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Regulation of Myogenesis by Cardiotrophin-1 and Tgfp Signalling

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Regulation of Myogenesis by Cardiotrophin-1 and Tgfp Signalling Regulation of Myogenesis by Cardiotrophin-1 and TGFp signalling Tetsuaki Miyake A dissertation submitted to the Faculty of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Programme in Biology York University Toronto, Ontario, Canada Jan 2010 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-64946-6 Our file Notre reference ISBN: 978-0-494-64946-6 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. •+• Canada Abstract Skeletal muscle differentiation is a process through which mono-nucleated myoblasts (MBs) form multi-nucleated myotubes (MTs). In this process, a highly orchestrated transcriptional regulatory network controls the program of muscle specific gene expression inducing specific genes in a spatially and temporally appropriate manner. Myogenic regulatory factors (MRFs) are muscle specific, sequence specific DNA binding transcription factors that play a central role in skeletal muscle differentiation. Two of the MRFs, MyoD and Myf5, are essential for myogenic lineage determination. MyoD is expressed in committed myogenic cells but the MBs do not differentiate until they receive appropriate cues. Induction of Myogenin (MyoG, another MRF), a critical downstream target of MyoD, is an absolute requirement for formation of myotubes. Therefore, myogenesis and particularly MyoD activity are inherently sensitive to external signals. Here, we attempted to characterize the roles of two soluble factors in muscle differentiation, namely Cardiotrophin-1 (CT-1) and Tumour growth factor-p* (TGFP). CT-1 is a member of the IL-6 family of cytokines. Although CT-1 is highly expressed in skeletal muscle during embryo development and in the adult, the role of CT-1 in skeletal muscle had not been characterized. It was found that CT-1 inhibits muscle differentiation and regeneration through activation of MEK signalling. Activated MEK physically interacts with MyoD and interferes with MyoD's trans-activation properties. In the presence of CT-1, myog induction is inhibited without affecting MyoD and Myf5 protein levels suggesting that CT-1 maintains the undifferentiated state of MBs by activation of MEK (Chapter III). iv TGFp is the prototypic member of the TGFp super-family of cytokines and a known potent inhibitor of myogenesis. We previously documented that an inhibitory Smad (I-Smad), Smad7, represses receptor regulated Smads (R-Smad) activation by TGF0; yet, Smad7 can not reverse TGFP's inhibitory effect on myogenesis. However, Smad7 enhances myogenesis by interacting with MyoD. These observations suggest that TGFp inhibits muscle differentiation independent of R-Smad activation and that Smad7 promotes myogenesis independent of inhibition of R-Smad activity. Since Smad7 masks the TGFp type I receptor from R-Smads in cytoplasm, we engineered a Smad7 fusion protein with a nuclear localization signal (NLS). This Smad7-NLS protein localizes predominantly in the nucleus and is incapable of inhibition of R- Smad activation by TGFP signalling. However, the Smad7-NLS enhanced muscle differentiation to a similar degree as the wild-type Smad7. Smad7 interacted with the N-terminal region of MyoD in the nucleus. Since MEK also associates with the N- terminal of MyoD, we speculated that Smad7 enhances MyoD's trans-activation properties through attenuation of MEK's inhibitory effect. We demonstrated that Smad7 and MEK functionally antagonize each other in regulating MyoD's trans- activation properties. Therefore, Smad7 promotes myogenesis by enhancing MyoD's trans-activation properties in the nucleus independent of R-Smad inhibition in cytoplasm (Chapter IV). Since Myostatin, another member of the TGFP super-family, activates the ERK pathway, we hypothesized that TGFp inhibits muscle differentiation through activation of MEK/ERK signalling instead of R-Smads. Blockade of R-Smad activation by a pharmacological inhibitor, SIS3, had no effect on the inhibitory effect v of TGFp on myogenesis; however, a pharmacological MEK inhibitor, U0126, partially reversed the inhibitory effect of TGFp. A known downstream target of MEK/ERK signalling c-Jun, which is an inhibitor of MyoD's trans-activation properties, was activated by phosphorylation and accumulated in the nucleus. As previously reported, ectopic expression of c-Jun strongly inhibited MyoD driven myog promoter reporter gene activity. Thus, TGFp inhibits myogenesis by the MEK/ERK/c-Jun pathway independent of activation of R-Smads (Chapter V). These observations position MEK activation as a nexus for convergence of signals that regulate skeletal muscle differentiation; Conservation of this mechanism to regulate the key transcriptional machinery involved in myogenesis by a variety of cytokines indicates a pivotal role for this common cellular pathway in skeletal muscle ontogeny and physiology. VI List of Abbrevations AP-1 Activator protein-1 AMSH Associated Molecule with the SH3 domain of STAM AS-C Achaete-Scute Complex BA Branchial Arche BDNF Brain-Derived Neurotrophic Factor bFGF basic Fibroblast Growth Factor bHLH basic Helix-Loop-Helix BMP Bone Morphogenic Protein BRCT BRCA1 C'-Terminal BRE BMP Response Element CBP CREB Binding Protein CD40 TNF receptor superfamily member-5 Cdc42 Cell division cycle 42 CDH15 M-Cadherin CDK Cyclin Dependent Kinase CES Core Enhancer Sequence ChIP Chromatin Immuno-Precipitation CKI CDK Inhibitor CLC Cardiotrophin-Like Cytokine c-Met hepatocyte growth factor receptor CNTF Ciliary NeuroTrophic Factor C0P9 Constitutive Photomorphogenic-9 CREB Cyclic AMP Responsive Element Binding Protein CRM1 exportin-1 CT Calcitonin CT-1 Cardiotrophin-1 CtBP C'-terminal Binding Protein CTGF/CCN2 Connective Tissue Growth Factor Cxcl12 Chemokine (C-X-C motif) Ligand 12 DML Dorsal Medial Lip DN Dominant Negative DRR Distal Regulatory Region dsDNA double stranded DNA DSL Delta, Serrate/ Jagged, and Lin-12 and Glp phenotype-2 Duspl Dual specificity phosphatase-1 E embryonic day (mouse) ECM Extracellular Matrix EGF Epidermal Growth Factor EMT Epithelial-Mesenchymal Transition ER81 Ets variant-1 ERK Extracellular signal-Regulated Kinase Evil Ectopic Viral integration site-1 FAK Focal Adhesion Kinase FERM Four-point-one, Ezrin, Radixin, and Moesin FGF Fibroblast Growth Factor FIK Fibroblast Intermediate conductance calcium-activated potassium channel FLRG Follistatin-Related Gene Fox03 Forkhead box 03 Vll FSTN Follistatin Furin paired basic amino acid cleaving enzyme GADD34 Growth Arrest and DNA Damege-34 GATA GATA binding protein GDF8 Growth Differentiation Factor-8 Gig1 Glucocorticoid-lnduced Gene-1 gp130 Glycoprotein 130 GR Glucocorticoid Receptor Grb2 SH2 domain of Growth factor Receptor Bound protein GSK3p Glycogen Synthase Kinase-36 HAT Histone Acetyltransferase HDAC Histone Deacetylase HER v-erb-b2 erythroblastic leukemia viral oncogene homolog Hes7 Hairy and enhancer of split-7 HGF Hepatocyte Growth Factor Hox Homeobox IBD Inflammatory Bowel Disease IFN Interferon IGF1 Insulin-like Growth Factor-1 IGFBP5 Insulin-like Growth Factor-Binding Protein-5 IL Interleukin Itch Itchy E3 ubiquitin protein ligase homolog Jab1/CSN5 COP9 constitutive photomorphogenic homolog subunit-5 JAK Janus Kinase JNK c-Jun N'-terminal Kinase kb kilo-base KLF10/TIEG1 Kruppel-Like Factor-10 LEF Lymphoid Enhancer-binding Factor LIF Leukemia Inhibitory Factor LIFRp LIF 3-Receptor LIM a-actinin-2-associated LIM protein LIMK1 LIM Kinase-1 LRP1 Lipoprotein-receptor Related Protein MAML1 Mastermind-Like-1 MAPC Multipotent Adult Progenitor Cell MAPK Mitogen-Activated Protein Kinase MB Myoblast MCM1 yeast mating type decisions MDSC Muscle-Derived Stem Cell MEF2 Myocyte Enhancer Factor-2 MefgflO Multiple epidermal growth factor repeat transmembrane protein MEKK MAPK Kinase Kinase MH2 MAD (Mothers against decapentaplegic)
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