Abstract
The Raf/MEK/MAPK signaling module elicits a strong negative impact on skeletal myogenesis that is reflected by a complete loss of muscle gene transcription and differentiation in multinucleated myocytes. Recent evidence indicates that Raf signaling also may contribute to myoblast cell cycle exit and cytoprotection. To further define the mechanisms by which Raf participates in cellular responses, a stable line of myoblasts expressing an estrogen receptor-Raf chimeric protein was created. The cells (23A2RafER(DD)) demonstrate a strict concentration-dependent increase in chimeric Raf protein synthesis and downstream phosphoMAPK activation. Initiation of low-level Raf activity in these cells augments contractile protein expression and myocyte fusion. By contrast, induction of high level Raf activity in 23A2RafER(DD) myoblasts inhibits the formation of myocytes and muscle reporter gene expression. Interestingly, treatment of myoblasts with conditioned medium isolated from Raf-repressive cells inhibits all of the aspects of myogenesis. Closer examination indicates that the transforming growth factor-beta(1) (TGF-beta(1)) gene is up-regulated in Raf-repressive myoblasts. The cells also direct elevated levels of Smad transcriptional activity, suggesting the existence of a TGF-beta(1) autocrine loop. However, extinguishing the biological activity of TGF-beta(1) does not restore the myogenic program. Our results provide evidence for the involvement of Raf signal transmission during myocyte formation as well as during inhibition of myogenesis.
Highlights
Overexpression studies and application of specific kinase inhibitors has led to the identification of several essential signaling pathways in skeletal myoblasts
Activated Raf signal transmission represents a powerful disruptor of normal myocyte formation and muscle gene expression
We have demonstrated that forms of activated Raf that initiate activation of differential downstream signaling intermediates cause varying responses on myogenin gene expression that may be correlated to overall signal strength (33). 23A2RafERDD myoblasts were created to provide additional insight into the effects of Raf signal intensity on myocyte formation
Summary
Overexpression studies and application of specific kinase inhibitors has led to the identification of several essential signaling pathways in skeletal myoblasts. Inhibition of MEK/ERK signal transduction does not reinstate the full complement of muscle gene transcription and myoblast fusion to myoblasts expressing constitutively active Ras or Raf (17, 22). A Raf kinase allele that fails to interact with MEK and cause ERK1/2 phosphorylation retained the ability to activate NFB-directed transcription and promote neuronal differentiation (29). These results argue that Raf participates in several downstream signaling cascades. Because many growth factors exhibit contrasting effects on skeletal myogenesis yet utilize many of the same intracellular signaling pathways, it is likely that signal intensity plays a critical role in the decision to complete terminal differentiation To this end, myoblasts that express an inducible activated Raf allele were created. Raf signaling modules are both positive and negative mediators of myogenesis that is a direct reflection of signal strength
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