MICROTUBULE REGULATION IN CYSTIC FIBROSIS PATHOPHYSIOLOGY By: SHARON MARIE RYMUT Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Thomas J Kelley Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY August 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/ dissertation of Sharon Marie Rymut candidate for the Doctor of Philosophy degree* Dissertation Advisor: Thomas J Kelley Committee Chair: Paul N MacDonald Committee Member: Ruth E Siegel Committee Member: Craig A Hodges Committee Member: Danny Manor Committee Member: Rebecca J Darrah Date of Defense: April 29, 2015 * We also certify that written approval has been obtained for any proprietary material contained therein. ii Dedication There are five chapters in this dissertation. To Mom, Dad, Joe, Marie and Susan, I dedicate one chapter to each of you. You can fight about which chapter you want later. iii Table of Contents Table of Contents................................................................................................................iv List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii Acknowledgements ............................................................................................................. x List of Abbreviations ........................................................................................................ xii ABSTRACT ........................................................................................................................ 1 CHAPTER 1: INTRODUCING CYSTIC FIBROSIS AND INFLAMMATORY SIGNALING ....................................................................................................................... 3 1.1 Overview of cystic fibrosis ...................................................................................... 4 1.1.1 Early days of cystic fibrosis ............................................................................... 4 1.1.2 Discovering CFTR ............................................................................................. 6 1.1.3 Importance of CFTR Correctors ........................................................................ 7 1.2 Cystic fibrosis related inflammation ........................................................................ 9 1.2.1 Overview ........................................................................................................... 9 1.2.2 Inflammatory signaling mechanism ................................................................ 11 1.2.3 Ibuprofen and clinical significance .................................................................. 13 1.3 CF Inflammation and Cholesterol Regulation ....................................................... 15 1.3.1 Overview ......................................................................................................... 15 1.3.2 Perinuclear cholesterol accumulation .............................................................. 15 1.3.3 SRE regulation and isoprenoid/cholesterol synthesis ...................................... 16 1.3.4 RhoA ................................................................................................................ 17 1.3.5 Statins .............................................................................................................. 18 1.3.6 Additional CF consequences due to perturbed cholesterol homeostasis ......... 19 1.4 Evidence of disrupted intracellular transport in CF ............................................... 21 1.4.1 Microtubule regulation and dynamics ............................................................. 21 1.4.2 Microtubule structure and function ................................................................. 22 1.4.3 Microtubule Acetylation .................................................................................. 23 1.4.4 Microtubule polymerization ............................................................................ 28 1.4.5 Summary .......................................................................................................... 30 1.5 Statement of Purpose .............................................................................................. 30 iv CHAPTER 2: DECIPHERING A ROLE FOR β-ARRESTIN-2 ..................................... 41 2.1. Abstract ................................................................................................................. 42 2.2 Introduction ............................................................................................................ 43 2.3 Methods .................................................................................................................. 45 2.4 Results .................................................................................................................... 47 2.4.1 Loss of GRK2 function does not change CF consequences ............................ 47 2.4.2 Microtubule disruption increases βarr2 expression ......................................... 48 2.5 Discussion .............................................................................................................. 48 CHAPTER 3: REDUCED MICROTUBULE ACETYLATION IN CYSTIC FIBROSIS EPITHELIAL CELLS ...................................................................................................... 55 3.1 Abstract .................................................................................................................. 56 3.2 Introduction ............................................................................................................ 56 3.3 Materials and Methods ........................................................................................... 58 3.4 Results .................................................................................................................... 63 3.4.1 Ac-tub levels in CF cell models and tissues .................................................... 63 3.4.2 Impact of HDAC6 inhibition on microtubule acetylation and cholesterol trafficking in CF cells ................................................................................................ 64 3.4.3 HDAC6-mediated control of CF-related signaling and NF-κB activation ...... 66 3.4.4. Mechanism linking CFTR to reduced Ac-tub content .................................... 67 3.4.5 Linking ER stress to microtubule acetylation.................................................. 71 3.5 Discussion .............................................................................................................. 71 CHAPTER 4: REDUCED EPAC1 ACTIVITY LEADS TO SLOWED MICROTUBULE POLYMERIZATION IN CYSTIC FIBROSIS EPITHELIAL CELLS ........................... 95 4.1 Abstract .................................................................................................................. 96 4.2 Introduction ............................................................................................................ 97 4.3 Materials and Methods ......................................................................................... 100 4.4 Results .................................................................................................................. 104 4.4.1 Effect of CFTR function on tubulin polymerization ..................................... 104 4.4.2 Microtubule reformation in cultured and primary CF epithelial cells ........... 105 4.4.3 Reduced EPAC1 activation in CF as a mechanism leading to reduced tubulin polymerization rates in CF cells .............................................................................. 106 4.4.4 EPAC1-mediated signaling regulates microtubule polymerization rates ...... 107 v 4.4.5 Mobilization of intracellular cholesterol in response to EPAC1 activation .. 108 4.4.6 Soluble adenylyl cyclase (sAC) control of EPAC1 activation and cholesterol transport ................................................................................................................... 109 4.4.7 Involvement of EPAC1 signaling in sAC-mediated control of cholesterol transport ................................................................................................................... 110 4.4.8 Partial recovery of microtubule polymerization after HDAC6 inhibition ..... 111 4.4.9 Effect of microtubule dysregulation on CF cellular phenotypes ................... 111 4.5 Discussion ............................................................................................................ 112 CHAPTER 5: SUMMARY, FUTURE DIRECTIONS, AND CONCLUSION ............. 137 5.1 Summary .............................................................................................................. 138 5.2 Implications of microtubule dysregulation in cystic fibrosis pathology .............. 138 5.2.1 Microtubule acetylation .................................................................................. 139 5.2.2 Microtubule polymerization .......................................................................... 140 5.2.3 βarr2 as a microtubule associated protein (MAP) ......................................... 141 5.3 Impact of restoring microtubule regulation on organelle transport...................... 142 5.4 Mechanisms for altered microtubule dynamics ..................................................