-mediated Induction of CYP2B Genes 67 1.8.2.4 PXR-mediated Induction of CYP3A Genes 68 1.8.2.5 PPAR-mediated Induction of CYP4A Genes 69 1.8.2.6 LXR- and FXR-mediated Regulation of CYP7A Gene Expression 70 1.8.3 Down-regulation of CYPs by Inflammatory Mediators 70 1.8.4 Regulation of CYP2J2 Gene Expression 71 1.9 Activator Protein-1 73 1.9.1 AP-1 Components, Dimerisation and DNA Binding 73 1.9.2 Regulation of AP-1 Activity 77 1.9.3 Role of AP-1 in Cellular Physiology and Pathophysiology 79 Chapter 2 Materials and Methods 82 2.1 Materials 82 2.1.1 Reagents and Chemicals 82 2.1.2 Plasmids and Reagents for Molecular Biology 83 2.1.3 Reagents for Cell Culture 83 2.1.4 Reagents for Protein Electrophoresis and Immunoblotting 84 2.2 General Molecular Techniques 84 2.2.1 Preparation of Competent E.coli Cells and Transformation of Plasmids 84 2.2.2 Culture of E.coli Cells and Purification of Plasmids for Transfection 85 2.2.3 DNA Sequencing 87 2.2.4 Electrophoresis and Purification of DNA 88 2.3 Preparation of CYP2J2 Promoter Reporter Constructs 89 vii Table of Contents 2.4 Cell Culture 91 2.4.1 Experimental Conditions 91 2.4.2 Cell Line and Culture Conditions 92 2.4.3 Passaging of Cells 92 2.4.4 Hypoxic Treatment of Cells and Harvesting of Cells for Extraction of Total RNA, Total Cell Lysates and Nuclear Extracts 92 2.4.5 Assessment of Cell Viability 93 2.5 RNA Extraction 94 2.5.1 Experimental Conditions 94 2.5.2 RNA Extraction Procedure 94 2.5.3 Quantitation of RNA by Spectrophotometry 95 2.5.4 Electrophoresis of RNA Samples 95 2.6 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 95 2.6.1 Experimental Conditions 95 2.6.2 Semi-quantitative RT-PCR of CYP2J2, c-Fos and c-Jun 96 2.6.3 Competitive RT-PCR for Quantification of CYP2J2 mRNA 98 2.6.3.1 Preparation of a Recombinant CYP2J2 RNA Internal Standard 98 2.6.3.2 Quantitative Competitive RT-PCR for CYP2J2 100 2.7 Protein Analysis 101 2.7.1 Isolation of Total Cell Lysates for Protein Analysis 101 2.7.2 Immunoblotting 102 2.8 Transient Transfection Analysis 103 2.8.1 Transient Transfection of HepG2 Cells 103 2.8.2 Luciferase Reporter Gene Assay 103 2.8.3 E-galactosidase Assay 104 2.9 Electrophoretic Mobility Shift Assay (EMSA) 105 2.9.1 Preparation of Nuclear Extracts 105 2.9.2 Preparation of Double-stranded Probes for use in EMSA 106 2.9.3 EMSA 109 Chapter 3 Regulation of the Expression of CYP2J2, and the AP-1 Proteins c-Fos and c-Jun, in Hypoxia and Reoxygenation 110 3.1 Introduction 110 3.2 Viability of HepG2 Cells Following Exposure to Hypoxia 112 viii Table of Contents 3.3 Analysis of CYP2J2 mRNA Levels in HepG2 Cells Following Exposure to Hypoxia and Reoxygenation 114 3.4 Identification of Multiple Potential Binding Sites for the Hypoxia-responsive Transcription Factor AP-1 within the 5’-flanking Region of the CYP2J2 Gene 118 3.5 Analysis of
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