Cross-talk between glycogen synthase kinase 3ß (GSK3ß) and p38MAPK regulates myocyte enhancer factor 2 (MEF2) activity in skeletal and cardiac muscle.

Characterizing the signaling network that controls MEF2 transcription factors is crucial for understanding skeletal and cardiac muscle gene expression. Glycogen synthase kinase 3ß (GSK3ß) regulates MEF2 activity indirectly through reciprocal regulation of p38MAPK. Cross-talk between GSK3ß and p38MAPK regulates MEF2 activity in skeletal and cardiac muscle. Understanding cross-talk in the signaling network converging at MEF2 control has therapeutic implications in cardiac and skeletal muscle pathology. Glycogen synthase kinase 3ß (GSK3ß) is a known regulator of striated muscle gene expression suppressing both myogenesis and cardiomyocyte hypertrophy. Since myocyte enhancer factor 2 (MEF2) proteins are key transcriptional regulators in both systems, we assessed whether MEF2 is a target for GSK3ß. Pharmacological inhibition of GSK3ß resulted in enhanced MEF2A/D expression and transcriptional activity in skeletal myoblasts and cardiac myocytes. Even though in silico analysis revealed GSK3ß consensus (S/T)XXX(S/T) sites on MEF2A, a subsequent in vitro kinase assay revealed that MEF2A is only a weak substrate. However, we did observe a posttranslational modification in MEF2A in skeletal myoblasts treated with a GSK3ß inhibitor which coincided with increased p38MAPK phosphorylation, a potent MEF2A activator, indicating that GSK3ß inhibition may de-repress p38MAPK. Heart specific excision of GSK3ß in mice also resulted in up-regulation of p38MAPK activity. Interestingly, upon pharmacological p38MAPK inhibition (SB203580), GSK3ß inhibition loses its effect on MEF2 transcriptional activity suggesting potent cross-talk between the two pathways. Thus we have documented that cross-talk between p38MAPK and GSK3ß signaling converges on MEF2 activity having potential consequences for therapeutic modulation of cardiac and skeletal muscle gene expression.

Glycogen synthase kinase 3ß represses MYOGENIN function in alveolar rhabdomyosarcoma.

MYOGENIN is a member of the muscle regulatory factor family that orchestrates an obligatory step in myogenesis, the terminal differentiation of skeletal muscle cells. A paradoxical feature of alveolar rhabdomyosarcoma (ARMS), a prevalent soft tissue sarcoma in children arising from cells with a myogenic phenotype, is the inability of these cells to undergo terminal differentiation despite the expression of MYOGENIN. The chimeric PAX3-FOXO1 fusion protein which results from a chromosomal translocation in ARMS has been implicated in blocking cell cycle arrest, preventing myogenesis from occurring. We report here that PAX3-FOXO1 enhances glycogen synthase kinase 3ß (GSK3ß) activity which in turn represses MYOGENIN activity. MYOGENIN is a GSK3ß substrate in vitro on the basis of in vitro kinase assays and MYOGENIN is phosphorylated in ARMS-derived RH30 cells. Constitutively active GSK3ß(S9A) increased the level of a phosphorylated form of MYOGENIN on the basis of western blot analysis and this effect was reversed by neutralization of the single consensus GSK3ß phosphoacceptor site by mutation (S160/164A). Congruently, GSK3ß inhibited the trans-activation of an E-box reporter gene by wild-type MYOGENIN, but not MYOGENIN with the S160/164A mutations. Functionally, GSK3ß repressed muscle creatine kinase (MCK) promoter activity, an effect which was reversed by the S160/164A mutated MYOGENIN. Importantly, GSK3ß inhibition or exogenous expression of the S160/164A mutated MYOGENIN in ARMS reduced the anchorage independent growth of RH30 cells in colony-formation assays. Thus, sustained GSK3ß activity represses a critical regulatory step in the myogenic cascade, contributing to the undifferentiated, proliferative phenotype in alveolar rhabdomyosarcoma (ARMS).