FIGURE 6.

TAK1 regulates myogenic differentiation through the activation of p38 MAPK. A, TAK1^+/+ and TAK1^−/− MEF were transduced with Ad.MyoD for 24h followed by incubation in DM for the indicated time intervals. Analysis of cell extracts by Western blot showed that the phosphorylation of p38 MAPK protein was completely blocked in TAK1^−/− MEF compared with TAK1^+/+. There was no difference in total p38, phosphorylated AMPK, and total or phosphorylated IκBα levels between TAK1^+/+ and TAK1^−/− MEFs. B, C2C12 myoblasts were stably transfected with vector alone or plasmid expressing either dominant negative TAK1 (dnTAK1) or TAK1 shRNAs and incubated in differentiation medium for 72 h. The levels of total and phospho-p38 MAPK and TAK1 were measured by Western blot. Representative immunoblots presented here show that overexpression of either dnTAK1 protein or TAK1 shRNAs inhibited the levels of phosphorylated p38 in C2C12 cultures. C, TAK1^+/+ and TAK1^−/− MEF were transiently transfected with either pcDNA3-MyoD alone or with pcDNA3-caMKK6 plasmid for 24 h followed by incubation in differentiation medium for additional 72 h. Myotube formation was monitored by immunocytochemistry using MF-20 antibody and 4′,6-diamidino-2-phenylindole. Representative photomicrographs presented here show that transfection with caMKK6 restored the myotube formation in TAK1^−/− MEF cultures. D, shown is quantification of the differentiation index in TAK1^+/+ and TAK1^−/−MEF cultures transfected with MyoD along with caMKK6 or without caMKK6. *, p < 0.01, values significantly different from TAK1^−/− MEF cultures without caMKK6. E, levels of CK measured using a CK activity assay kit and MyHCf and phospho-p38 protein (Western blot) were also found to be significantly increased in TAK1^−/− MEF transfected with caMKK6. *, p < 0.05, values significantly different from TAK1^+/+ MEFs without caMKK6. #, p < 0.05, values significantly different from TAK1^−/− MEFs transfected with no caMKK6.