Effect Of Cyclic Stretch On P38MAPK Signaling Pathway During Stretch-induced Diffetentiation Process Of C2C12Myoblasts

Objective:To investigate the effect of cyclic stretch on p38MAPK(Mitogen-Activated Protein Kinase) signaling pathway during stretch-induced differentiation process of C2C12myoblasts.Methods:C2C12cells were seeded in GM at3×105cells/well on Bio-flex Collagen Type I coated6-well plates (Flexcell International, USA) and grown to85%confluence (2-3days). The medium was replaced with DM and cells were subsequently subjected to cyclic mechanical stretch at an optimal magnitude (10%) and frequency (0.5Hz) using the computer-controlled Flexcell Strain Unit. The effects of cyclic stretch were examined following a2,6,12, or24h exposure to cyclic stretch. Unstretched control (untreated) cells in each assay were cultured in DM on Bioflex Collagen Type I coated6-well plates, but not exposed to cyclic stretch. Using western blot analysis with antibodies specific to p38MAPK phosphorylated forms and the total protein levels of the p38MAPK, we ascertained whether static stretch activated p38MAPK signaling pathway in our myoblast culture system.Results:The differentiation specific protein—myogenin was induced after continuously stretched for24h, whereas its expression was undetectable in control groups; To study in parallel the implication of p38in this process, protein extracts were prepared from C2C12cells and p38MAPK activity analyzed using western blotting. The activity of p38(detected by immunoblotting with an antibody specific for its activated form) was induced during the onset of differentiation, whereas no phosphorylation of p38was detected in cells cultured in control groups, confirming a correlation between p38activation and stretch-induced muscle differentiation. Treatment of cells with the specific p38MAPK inhibitor SB203580could decrease the expression of myogenin, but not completely abolish the myogenin expression after being stretched.Conclusions:These results indicated that p38MAPK is activated during stretch-induced C2C12cell differentiation. p38MAPK signaling pathway plays an important role during stretch-induced C2C12cell differentiation, but is not activated exclusively in this process.