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Establishing the Role of the Pancreatic Transcription Factor MIST1 in XBP1-Mediated Maintenance of Pancreatic Acinar Cell Homeostasis

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Establishing the Role of the Pancreatic Transcription Factor MIST1 in XBP1-Mediated Maintenance of Pancreatic Acinar Cell Homeostasis Purdue University Purdue e-Pubs Open Access Dissertations Theses and Dissertations Fall 2013 Establishing The Role Of The aP ncreatic Transcription Factor Mist1 In Xbp1-Mediated Maintenance Of Pancreatic Acinar Cell Homeostasis David Alan Hess Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_dissertations Part of the Cell Biology Commons Recommended Citation Hess, David Alan, "Establishing The Role Of The aP ncreatic Transcription Factor Mist1 In Xbp1-Mediated Maintenance Of Pancreatic Acinar Cell Homeostasis" (2013). Open Access Dissertations. 136. https://docs.lib.purdue.edu/open_access_dissertations/136 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By David A. Hess Entitled Establishing the role of the pancreatic transcription factor MIST1 in XBP1-mediated maintenance of pancreatic acinar cell homeostasis Doctor of Philosophy For the degree of Is approved by the final examining committee: R. Claudio Aguilar Chair Stephen F. Konieczny Julia Kirshner Sophie Lelievre To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________Stephen F. Konieczny ____________________________________ Approved by: Peter J. Hollenbeck 5/31/2013 Head of the Graduate Program Date ESTABLISHING THE ROLE OF THE PANCREATIC TRANSCRIPTION FACTOR MIST1 IN XBP1-MEDIATED MAINTENANCE OF PANCREATIC ACINAR CELL HOMEOSTASIS A Dissertation Submitted to the Faculty of Purdue University by David Alan Hess In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2013 Purdue University West Lafayette, Indiana ii TABLE OF CONTENTS Page LIST OF TABLES .......................................................................................................... vi LIST OF FIGURES ........................................................................................................ vii LIST OF ABBREVIATIONS ........................................................................................... xi ABSTRACT ................................................................................................................. xiii CHAPTER 1. INTRODUCTION ....................................................................................... 1 1.1 Regulated secretion is a necessary component of metazoan survival .................. 1 1.2 Quality control mechanisms ensure proper protein folding following synthesis ..... 7 1.3 The unfolded protein response (UPR) is a multi-component compensatory mechanism that maintains cellular homeostasis ........................................................11 1.4 XBP1 is the primary transcription factor utilized by the IRE1 branch of the UPR ..16 1.5 MIST1 is a transcription factor linked to proper functioning of secretory cells.......21 1.6 The unfolded protein response and MIST1 both play roles in maintenance and recovery of acinar cell identity following damage in the exocrine pancreas ................25 CHAPTER 2. MATERIALS AND METHODS ................................................................ 35 2.1 Mouse strains ......................................................................................................35 2.2 Mouse genotyping ...............................................................................................35 2.3 Tamoxifen administration for Cre-mediated induction ..........................................35 2.4 Tissue section preparation, immunohistochemistry, and immunofluorescent staining ......................................................................................................................37 2.5 Electron microscopy ............................................................................................38 2.6 RT-qPCR .............................................................................................................40 iii Page 2.7 Cell Culture ..........................................................................................................40 2.8 Generation of shCtrl, shXbp1, and shMist1 viruses and cell lines ........................42 2.9 Luciferase assays ................................................................................................43 2.10 Bioinformatic analysis of candidate MIST1 effectors ..........................................43 CHAPTER 3. XBP1 IS A NECESSARY COMPONENT OF THE PANCREATIC ACINAR CELL UNFOLDED PROTEIN RESPONSE ....................................................45 3.1 Introduction ..........................................................................................................45 3.2 Generation and testing of Mist1CreER/+; Xbp1fl/fl mice .............................................50 3.3 Xbp1 ablation causes ER stress and activation of an incomplete UPR in Mist1CreER/+; Xbp1ΔEx2 acinar cells ..............................................................................57 3.4 Loss of Xbp1 triggers progressive cell damage in acinar cells .............................60 3.5 Acinar cells lacking Xbp1 die approximately four weeks after ablation .................63 3.6 Discussion ...........................................................................................................65 CHAPTER 4. PANCREATIC DAMAGE IN Mist1CreER/+; Xbp1ΔEx2 MICE TRIGGERS A COORDINATED, MULTI-CELL COMPARTMENT REGENERATIVE RESPONSE ......71 4.1 Introduction ..........................................................................................................71 4.2 Xbp1ΔEx2 pancreata develop a pronounced inflammatory response and activate embryonic signaling components ...............................................................................75 4.3 Pancreata in post-crisis Xbp1ΔEx2 mice have diminished UPR activation and recover normal exocrine parameters .........................................................................79 4.4 Exocrine regeneration following loss of Xbp1ΔEx2 acini is accompanied by proliferation of both the acinar and centroacinar compartments .................................85 4.5 Recovered pancreata retain abnormal long-term histological features but show no evidence of increased pancreatic malignancies .........................................................89 4.7 Discussion ...........................................................................................................95 CHAPTER 5. MIST1 FUNCTIONS AS A TRANSCRIPTIONAL SCALING FACTOR IN THE XBP1-MEDIATED BRANCH OF THE UNFOLDED PROTEIN RESPONSE .........99 iv Page 5.1 Introduction ..........................................................................................................99 5.2 Induction of ER stress leads to activation of MIST1 ........................................... 103 5.3 Mist1 is specifically targeted by XBP1 under conditions of ER stress ................ 109 5.4 Proposed MIST1 targets are induced during ER stress ..................................... 114 5.5 A subset of putative MIST1 targets are expressed in a MIST1-dependent fashion following ER stress .................................................................................................. 120 5.6 Discussion ......................................................................................................... 120 CHAPTER 6. HIGH-LEVEL KRASG12D EXPRESSION IN DEVELOPING PANCREATIC ACINAR CELLS LEADS TO ACINAR-DUCTAL METAPLASIA THAT IS MITIGATED BY MIST1 .................................................................................................................... 124 6.1 Introduction ........................................................................................................ 124 6.2 Generation of Mist1Kras/+ mice ............................................................................ 132 6.3 Mist1Kras/LacZ mice develop extensive ADM that can be rescued by expression of a MIST1myc transgene .............................................................................................. 132 6.4 Mist1Kras/+ pancreata develop increasing numbers of proliferative ductal structures during embryogenesis ............................................................................................. 134 6.5 Ductal complex size is correlated with expression of MIST1 .............................. 142 6.6 Adult Mist1Kras/+ acinar cells are predisposed to ductal complex and cyst formation ................................................................................................................................ 142 6.7 Discussion ......................................................................................................... 145 CHAPTER 7. SUMMARY AND FUTURE DIRECTIONS ............................................. 150 LIST OF REFERENCES ............................................................................................. 156 APPENDICES ............................................................................................................. 173 Appendix A: ChIP Enrichment Scores and E-box Analysis for all MIST1 Candidate Effectors .................................................................................................................
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