Abstract
In skeletal muscle, the transformation of fast into slow fiber type is accompanied by shifts in fiber type-specific gene expression that includes down-regulation of the adult fast fiber myosin heavy chain IId/x (MyHCIId/x) gene. Here, we report that the mitogen-activated protein kinases (MAPKs) p38alpha/beta regulate MyHCIId/x gene expression. Electrical stimulation of rabbit skeletal muscle cells with a slow fiber type activity pattern and treatment of C2C12 myotubes with Ca(2+)-ionophore inhibited p38alpha/beta MAPKs and reduced fast fiber type MyHC protein expression and promoter activity. Pharmacological inhibition of p38alpha/beta also down-regulated MyHCII gene expression. In controls, binding of the myocyte enhancer factor-2 (MEF-2) isoforms C and D as a heterodimer to a proximal consensus site within the MyHCIId/x promoter and recruitment of a transcriptional coactivator, the CREB-binding protein CBP, were observed. Overexpression of wild type MEF-2C but not of a MEF-2C mutant that cannot be phosphorylated by p38 induced promoter activity. Mutation of the MEF-2-binding site decreased the inducing effect of overexpressed CBP. Inhibition of p38alpha/beta MAPKs abolished CBP binding, whereas enforced induction of p38 by activated MAPK kinase 6 (MKK6EE) enhanced binding of CBP and increased promoter activity. Furthermore, knockdown of endogenous CBP by RNA interference eliminated promoter activation by MEF-2C or MKK6EE. In electrical stimulated and Ca(2+)-ionophore-treated myotubes, CBP was absent in complex formation at that site. Taken together, the data indicate that p38alpha/beta MAPKs-mediated coactivator recruitment at a proximal MEF-2 site is important for MyHCIId/x gene regulation in skeletal muscle.
Highlights
Skeletal muscle fibers have been classified into fiber types based on their contraction speed, force development, fatigabil
The possible role of p38 mitogen-activated protein kinase (MAPK) in regulating the adult fast MyHCIId/x isoform was investigated in C2C12 and primary skeletal myotubes
Our finding that p38 MAPK is essential for the maintenance of high levels of basal MyHCIId/x promoter activity extends the role of p38 MAPK in skeletal muscle cells
Summary
MyHC, myosin heavy chain; CBP, CREB-binding protein; CREB, cyclic AMP-responsive element binding protein; CsA, cyclosporin A; EMSA, electrophoretic mobility shift assay; MAPK, mitogenactivated protein kinase; MEF-2, myocyte enhancer factor 2; MKK, MAPK kinase; MKP-1, MAPK phophatase-1; DMEM, Dulbecco’s modified Eagle’s medium; DM, differentiation medium; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; siRNA, small interfering RNA; P-p38, phosphorylated p38. P38␣/ MAPKs Regulate MyHCIId/x Promoter Activity (MKK6EE) reportedly promotes expression of fast but not slow MyHC protein while enhancing differentiation of C2C12 muscle cells [16]. The interaction of p300 with MEF-2C in differentiating C2C12 cells and subsequent acetylation of the transcription factor resulted in enhanced DNA binding and transcriptional activity [30]. Despite their high degree of homology, p300 and CBP are not completely redundant but play unique and distinct roles in gene regulation [28]. Activated p38␣/ MAPKs mediated MyHCIId/x gene expression via recruitment of transcriptional coactivator CBP to a MEF-2C/D heterodimer complex at a proximal MEF-2-binding site in the promoter. Our data reveal a new role for p38␣/ MAPKs in regulating MyHCIId/x gene activity during fiber type transformation in skeletal muscle
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