BIOMECHANICAL FORCES UPREGULATE MYOGENIC GENE INDUCTION IN THE PRESENCE OR ABSENCE OF INFLAMMATION - A POSSIBLE ROLE OF IGFR1-PI3K-AKT PATHWAY DISSERTATION Presented in Partial Fulfillment for the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University by Ravi Chandran D.D.S., M.D.S. * * * * * The Ohio State University 2007 Approved by Dissertation Committee: Dr. Sudha Agarwal, Advisor _____________________________ Dr. Ning Quan Dr. Peter Reiser Advisor Oral Biology Graduate Dr. Denis Guttridge Program ABSTRACT C2C12 myoblasts proliferate in response to mitogens and, upon mitogen withdrawal, differentiate into multinucleated myotubes. Over the past decade, several studies have unraveled important mechanisms by which the four myogenic regulatory factors, MRF’s (Myod1, Myf5, myogenin, and MRF4/Myf6) control the specification and the differentiation of the muscle lineage. The members of bHLH transcription factors act synergistically with the myocyte enhancer binding factor-2 (MEF2) family of co-factors to induce synthesis of muscle restricted target genes. It is well established that Myod1 and Myf5 are required for commitment to the myogenic lineage, whereas myogenin plays a critical role in the expression of the terminal muscle phenotype previously established by Myod1 and Myf5, and MRF4 partly subserves both roles. In this study, we first induced C2C12 myoblasts to differentiate by transferring from growth medium (GM) to differentiation medium (DM) and characterizing the phenotype of C2C12 cells and examining the expression patterns of crucial myogenic factors and its targets genes. Later, by means of an in vitro model system, cyclic equibiaxial stretching and treatment with a proinflammatory cytokine, TNF-α, we have examined the effects of tensile forces imposed on C2C12 myoblasts. The expression pattern of Myod1 showed a considerable presence in the undifferentiated myoblast and during the early stages of differentiation with a modest increase over the final days of myotube formation. Concomitantly, expression of the Mef2a transcription factor and the cell cycle inhibitor, Cdkn1a, were increased, marking the onset of myogenesis and cell cycle withdrawal. The expression of myogenin was little to none during the first 2 days with levels rising rapidly during the next 3 days of differention. As expected, myosin heavy chain isoforms and α-tropomyosin showed a rapid increase following the expression of myogenin and, by day 4, multinucleated myotubes had formed, staining positively for myosin heavy chain protein. ii TNF-α inhibits skeletal muscle differentiation via activation of the NF-κB signal transduction pathway and its subsequent induction of Nos2a and NO production, leading to MYOD1 protein loss. Therefore, we used Nos2a mRNA expression as a biomarker to optimize the dose and magnitude of CTS required for the inhibition of inflammatory responses. The results demonstrate that C2C12 myoblasts respond to mechanical signals in a magnitude-dependent manner, inhibiting rhTNF-α-induced Nos2a expression to various degrees in a magnitude-dependent manner. Next we examined salient members of the bHLH and MEF2 family of proteins to delineate the mechanisms of CTS-mediated regulation of skeletal muscle phenotype in the presence or absence of inflammation. At low physiological levels, CTS activates one of the earliest events in muscle cell differentiation by augmenting rapid and significantly greater expression, synthesis, and nuclear translocation of Myod1. Further, the nuclear Myod1 and myogenin levels are upregulated by CTS even in the presence of TNF-α, and mRNA expression of these genes is significantly increased. The function of bHLH and MEF2 transcription factors, in context of the myogenic differentiation, is to upregulate induction of skeletal muscle structural proteins. CTS markedly upregulates the transcriptional activation of Myh1, Myh2 and Myh4, and synthesis of MYHC and TPM1 proteins. It is well accepted that muscle cells are responsive to biomechanical signals such as those experienced during exercise and can upregulate skeletal muscle hypertrophy. Mechanical signals have been shown to phosphorylate and activate AKT and its downstream target mTOR resulting in increased proteins synthesis in myotubes. Similiarly, mechanical stretch activates epidermal growth factor receptor and angiotensin II type 1 receptor, resulting in AKT phosphorylation. The effects of TNF-α alone or in addition to CTS on IGFR1-PI3K-AKT pathway and the early events leading to AKT activation were examined next. We found that GSK3β was phosphorylated by CTS as opposed to the unstretched groups. A delay in activity, as compared to AKT phosphorylation, possibly could be a result of the intermediate steps that are involved before GSK3β phosphorylation. Further, our results show that after blocking the PI3K activity, using LY294002, the CTS mediated phosphorylation of AKT is blocked at the iii Ser 473. Finally by using different inhibitors, such as AG1024 that blocks the actions of IGFR1 and IR kinases and pertussis toxin that blocks the effects of G-protein coupled receptor, we examined if mechanotransduction occurs at the receptor level by activating multiple substrates including growth factor, insulin and G- coupled receptors. While numerous studies have examined the mechanoresponsiveness of muscle tissues in vivo, only a few studies have elucidated the mechanisms of stretch-mediated upregulation of myogenesis in vitro. Elucidation of such intracellular mechanisms is critical because it provides pieces of missing information to the response of muscle tissues during exercise and other mechanical activities. Biomechanical signal mediated myogenesis is likely to be beneficial if the augmented myogenesis could be reproduced in vivo. iv Dedicated to my family for their unconditional love and unwavering support v ACKNOWLEDGMENTS I would like to first thank my advisor, Dr. Sudha Agarwal. She not only guided me at work and in research but also helped me grow as a person. Her constant motivation to excel was the driving force that made this dissertation a reality. Many thanks to the members of my dissertation committee, Drs. Ning Quan, Peter Reiser and Denis Guttridge, for sharing wisdom, showing encouragement and for their able guidance and thoughtful comments. I would like to express my gratitude to Dr. Thomas Knobloch, for his expert assistance with molecular techniques and offering valuable and insightful feedback on manuscripts. Through my association with him I have gained immense knowledge to fine tune my thought process. I would like to thank all present and past members of the Agarwal’s lab without whose intellectual and technical assistance my research would not have progressed. Specifically, I am grateful to Dr. Mirela Anghelina for helping me with cell biology and Dr. Ewa Wypasek for teaching me the basics of western blotting and immunostaining procedures. Also special thanks to Dr. Agata Zyga, for sharing the workspace, equipments and C2C12 cell cultures along with me as well as being a source of inspiration and an entertainment during work. My former labmates Dr. Ling-Li He, Dr. Sabahattin Bicer, Leonid Polinkovsky, Ryan Bailey and Kelly Moneysmith were also indispensable team players all along. I would also like to thank Dr. Denis Guttridge and his graduate student Nadine Bakkar for providing C2C12 cells and helping with the cell culture techniques. A special thanks to Drs. Micheal Beck and Michael Bailey for helping me with statistics. vi My friends Bjoern Rath, Jin Nam, Danen Sjostrom, Priyangi Perera, Niki Kritikos, Sathya Naryanan, Pattabi Raman, Praveen Gajendrareddy, Sivaraman Prakasam and Jason Nasse for their boundless support and encouragement. I would like to thank my parents, my brother and his beloved wife. Each and every one contributed immensely towards my success. One other person I am deeply indebted to is my longtime pal Shashi Madhavan. If not for him, I wouldn’t have dared to dream. He has been nothing but supportive all along. His patience, kindness and unwavering affection has helped me achieve one of my biggest goals in life. vii VITA September 30, 1977 ……………………... Born; Arni, India 2000 ………………………………………Doctor of Dental Surgery Dr. M.G.R. Medical University India 2003……………………………………….Master of Dental Surgery Oral & Maxillo-facial Surgery Dr. M.G.R. Medical University India 2003 to present ……………………………Graduate Fellow / Research Assistant Department of Oral Biology College of Dentistry The Ohio State University PUBLICATIONS 1. “Neurotransmitter phenotypes of intermediate zone reticular formation projections to the motor trigeminal and hypoglossal nuclei in the rat”. J Comp Neurol. 2005, 488: 28-47. Travers JB, Yoo JE, Chandran R, Herman K, Travers SP. 2. “Biomechanical signals upregulate myogenic gene induction in the presence or absence of inflammation”. Am J Physiol Cell Physiol 293: C267–C276, 2007. Chandran R, Knobloch TJ, Anghelina M, Agarwal S. FIELDS OF STUDY Major Field: Oral Biology. Specific Field of Study: Biomechanical signal transduction, skeletal muscle differentiation, muscle inflammation, repair, regeneration and muscle decay. viii TABLE OF CONTENTS Page ABSTRACT …………………………………………..………..…………………….. ii DEDICATION…………………………………………………….………...………… v ACKNOWLEDGEMENTS………………………………………….…………...…… vi VITA………………………………………………………………………...…….…... viii LIST OF TABLES……………………………………………………………..…….... xii LIST OF FIGURES…………………………………………………………..…..….... xiii LIST OF ABBREVIATIONS………………………………………………..…………