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Abstract
BackgroundMyogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK) pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified.MethodsRNA interference (RNAi), chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells.ResultsIn a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated.ConclusionsOur findings reveal that Dbn1 expression is a target of p38 MAPK signaling during myogenesis and that drebrin promotes myoblast differentiation.
Highlights
Myogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes
Dbn1 is induced during myogenic differentiation In an effort to identify genes regulated by p38 signaling during myoblast differentiation, microarray analyses were performed on C2C12 cells cultured at low density in GM or differentiation medium (DM) at various time points, plus or minus the p38 inhibitor SB203580
Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that Dbn1 is expressed at very low levels in proliferating primary myoblasts in GM, is expressed at higher levels at the time of shift to DM and increases in abundance over the 2 days in DM (Figure 1A)
Summary
Myogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. In addition to the acquisition of a muscle-specific transcriptional program, the changes in myoblast morphology that occur during differentiation indicate that dramatic alterations in the F-actin cytoskeleton are required for the formation of myofibers. At least some aspects of the cytoskeletal rearrangement and morphological changes that occur during differentiation are likely to be mediated by transcriptional induction of regulators of these processes, as expression of MyoD in fibroblasts induces expression of muscle-specific genes and elongation and fusion into multinucleated myotubes [4]
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