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The Endoplasmic Reticulum Chaperone Erdj4 Is Required for Survival, Glucose Metabolism and B Cell Development

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The Endoplasmic Reticulum Chaperone Erdj4 Is Required for Survival, Glucose Metabolism and B Cell Development The endoplasmic reticulum chaperone ERdj4 is required for survival, glucose metabolism and B cell development A dissertation submitted to the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Immunobiology Graduate Program of the College of Medicine 2012 by Jill Marie Fritz B.A., Miami University, Oxford, OH Advisory Committee: Timothy Weaver, M.S., Ph.D., Chair George Deepe, M.D. Fred Finkelman, M.D. H. Leighton Grimes, Ph.D. Christopher Karp, M.D. Francis McCormack, M.D. ABSTRACT The ER-localized DnaJ homologue 4 (ERdj4) is a soluble ER chaperone induced by the unfolded protein response (UPR) to assist in the removal of unfolded/misfolded proteins from the ER lumen for proteasomal degradation. To elucidate the function of ERdj4 in vivo, ERdj4 gene trap (ERdj4GT/GT) mice were generated from embryonic stem cells harboring a gene trap cassette inserted into the ERdj4 locus. ERdj4GT/GT mice expressed hypomorphic levels of ERdj4 with a 10-100 fold reduction in all tissues and cell types examined. Approximately 30-50% of ERdj4GT/GT mice died perinatally in association with growth retardation and hypoglycemia. ERdj4GT/GT neonates exhibited signs of delayed pancreatic development, including abnormal distribution of pancreatic α- and β-cells and reduced insulin and glucagon in the pancreas. Surviving adult ERdj4GT/GT mice were glucose intolerant, resulting from hypoinsulinemia rather than insulin resistance. Pancreatic β-cells exhibited increased ER stress, including ER dilation and upregulation of the UPR. Proinsulin accumulated in the ER of β-cells from ERdj4GT/GT mice consistent with our previous finding that proinsulin is a substrate for ERdj4. The insulin processing enzymes, including Pcsk1, Pcsk2 and CPE, also associated with ERdj4, contributing to defects in proinsulin biosynthesis in ERdj4GT/GT mice. ERdj4 deficiency also resulted in pancreatic α-cell hyperplasia in association with increased ER stress. Since previous studies had demonstrated that the UPR is required for both early and late B lymphopoiesis, we hypothesized that ERdj4 deficiency would affect B cell development. Pro-B, pre-B, immature and mature B cell populations were significantly reduced in the bone marrow of ERdj4GT/GT mice in association with increased pro-B cell death. Further, mature B cell populations were reduced in the spleen and peritoneal cavity. ERdj4GT/GT donor cells transplanted into ERdj4+/+ recipients rescued all stages of B cell development, confirming a defect in the microenvironment. Osteogenic cells support B cell development and the UPR is required for osteoblast differentiation and function. Bone-lining cells, which differentiate from osteoblasts, were reduced in ERdj4GT/GT mice in association with increased ER stress. Further, primary bone cells from ERdj4GT/GT mice had reduced expression of osteogenic-specific markers. These data suggest that the loss of ERdj4 affects the maturation/survival of osteogenic cells, which in turn leads to a defect in B lymphopoiesis. Collectively, this work reveals an important role for ERdj4 in survival, glucose metabolism and B cell development. iii iv ACKNOWLEDGMENTS First and foremost, I wish to express sincere gratitude and appreciation to my scientific thesis advisor, Dr. Timothy Weaver. Without his dedication, guidance and encouragement, this work would not be possible. I also wholeheartedly thank my committee members Dr. George Deepe, Dr. Fred Finkelman, Dr. Lee Grimes, Dr. Chris Karp and Dr. Frank McCormack for their insight and support during my scientific training. I sincerely appreciate their efforts and direction in completing this work. I acknowledge all the members of the Weaver lab, both past and present, for their friendship, motivation and expertise. I would especially like to thank Dr. Henry Akinbi for his careful supervision and support, especially during the first few years of my thesis project. Pursuing my Ph.D. would not have been possible without my undergraduate mentor, Dr. John Stevenson, who introduced me to the exciting world of scientific research. This thesis is dedicated to my mother, Barbara A. Fritz and my father, Gerald W. Fritz, as well as my grandma, Barbara H. Bridge and my grandpa Edward D. Bridge. They instilled within me their passion, persistence and inquisitive mind. Their unconditional love and support make it possible to achieve any goal. v TABLE OF CONTENTS CONTENT PAGE List of tables and figures xiii List of abbreviations xvi CHAPTER I Molecular Chaperones and the UPR in 1 Development and Metabolism: Background, Significance and Hypothesis 1. Molecular Chaperones in the Endoplasmic Reticulum 2 1a. BiP: an ER-localized HSP70 chaperone 2 1b. DnaJ family of cochaperones 3 1bi. Structure and classification 5 1bii. Functionality 5 1biii. ER-localized DnaJ proteins 6 1biv. ERdj4 8 2. The Unfolded Protein Response 9 2a. Discovery 9 2b. Initial discovery of UPR signaling components in yeast 10 2c. The mammalian UPR 10 2ci. The ER stress sensors: ATF6, PERK and IRE1 11 2cii. ER stress-induced apoptosis 14 3. β-cell Stress, the UPR and Diabetes 14 3a. Inducers of ER stress in β-cells 15 3ai. Glucose 15 vi 3aii. Free fatty acids 16 3aiii. Proinflammatory cytokines 16 3aiv. Misfolded protein 18 3av. Islet amyloid peptide 18 3b. The UPR in β-cell dysfunction and diabetes 18 3bi. PERK/eIF2α 18 3bii. IRE1α/XBP1 19 3biii. ATF6α 21 3biv. ER chaperones 21 3c. The UPR and insulin resistance 22 4. B Cell Subsets and Effector Functions 22 4a. B1 cells 23 4b. B2 cells: Follicular and marginal zone B cells 24 4bi. Follicular B cells 24 4bii. Marginal zone B cells 24 5. B Cell Development 25 5a. B1 cell development 25 5b. B2 cell development 28 5bi. Early B2 cell differentiation in the bone marrow 28 5bii. Transcriptional regulation of early B2 cell development 30 5biii. Transitional B cells 31 5biv. Follicular verses marginal zone B cell fate 32 5c. The UPR in B cell development 34 vii 6. Growth Factors and Cellular Niches Required for B Cell Development 35 6a. B cell growth factors 36 6ai. CXCL12 36 6aii. IL-7 36 6aiii. FLT3L 38 6aiv. RANKL 38 6av. SCF 39 6b. Bone marrow niches that support B cell development 40 6bi. CAR niche: reticular cells expressing CXCL12 40 6bii. IL-7-expressing cells 40 6biii. Dendritic cells 41 6biv. Osteoblasts 41 7. The UPR in Osteoblast Differentiation 42 7a. PERK/eIF2α 42 7b. ATF4 44 7c. ATF6 45 7d. IRE1α/XBP1 45 8. Summary and Hypothesis 46 References 48 CHAPTER II Deficiency of ERdj4 is associated with perinatal 66 lethality, constitutive ER stress and glucose intolerance viii Abstract 68 Introduction 69 Results 72 Generation of ERdj4 gene trap mice 72 Elevated ER stress and increased susceptibility to cell death 72 in ERdj4GT/GT MEFs Hypoglycemia and growth retardation in ERdj4GT/GT mice 73 Constitutive ER stress is associated with histological 74 abnormalities in pancreatic islets of ERdj4GT/GT mice ERdj4 deficiency impairs insulin biosynthesis 75 Hypoinsulinemia causes glucose intolerance in ERdj4GT/GT 76 mice Discussion 77 Materials and Methods 81 Acknowledgements 87 Figure Legends 88 Figures 93 References 105 CHAPTER III Deficiency of the ER stress chaperone ERdj4 is 110 associated with defects in osteogenesis and B cell development Abstract 112 ix Introduction 113 Results 117 Bone-lining cells are reduced in ERdj4GT/GT mice 117 ERdj4 deficiency affects early B lymphopoiesis 119 ERdj4 deficiency in the bone marrow microenvironment 119 impairs B cell development Mature B cell populations are reduced in ERdj4GT/GT mice 120 Plasma cell differentiation and antibody secretion are normal 121 in ERdj4GT/GT mice Discussion 122 Materials and Methods 126 Acknowledgements 130 Figure Legends 131 Figures 136 References 146 CHAPTER IV Discussion and Future Directions 151 Summary 152 1. Gene trapping ERdj4: a blessing in disguise? 154 2. What is the function of ERdj4 in perinatal survival? 155 3. ER stress and Type 2 Diabetes 157 3a. Do physiological stressors cause diabetes in ERdj4GT/GT mice? 158 3b. What causes β-cell failure in ERdj4GT/GT mice? 159 x 3c. Does rescue of ERdj4 expression in pancreatic β-cells result in 160 normal insulin secretion and glucose metabolism in ERdj4GT/GT mice? 3d. Is the chaperone activity of ERdj4 important in α-cells? 163 3di. Does loss of ERdj4 affect glucagon biosynthesis? 163 3dii. What causes α-cell hyperplasia in ERdj4GT/GT mice? 164 4. The role of ERdj4 in the bone marrow microenvironment 165 4a. Does the loss of ERdj4 in osteogenic cells impair B cell development 165 in ERdj4GT/GT mice? 4ai Do ERdj4GT/GT osteogenic cells support B cell maturation 165 in vitro? 4aii. Does administration of PTH rescue the defect in B cell 166 development in ERdj4GT/GT mice? 4aiii. Would rescue of ERdj4 expression in osteogenic cells result in 167 normal B cell development in ERdj4GT/GT mice? 4b. Which cell(s) of the osteogenic lineage affects B cell development? 168 4c. How does ERdj4 deficiency affect peripheral B cells? 169 4d. How does the loss of ERdj4 affect postosteoblasts and skeletal 170 development? 4di. Quantitation of osteogenic cells from ERdj4GT/GT mice 170 4dii. Why are postosteoblasts reduced in ERdj4GT/GT mice? 171 4diii. Does the loss of postosteoblasts affect skeletal development 171 in ERdj4GT/GT mice? 5. Molecular chaperones in the treatment of ER stress-related disease 172 xi 6. ERdj4 in cellular function and homeostasis 173 References 175 xii LIST OF TABLES AND FIGURES CHAPER I PAGE Figure 1 The BiP/HSP70 chaperone cycle 4 Figure 2 The mammalian
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