Abstract
Activated Raf is a potent inhibitor of skeletal muscle gene transcription and myocyte formation through stimulation of downstream MAPK. However, the molecular targets of elevated MAPK with regard to myogenic repression remain elusive. We examined the effects of activated Raf on myogenin gene expression in avian myoblasts. Overexpression of activated Raf in embryonic chick myoblasts prevented myogenin gene transcription and myocyte differentiation. Treatment with PD98059, an inhibitor of MAPK kinase (MEK), restored myogenin expression but did not reinstate the myogenic program. Using a panel of myogenin promoter deletion mutants, we were unable to identify a region within the proximal 829-bp promoter that confers responsiveness to MEK. Interestingly, our experiments identified MEF2A as a target of Raf-mediated inhibition in mouse myoblasts but not in avian myogenic cells. Embryonic myoblasts overexpressing activated Raf were unable to drive transcription from a minimal myogenin promoter reporter, containing a single E-box and MEF2 site, to levels comparable with controls. Unlike mouse myoblasts, forced expression of MEF2A did not synergistically enhance transcription from the myogenin promoter in chick myoblasts, indicating that additional molecular determinants of the block to myogenesis exist. Results of these experiments further exemplify specie differences in the mode of Raf-mediated inhibition of muscle differentiation.
Highlights
One of the downstream kinase cascades responsible for transmission of Ras function includes the archetypical signaling axis Raf/MEK/mitogen-activated protein kinases (MAPK) [9]
Because myogenin is critical for full differentiation of the myocyte and overexpression of activated kinases often leads to loss of myogenic regulatory factors (MRFs) gene expression [20, 21], we reasoned that Raf signaling pathways may inhibit skeletal myogenesis by disruption of myogenin expression and function
Activated Raf Inhibits Myogenin Expression—Early work demonstrated that constitutive Ras activity leads to a loss of MRF expression in myocytes, which may contribute to the inhibitory effects of the kinase [21]
Summary
One of the downstream kinase cascades responsible for transmission of Ras function includes the archetypical signaling axis Raf/MEK/MAPK [9] Initiation of this pathway through “mis-expression” of activated Raf leads to repression of the myogenic gene program and inhibition of myocyte formation [10]. Because myogenin is critical for full differentiation of the myocyte and overexpression of activated kinases often leads to loss of MRF gene expression [20, 21], we reasoned that Raf signaling pathways may inhibit skeletal myogenesis by disruption of myogenin expression and function. Additional transcriptional intermediates are involved in full repression of myogenin gene expression by Raf kinase as MEF2C overexpression is unable to fully overcome the detrimental effects of the Raf/MEK/MAPK signaling module
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