MOLECULAR MECHANISMS OF NF-κB REGULATION OF SKELETAL MYOGENESIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nadine A. Bakkar, M.S. The Ohio State University 2008 Dissertation Committee: Approved by Denis Guttridge, PhD, Advisor Jill Rafael-Fortney, PhD _______________________ Gustavo Leone, PhD Advisor Michael Ostrowski, PhD Graduate Program in Molecular, Cellular and Developmental Biology i ii ABSTRACT NF-κB is a ubiquitous transcription factor involved in the regulation of innate immunity, cellular survival, proliferation, as well as differentiation. Its deregulation is associated with various diseases, and have thus been the target of developing therapeutic strategies. Skeletal muscle diseases are one field where this transcription factor is receiving recent attention, owing to its implication in muscular dystrophy, wasting and regeneration. In this dissertation, we focused on NF-κB regulation of myogenic differentiation, in an attempt to further understand the complex ways this transcription factor follows to regulate muscle development and extrapolate it to disease. In chapter 2, we focused on Myostatin (Mstn) a potent negative regulator of myogenesis that can inhibit myoblast proliferation and suppress synthesis of MyoD. NF-κB is similarly able to promote myoblast growth and induce loss of MyoD message. Given the similarities of these phenotypes, we examined potential Mstn and NF-κB signaling crosstalks in myoblasts and differentiated myotubes. Results show that Mstn does not activate NF-κB, nor does activated NF-κB induce Mstn expression. Furthermore, Mstn inhibition of differentiation can still occur in cells devoid of NF-κB activity. Such findings were confirmed in proliferating muscle precursors as well as mature muscle fibers and thus highlight the intrinsic differences between those two signaling pathways in the regulation of skeletal myogenesis, ii In chapter 3, we examined NF-κB regulation of skeletal myogenesis using genetic knockout models. Previous studies had attempted to investigate such a regulation, yet results remained perplexing, with both pro- and anti-myogenic roles of NF-κB documented. Using primary myoblasts and muscles devoid of NF-κB classical pathway components p65 or IKKβ, we show that this canonical signaling is a negative regulator of myogenesis and gets downregulated during differentiation. On the other hand, NF-κB alternative signaling, mediated by IKKα activating the RelB/p52 complex is turned on and regulates mitochondrial biogenesis. Such a pathway is hence involved in the energy production and subsequent maintenance of newly formed myotubes. Consequently, our findings help to resolve the conundrum of NF-κB signaling in myogenesis by showing the existence of two opposing NF-κB pathways that function at temporally distinct stages of differentiation: classical signals inhibit premature myogenesis while alternative pathway activation regulate energy production and maintenance of nascent myofibers. Collectively, results presented in this dissertation highlight the various branches through which NF-κB signals to regulate skeletal myogenesis, emphasizing the need to take this complex regulation into account in clinical strategies aimed to modulate its activity. iii Dedicated to my family and friends. iv ACKNOWLEDGMENTS First, I would like to thank my advisor, Denis Guttridge. I think I was pretty lucky in having you as a benchmate/lab partner during the first year that I joined sharing experiment tips as well as restaurant reviews, political discussions, french lessons, or independent movie recommendations. And although you later moved to your office, I am still able to chat with you about anything. More importantly, you’ve taught me to think outside “the box” and to keep an open mind and question known dogmas. And although I hate to admit it in public to my friends, I do enjoy getting new data to step into your office and discuss them with you. You’ve helped me grow as a scientist while making sure I keep a healthy balance between scientific career and personal life. To past and present members of the Guttridge lab, it’s been a real pleasure working with you guys as well as organizing floor parties and going out. You made me feel like I belong to a group and we have shared more than I could ever describe, from nerdy science talks over lunch to teaching me american slang and shopping . Thank you for making the work atmosphere so enjoyable. Special thanks go to Erin, Jay and Mike for being above all my first American friends; Jingxin, Kate, and Huating for their constant guidance; Jen, Tara, Wei, Jeff, Swarnali, Lori and Erik for all the good times and the laughs in the lab and outside. v I would also like to thank Drs Jill Rafael-Fortney, Gustavo Leone and Michael Ostrowski for taking time out of their busy schedules to serve on my committee, as well as their helpful advice. To all my friends for always being there and making Columbus my home away from home; thank you.. Ihab, I cannot thank you enough for your unconditional and constant support; Sama, Joe, Danielle, Sleiman, Mirna, Fadi, thank you for always being there to share the good times and the bad ones...Francisco, thank you for all the laughs, coffee breaks and Spanish lessons…Myrna and Lyne, even though you are not physically around, thank you for being “there”…To all my friends all over the world, Hilda, Wassim, Nesrine, Hisham, Mazen to name a few, thank you for being my friends… And finally, I cannot but thank my mother Nawal, my father Ali and my brother Walid for believing in me, supporting me in every possible way, and always ALWAYS being proud of me. vi VITA May 8th, 1978………………………………Born- Beirut, Lebanon June 1999…………………………………..Bachelor of Science in Biology The American University of Beirut Beirut, Lebanon June 2001 …………………………….........Master of Science in Biology The American University of Beirut Beirut, Lebanon Sept 2001-Aug 2002……………………….Research Associate, Dept of Pediatrics The American University of Beirut Beirut, Lebanon Sept 2002-Present……………………… PhD Candidate, Molecular Cellular and Developmental Biology Graduate Program, The Ohio State University, Ohio, USA PUBLICATIONS Bakkar N, Wang J, Ladner KJ, Wang H, Dahlman JM, Carathers M, Acharyya S, Rudnicki MA, Hollenbach AD, Guttridge DC. IKK/NF-kappaB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis. J Cell Biol. 2008 Feb 25;180(4):787-802. Wang H, Hertlein E, Bakkar N, Sun H, Acharyya S, Wang J, Carathers M, Davuluri R, Guttridge DC.NF-kappaB regulation of YY1 inhibits skeletal myogenesis through transcriptional silencing of myofibrillar genes. Mol Cell Biol. 2007 Jun;27(12):4374-87. vii Acharyya S, Villalta SA, Bakkar N, Bupha-Intr T, Janssen PM, Carathers M, Li ZW, Beg AA, Ghosh S, Sahenk Z, Weinstein M, Gardner KL, Rafael-Fortney JA, Karin M, Tidball JG, Baldwin AS, Guttridge DC. Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. J Clin Invest. 2007 Apr;117(4):889-901. Bakkar N, Wackerhage H, Guttridge DC. Myostatin and NF-κB regulate skeletal myogenesis through distinct signaling pathways. Signal Trasduction. 2005 4: 202-210. Hertlein E, Wang J, Ladner KJ, Bakkar N, Guttridge DC. RelA/p65 regulation of IkappaBbeta. Mol Cell Biol. 2005 Jun;25(12):4956-68. Mikati MA, Holmes GL, Werner S, Bakkar N, Carmant L, Liu Z, Stafstrom CE. Effects of nimodipine on the behavioral sequalae of experimental status epilepticus in prepubescent rats. Epilepsy Behav. 2004 Apr;5(2):168-74. Mikati MA, Shamseddine A, Sabban M, Dbaibo G, Kurdi R, Abi Habib R, and Bakkar N. Time course of changes in apoptotic signal transduction factors during and after experimental status epilepticus. AES Proceedings. Epilepsia 2002. Vol 43 s7:1-375. Abstract 1.044 Gali-Muhtasib H, Bakkar N. Modulating cell cycle: current applications and prospects for future drug development. Curr Cancer Drug Targets. 2002 Dec;2(4):309-36. FIELD OF STUDY Major Field: Molecular, Cellular and Developmental Biology viii TABLE OF CONTENTS ABSTRACT.................................................................................................................................... ii ACKNOWLEDGMENTS .............................................................................................................. v VITA............................................................................................................................................. vii LIST OF FIGURES ....................................................................................................................... xi LIST OF ABBREVIATIONS......................................................................................................xiii CHAPTER 1 ................................................................................................................................... 1 INTRODUCTION ....................................................................................................................1 1.1 NF-κB Signaling................................................................................................... 1 NF-κB and IκB Family Members...................................................................... 1 The IKK Complex.............................................................................................. 4 Upstream Activators of NF-κB.........................................................................