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Université Libre de Bruxelles Faculté de Médecine Laboratoire de Médecine Expérimentale
« Identification and characterization of the endoplasmic reticulum (ER)-stress pathways in pancreatic beta-cells. » Pierre Pirot Promoter : Décio Laks Eizirik Co-Promoter : Alessandra Kupper Cardozo
Thèse présentée en vue de l’obtention du Grade de Docteur en Sciences Biomédicales.
I dedicate this thesis to Guy, Marie-Claire and Félicien, my beloved parents and brother.
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Table of contents Composition of the thesis jury ……....................................................................................
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List of reports constituting this thesis..................................................................................
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Summary .............................................................................................................................
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Résumé …...........................................................................................................................
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List of abbreviations ...........................................................................................................
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1. Introduction ....................................................................................................................
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1.1 The endoplasmic reticulum ......................................................................................
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1.1.1 Physiological role ..............................................................................................
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1.1.2 Stress of the endoplasmic reticulum ..................................................................
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a) The unfolded protein response (UPR) ................................................................
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b) Apoptosis ............................................................................................................
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1.2 Diabetes Mellitus.......................................................................................................
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1.2.1 Definition and epidemiology .............................................................................
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1.2.2. Type 1 diabetes mellitus (T1DM) ....................................................................
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1.2.3.1. Genetics of T1DM .....................................................................................
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1.2.2.2. T1DM onset: environmental triggers, recruitment and activation of immune cells ...........................................................................................................
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1.2.2.3. Mediators of beta-cell destruction in T1DM ….........................................
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1.2.3. Type 2 diabetes mellitus (T2DM) and beta-cell death .....................................
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1.3 Endoplasmic reticulum and pancreatic beta-cell ......................................................
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1.3.1 Physiological role ..............................................................................................
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1.3.2 ER stress and diabetes mellitus .........................................................................
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1.3.2.1 Lessons from mouse mutants .....................................................................
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a) The Akita mice …...............................................................................................
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b) Mutations of the PERK-eIF2a pathway .............................................................
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1.3.2.2. ER stress and human diabetes ...................................................................
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1.3.2.3. Pathophysiological ER stress inducers ......................................................
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a) ER stress and T1DM: role for cytokines and nitric oxide ................................
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b) ER stress and T2DM: role for free fatty acids and chronic hyperglycemia ….
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1.4. Chemical ER stress inducers: the SERCA blockers ................................................
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1.5. The APOCHIP ….....................................................................................................
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1.5.1. Background .......................................................................................................
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1.5.2. Microarray procedure .......................................................................................
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a) Target preparation and hybridization .................................................................
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b) Scanning .............................................................................................................
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c) Data extraction, normalisation and statistical analysis .......................................
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2. Aims of the study ...........................................................................................................
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3. Results 3.I. Interferon-g potentiates endoplasmic reticulum stress-induced beta-cell death by reducing beta-cell defense mechanisms .........................................................................
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3.II. Transcriptional regulation of the endoplasmic reticulum (ER) stress gene Chop in pancreatic insulin producing cells ..............................................................................
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3.III. Global profiling of genes modified by endoplasmic reticulum (ER) stress in pancreatic beta-cells reveals the early degradation of insulin mRNAs ..........................
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4. General discussion and conclusions ...............................................................................
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5. Future experiments and perspectives .............................................................................. 114 6. Thesis annexe summary .................................................................................................
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7.Curriculum vitae .............................................................................................................. 119 8. Acknowledgments ..........................................................................................................
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9. References ......................................................................................................................
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Composition of the thesis jury: •
Mr. Philippe Lebrun (President) Laboratory of Pharmacology. Université Libre de Bruxelles (ULB), Faculty of Medicine. Brussels, Belgium.
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Mr. Decio L. Eizirik (Secretary) Laboratory of Experimental Medicine. Université Libre de Bruxelles (ULB), Faculty of Medicine. Brussels, Belgium.
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Mrs. Alexandra K. Cardozo (Co-promoter) Laboratory of Experimental Medicine. Université Libre de Bruxelles (ULB), Faculty of Medicine. Brussels, Belgium.
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Mr. Harry Heimberg (Foreign expert) Diabetes Research Center. Vrije Universiteit Brussel (VUB). Brussels, Belgium
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Mr. Peter Vandenabeele (Foreign expert) Department for Molecular Biomedical Research. Flanders Institute for Biotechnology (VIB). Gent, Belgium.
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Mr. Patrick Robberecht (ULB jury member) Department of Biological Chemistry and Nutrition. Université Libre de Bruxelles (ULB), Faculty of Medicine. Brussels, Belgium.
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Mr. Cédric Blanpain (ULB jury member) Interdisciplinary Research Institute (IRIBHM). Université Libre de Bruxelles (ULB), Faculty of Medicine. Brussels, Belgium.
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Mr. André Herchuelz (ULB jury member) Laboratory of Pharmacology. Université Libre de Bruxelles (ULB), Faculty of Medicine. Brussels, Belgium.
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List of reports constituting this thesis: I. Pirot P, Eizirik DL, Cardozo AK, Interferon-γ potentiates endoplasmic reticulum stress-induced death by reducing pancreatic beta cell defence mechanisms. Diabetologia. 2006 49:1229-36. II. Pirot P, Ortis F, Cnop M, Ma Y, Hendershot LM, Eizirik DL, Cardozo AK, Transcriptional regulation of the endoplasmic reticulum (ER) stress gene Chop in pancreatic insulin producing cells. Diabetes. 2007 56:1069-77. III. Pirot P, Naamane N, Libert F, Magnusson NE, Ørtonft TF, Cardozo AK, Eizirik DL, Global profiling of genes modified by endoplasmic reticulum stress in pancreatic beta cells reveals the early degradation of insulin mRNAs. Diabetologia. 2007 50:1006-14.
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Summary The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK. Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the proinflamatory cytokines interleukin-1β (IL-1β) and interferon−γ (IFN-γ), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL-1β alone
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triggers ER stress but does not induce beta-cell death, while IFN-γ neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown. Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis. In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (90%. The apoptotic index was calculated as ð½% apoptotic cells in experimental condition% apoptotic cells in control ½100 % dead cells in controlÞ100 [17, 18]. UPRE luciferase reporter assay The reporter plasmid containing the luciferase gene under the control of five UPREs was kindly provided by Prof. Prywes (Columbia University, New York, NY). INS-1E cells were plated at a density of 140,000 cells/condition in 24-well plates and co-transfected with the UPRE and pRLCMV plasmids (internal control for transfection efficiency) as described elswhere [19]. Twenty-four hours after transfection the cells were treated with IFN-γ and CPA
as described above. Luciferase activities in the cell lysates were determined in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA) using the dual-luciferase reporter assay system according to the manufacturer’s instructions (Promega, Madison, WI, USA). Test values were corrected for luciferase value of the internal control plasmid (pRL-CMV). Statistical analysis Data are shown as mean±SEM, and comparisons between groups were made by paired t test or by ANOVA followed by t test with the Bonferroni correction, as indicated. A p value of ≤ 0.05 was considered statistically significant.
Results CPA induces ER stress and UPR in INS-1E cells To validate the use of CPA as an ER stress-inducing agent, we initially characterised different components of the UPR/ ER stress response induced by CPA in INS-1E cells. For this purpose INS-1E cells were exposed for 6 or 12 h to different concentrations of CPA. A low concentration of CPA (6.25 μmol/l) was sufficient to trigger the UPR response, as indicated by induction of Chop (Fig. 1a), Bip (Fig. 1b) and Xbp1 (Fig. 1c) mRNA expression. These effects were more pronounced in INS-1E cells exposed to 25 μmol/l CPA (Fig. 1). IRE-1α-mediated splicing of Xbp1 in INS-1E cells under basal conditions (upper band 600 bp, Fig. 1d) was 33.2±2.5% and 32.8±1.5% of the total Xbp1 mRNA at 6 and 12 h, respectively. Exposure to 6.25 and 25 μmol/l CPA for 6 h increased the levels of Xbp1s to 44.9± 2.2 and 77±2.3%, respectively (p
