RESUMO
Embryonic chick skeletal muscle undergoes profound hypertrophy in response to ectopic IGF-I resulting in two- to three-fold increase in total muscle mass. IGF-I likely causes several changes in gene expression profiles to elicit the robust effect. To identify genes differentially affected by IGF-I, total RNA was isolated from the hindlimbs of chick embryos infected with RCAS or RCAS-IGF-I and used in a subtractive library screen. CMD4 was identified as a novel, avian-specific gene expressed in muscle. In situ mRNA analysis reveals that the gene product is expressed in multiple tissues including skeletal muscle. Ectopic expression of IGF-I within the hindlimb results in a reduction in CMD4 mRNA to levels below conventional detection limits. A chimeric CMD4-yellow fluorescent protein (CMD4-YFP) demonstrates an indiscriminant localization pattern throughout the cytoplasm and nucleus of myoblasts. By contrast to control C2C12 myoblasts, a stable muscle cell line that expresses CMD4-YFP (C2C12-CMD4-YFP) is unable to form the large multinucleated cells characteristic of mature myofibers. The differentiation defective myoblasts do not express myosin heavy chain but the relative amounts of myogenin, desmin and troponin proteins do not differ from controls. The transcriptional activity of the myogenic regulatory factors (MRFs) remains unchanged by CMD4 expression. We report the identification of an IGF-I inhibited gene present in skeletal muscle. While the mechanism of CMD4-mediated inhibition of muscle development remains elusive, we propose that loss of CMD4 gene expression may be required for optimal muscle hypertrophy in the chick embryo.
Assuntos
Proteínas Aviárias/genética , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Insulin-Like I/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Animais , Proteínas Aviárias/fisiologia , Sequência de Bases , Diferenciação Celular , Embrião de Galinha , Biblioteca Gênica , Fator de Crescimento Insulin-Like I/genética , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Músculo Esquelético/citologia , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , TransfecçãoRESUMO
Raf kinase is the upstream activator of MEK1/2 leading to phosphorylation and activation of ERK1/2. Sustained activation of Raf represses skeletal muscle-specific reporter gene transcription and formation of multinucleated myofibers. Inhibition of myogenesis by activated Raf involves downstream ERK1/2 as well as undefined mediators. To identify Raf-interacting proteins that may influence repression of muscle formation, a yeast two-hybrid screen was performed using a MEK1-binding defective Raf (RafBXB-T481A) as bait. Twenty cDNAs coding for Raf-interacting proteins were identified including Ran binding protein 9 (RanBP9), a protein previously reported to interact with receptor tyrosine kinases. Forced expression of RanBP9 in myogenic cells did not alter myogenesis. Co-expression of RanBP9 with constitutively active RafBXB, but not RafBXB-T481A, synergistically inhibited MyoD-directed muscle reporter gene transcription. Knockdown of RanBP9 expression did not restore the differentiation program to Raf-expressing myoblasts. Thus, RanBP9 physically associates with Raf but does not substantially contribute to the inhibitory actions of the kinase.