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Endoplasmic Reticulum Stress in Pancreatic Β-Cells

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Endoplasmic Reticulum Stress in Pancreatic Β-Cells ENDOPLASMIC RETICULUM STRESS IN PANCREATIC β-CELLS ER STRESS AND CYTOKINE-MEDIATED β-CELL DYSFUNCTION AND APOPTOSIS ANALYSIS OF THE ER STRESS RESPONSE IN A PANCREATIC β-CELL LINE EXPRESSING A FOLDING-DEFICIENT PROINSULIN-EGFP FUSION PROTEIN By Taila Hartley A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto © Copyright by Taila Hartley 2009 ENDOPLASMIC RETICULUM STRESS IN PANCREATIC β-CELLS: ER STRESS AND CYTOKINE-MEDIATED β-CELL DYSFUNCTION AND APOPTOSIS ANALYSIS OF THE ER STRESS RESPONSE IN A PANCREATIC β-CELL CLONE EXPRESSING A FOLDING-DEFICIENT PROINSULIN-EGFP FUSION PROTEIN Taila Hartley Master of Science, Graduate Department of Biochemistry, University of Toronto, 2009 ABSTRACT Endoplasmic reticulum (ER) stress has been implicated in pancreatic β-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress- induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated β-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented. ii ACKNOWLEDGEMENTS This thesis would not have been possible without the input and encouragement of numerous individuals, and to them I would like to express my sincere gratitude. First and foremost, I would like to thank my supervisor, Dr. Allen Volchuk, for his constant enthusiasm and open door over the last two years. His support and encouragement in not only experimental work but in written work and presentations as well, have allowed me to complete these projects to the best of my ability while at the same time gaining valuable experiences. In particular, I would like to thank Allen for the chance to present at the American Society for Cell Biology Conference (2008) in San Francisco and the chance to help write a review paper published in the American Journal of Physiology. I would also like to thank my committee members, Dr. Williams and Dr. Grinstein for their helpful ideas and suggestions on my studies. These projects would not have been completed without the help of numerous technicians including Monika Sharma (UHN Microarray Facility), Doug Holmyard (Mount Sinai Electron Microscopy Facility) and Leanne Jamieson (Sick Kids Flow Cytometry Facility). I would also like to thank the entire Volchuk lab, past and present, for providing the best lab environment I have ever experienced. I have no doubt that Elida’s hard work a couple of years ago provided me with a solid base to build my project and made my life much easier. In addition, I need to thank Elida for her efforts in training me at the beginning of my project. Liling’s incredibly hard work to keep the lab functioning iii smoothly are only topped by all of the effort she devotes to helping everyone in the lab with new techniques and for that she is without a doubt the most valuable asset of the entire lab. Thanks Liling. Tracy, my best biochemistry buddy and the most phenomenal dance partner, your constant encouragement and help with everything from life to cloning have made this thesis possible. Ravi, the source of all answers whether about techniques or politics, thanks for being a great labmate. Madura, the most generous person I know, it’s been incredible sharing the lab with you over the last year. I’d also like to thank the new students of the lab, Akansha and Irmgard, as well as the entire 10th floor of MaRS-TMDT for their encouragement and friendship. Finally, I’d like to thank my family and friends who have been a constant source of encouragement. Thanks mom and dad for keeping me out of trouble and reminding me of the value of education. To my sisters (Kendra, Yasmin and Tara) and brothers (Scott and Nick), thanks for always being only a phone call away. To Brendon, thanks for making anywhere we are feel like home and standing by me whatever decisions I make. Last, but certainly not least, I’d like to thank the Banting and Best Diabetes Centre for funding my work and giving me the opportunity to present my results on several different occasions. iv TABLE OF CONTENTS ABSTRACT II ACKNOWLEDGEMENTS II TABLE OF CONTENTS V LIST OF FIGURES VIII LIST OF ABBREVIATIONS X CHAPTER 1: INTRODUCTION 1 1.1 THE ENDOPLASMIC RETICULUM 2 1.2 MOLECULAR CHAPERONES OF THE ER 4 1.2.1 GRP78 6 1.3 ER-RESIDENT PROTEIN DEGRADATION 8 1.3.1 ERAD I 8 1.3.2 ERAD II 11 1.4 ER STRESS AND THE UNFOLDED PROTEIN RESPONSE 12 1.4.1 PERK SIGNALING DURING THE ER STRESS RESPONSE 14 1.4.2 IRE1 SIGNALING DURING THE ER STRESS RESPONSE 16 1.4.3 ATF6 SIGNALING IN THE ER STRESS RESPONSE 17 1.4.4 RECOVERY FROM THE UNFOLDED PROTEIN RESPONSE 18 1.5 ER STRESS-INDUCED APOPTOSIS 19 1.4.1 CHOP 20 1.4.1 JNK 22 1.4.2 CASPASE-12 23 1.5 BIOSYNTHESIS AND SECRETION OF INSULIN IN PANCREATIC Β-CELLS 24 1.5.1 INSULIN BIOSYNTHESIS 25 1.5.2 MECHANISMS OF INSULIN RELEASE 26 1.6 ER STRESS IN PANCREATIC β-CELLS 28 1.7 MECHANISMS OF β-CELL DYSFUNCTION IN TYPE 1 AND TYPE 2 DIABETES 30 1.7.1 CYTOKINES, INTERLEUKIN-1β, INTERFERON-γ AND NITRIC OXIDE 32 1.7.2 CYTOKINES, ER STRESS, AND PANCREATIC β-CELL APOPTOSIS 35 1.7.3 CYTOKINES AND β-CELL DYSFUNCTION 38 1.8 RATIONALE AND HYPOTHESIS 40 CHAPTER 2: MATERIALS AND METHODS 42 2.1 CELL CULTURE 43 2.2 CYTOKINE PREPARATION, CELL TREATMENT AND LYSES 43 2.3 INFECTION OF INS-1E WITH GRP78 ADENOVIRUS (AD-GRP78) 44 2.4 REVERSE TRANSFECTION OF SHORT INTERFERING RNA (SIRNA) 45 2.5 MEASUREMENT OF XBP-1 MRNA SPLICING 45 2.6 RNA ISOLATION AND REAL-TIME QUANTITATIVE POLYMERASE CHAIN REACTION (PCR) 46 2.7 ELECTRON MICROSCOPY 47 2.8 INSULIN SECRETION ASSAY AND RAT INSULIN RADIOIMMUNOASSAY (RIA) 48 2.9 WESTERN BLOT ANALYSIS 49 2.10 SUCROSE DENSITY FRACTIONATION 50 v 2.11 IMMUNOPRECIPITATION 51 2.12 APOPTOSIS ASSAYS 52 2.12.1 ELISAPLUS CELL DEATH DETECTION KIT 52 2.12.2 FLOW CYTOMETRY-BASED APO-BRDU TUNEL ASSAY 52 2.13 MICROARRAY ANALYSIS 53 2.14 CLONING OF SDF2-L1 54 2.15 TRANSIENT TRANSFECTION 57 2.15 DATA ANALYSIS 58 CHAPTER 3: ER STRESS AND CYTOKINE-INDUCED PANCREATIC β-CELL DYSFUNCTION AND APOPTOSIS 59 3.1 INTRODUCTION 60 3.2 RESULTS 62 3.2.1 CYTOKINES INDUCE APOPTOSIS IN INS-1E CELLS. 62 3.2.2 CYTOKINES ACTIVATE AN EARLY ER STRESS RESPONSE IN INS-1E CELLS. 63 3.2.3 GRP78 OVEREXPRESSION OR TREATMENT WITH CHEMICAL CHAPERONE DOES NOT PROTECT INS-1E CELLS FROM CYTOKINE-INDUCED β-CELL APOPTOSIS. 66 3.2.4 GRP78/BIP KNOCKDOWN INCREASES INS-1E SUSCEPTIBILITY TO CYTOKINE- INDUCED APOPTOSIS AND POTENTIATES THE EFFECTS OF CYTOKINES TO REDUCE PROINSULIN LEVELS. 67 3.2.5 GRP78 OVEREXPRESSION DOES NOT AMELIORATE CYTOKINE-INDUCED β-CELL DYSFUNCTION IN INSULIN BIOSYNTHESIS OR SECRETION. 70 3.2.6 EFFECT OF CYTOKINE TREATMENT ON RAT ISLET INSULIN BIOSYNTHESIS AND SECRETION. 71 3.3 DISCUSSION 74 3.4 SUMMARY AND FUTURE DIRECTIONS 79 CHAPTER 4: ANALYSIS OF THE ER STRESS RESPONSE IN A PANCREATIC β- CELL LINE EXPRESSING A FOLDING-DEFICIENT PROINSULIN-EGFP FUSION PROTEIN 82 4.1 INTRODUCTION 83 4.1.1 METHODS: GENERATION AND CHARACTERIZATION OF THE INSULIN 2 (C96Y)-EGFP STABLE INS-1 CELL LINE 85 4.2 RESULTS 87 4.2.1 THE ER STRESS RESPONSE TO INSULIN2 (C96Y)-EGFP EXPRESSION 90 4.2.2 MICROARRAY ANALYSIS OF INSULIN2 (C96Y)-EGFP EXPRESSION 90 4.2.3 INDUCTION OF APOPTOSIS FOLLOWING INS2 (C96Y)-EGFP EXPRESSION 97 4.3 DISCUSSION 102 4.4 SUMMARY AND FUTURE DIRECTIONS 109 APPENDIX 1: MICROARRAY RESULTS FROM THE ANALYSIS OF THE ER STRESS RESPONSE IN CLONE #4S2 112 APPENDIX 2: EXAMINING THE ROLE OF STROMAL DERIVED FACTOR 2-LIKE 1 IN PANCREATIC β-CELL ER STRESS AND APOPTOSIS 127 A2.1 RATIONALE AND HYPOTHESIS 128 A2.2 RESULTS 129 A2.2.1 SDF2L1 IS INDUCED IN MKR MICE, ANOTHER MODEL OF DIABETES 129 vi A2.2.2 GENERATION OF EXPRESSION VECTORS FOR A FUNCTIONAL ANALYSIS OF SDF2L1 130 A2.3 SUMMARY AND FUTURE DIRECTIONS 132 REFERENCES 134 vii LIST OF FIGURES CHAPTER 1: INTRODUCTION Figure 1.1 Functions of GRP78 in the ER. Figure 1.2 Signaling in the unfolded protein response. Figure 1.3 ER stress-induced apoptosis. Figure 1.4 Insulin biosynthesis and glucose-stimulated insulin secretion. Figure 1.5 Model of how cytokines may induce ER and potentially apoptosis in β-cells. CHAPTER 3: ER STRESS AND CYTOKINE-INDUCED PANCREATIC β - CELL DYSFUNCTION AND APOPTOSIS Figure 3.1 Cytokines induce cell death in the rat pancreatic β-cell line INS-1E. Figure 3.2 Cytokine exposure induces the UPR in INS-1E cells. Figure 3.3 Effect of GRP78 overexpression or PBA treatment on cytokine-induced apoptosis in INS-1E cells. Figure 3.4 siRNA-mediated silencing of GRP78 expression in INS-1E cells renders them more susceptible to cytokine-induced apoptosis.
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