FIGURE 7.

Role of MAPK in TWEAK-induced MMP-9 production in myotubes. C2C12 myotubes were preincubated for 2 h with the indicated concentrations of PD98059 (ERK1/2 inhibitor), SP600125 (JNK1 inhibitor), or SB203580 (p38 kinase inhibitor) followed by treatment with TWEAK (100 ng/ml) for 18 h. A, the transcript level of MMP-9 measured by quantitative real time PCR showed that pretreatment with SB203580 but not PD98059 or SP600125 inhibits the TWEAK-induced expression of MMP-9 in myotubes. B, analysis of culture supernatants by zymography reveled that SB203580 inhibits the TWEAK-induced production of MMP-9 in myotubes. C, C2C12 myoblasts were transfected with control or p38 MAPK siRNA and incubated in differentiation medium for 72 h followed by treatment with TWEAK (100 ng/ml) for 18 h. The production of MMP-9 protein in culture supernatants and the levels of p38 protein in cell extracts were measured by Western blotting. Data presented here demonstrate that siRNA-mediated knockdown of p38 protein inhibits the TWEAK-induced MMP-9 production in myotubes. D, C2C12 myoblasts were transfected with vector alone or dominant negative mutants of MKK3 and MKK6 (equal amounts) along with pNF-κB-SEAP plasmid (1:10 ratio). The myoblasts were differentiated into myotubes and treated with TWEAK (100 ng/ml) for 18 h, and SEAP activity in culture supernatants was measured. Data (mean ± S.D.) presented here show that DN-MKK3/6 significantly inhibits the TWEAK-induced MMP-9 production in myotubes. ^*, p < 0.05, values significantly different from TWEAK-treated myotubes transfected with vector alone.