Activated raf kinase inhibits muscle cell differentiation through a MEF2-dependent mechanism

Muscle cell development is dependent on the activity of cell type-specific basic-helix-loop-helix transcription factors, MyoD, Myf-5, myogenin, and MRF4 which collaborate with myocyte enhancer factor 2 proteins to activate muscle-specific gene expression. Growth factors and activated Ras prevent differentiation of myoblasts in culture but the downstream signalling pathways are not well understood. Here, we demonstrate that active Raf kinase (Raf-BxB) completely inhibits myogenic conversion of 10T1/2 cells mediated by Myf-5 and differentiation of L6 myoblasts as indicated by the absence of myotubes, lack of myogenin expression, and markedly reduced expression of myosin heavy chain. However, activated Raf inhibits transcriptional activation by Myf-5 only partially suggesting that other potential targets of Ras/Raf signalling may be involved. Significantly, we observed that elevated Raf kinase activity in L6 muscle cells suppresses the accumulation of MEF2 protein in nuclei, while MEF2 transcription appears unaffected. Moreover, forced expression of MEF2A in 10T1/2 cells rescues MyoD dependent myogenic conversion in the presence of constitutively active Raf kinase and partially restores transactivation of a myogenin promoter-dependent reporter gene in L6 muscle cells containing activated Raf kinase. From these observations we conclude that persistent activation of Raf signalling affects nuclear MEF2 functions which may explain why myogenin expression and myoblast differentiation are inhibited.