University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2013-05-01 Role of Map4k4 in Skeletal Muscle Differentiation: A Dissertation Mengxi Wang University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cell Biology Commons, Developmental Biology Commons, Molecular Biology Commons, and the Molecular Genetics Commons Repository Citation Wang M. (2013). Role of Map4k4 in Skeletal Muscle Differentiation: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/M2CW28. Retrieved from https://escholarship.umassmed.edu/ gsbs_diss/675 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. ROLE OF MAP4K4 IN SKELTAL MUSCLE DIFFERENTIATION A Dissertation Presented By MENGXI WANG Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY MAY 1, 2013 INTERDISIPLINATY GRADUATE PROGRAM ii ROLE OF MAP4K4 IN SKELETAL MUSCLE DIFFERENTIATION A Dissertation Presented By Mengxi Wang The signatures of the Dissertation Committee signify completion and approval as to style and content of the Dissertation _____________________________________________ Michael Czech, Ph.D., Thesis Advisor _____________________________________________ Anton Bennett, Ph.D., Member of Committee _____________________________________________ Mitsuo Ikebe, Ph.D., Member of Committee _____________________________________________ Yicktung Ip, Ph.D., Member of Committee _____________________________________________ Yong-Xu Wang, Ph.D., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee _____________________________________________ Anthony Imbalzano, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school. _______________________________________________ Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Interdisciplinary Graduate Program May 1, 2013 iii Acknowledgments I would like to thank all my teachers on my long educational journey to this final degree. First of all, I would like to express my deepest gratitude to my thesis advisor Dr. Michael P. Czech for his encouragement, guidance, advice and support, which helped me out from my difficult times. I would also like to thank him for all his efforts in training me in my growth as an independent scientist, and providing a great collaborative working environment in the laboratory. I would like to thank my TRAC meeting committee members, Dr. Anthony Imbalzano, Dr. Yong-Xu Wang, Dr. Mitsuo Ikebe and Dr. Fumihiko Urano for their generous help and support in my graduate research. This dissertation could not have been finished without their encouragement and excellent scientific and technical advice. I would also like to thank Dr. Tony Ip and Dr. Anton M. Bennett for agreeing to serve as dissertation committee members for my thesis defense and their thoughtful contributions on that day. I would like to thank all members of the Czech lab family, past and present, for their inspiration, kindness and support. In particular, I would like to thank Dr. Myriam Aouadi for helping me get started in the laboratory, teaching me mouse work techniques, and sharing her insightful ideas in my research. I would also like to thank Dr. Rachel Roth Flach for her inspirable scientific insights, kind encouragement and excellent teaching skills. I am grateful to Chang-An, Laura, Marina, Ozlem, Emilie, Matthieu, Michaela, Pranitha, Anouch and Adilson, who share joys and tears in life with me, cheer me up iv when I’m feeling down and provide helpful suggestions for my career. You all make the long days and nights (weekends and holidays) at lab full of fun. Special thanks go to my Chinese friends Liting, Liusong, Dan, Wei and others. They are like my family in the US and have been helping me through all the difficult times in my graduate years. I particularly want to thank Liting and Liusong for being there every Friday night to play Chinese poker and chit chat about life in the past five years. It is always the most desirable moment in a week. Finally, I would like to thank my parents Xunyi Wang and Huili Chen who are far away in China but have their hearts with me all the time, and my loving husband Han, for their unconditional love, support and encouragement in my scientific research and daily life, without which I would not have finished my graduate study. v Abstract Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis. Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The vi inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined. In the second part of my thesis, I assess the relationship between Map4k4 and IGF- mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation. The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy. vii Table of Contents Acknowledgments.............................................................................................................. iii Abstract ................................................................................................................................v List of Figures ......................................................................................................................x List of Frequently Used Abbreviations ............................................................................. xii Copyright Information ..................................................................................................... xiv CHAPTER I: Introduction ...................................................................................................1 Myogenesis...................................................................................................................... 1 Myogenic transcription factors .................................................................................... 2 Paired-homeobox transcription factors .................................................................... 2 Myogenic regulatory factors .................................................................................... 3 Interaction of MRFs with transcriptional cofactors ................................................