Knocking out p38α+p38β+p38γ is required to abort the myogenic program in C2C12 myoblasts and to impose uncontrolled proliferation.

The p38 MAPKs' family includes four isoforms, of which only p38α has been considered essential for numerous important processes including mice embryogenesis. It is also considered essential for myoblast to myotube differentiation, as exposure of myoblasts to p38α/β inhibitors or to siRNA that targets p38α suppresses the process. The functions of p38β and p38γ in myoblast differentiation are not clear. We knocked out p38α in C2C12 myoblasts, assuming that the resulting C2p38α(-/-) cells would not differentiate. They did, however, form mature fibers. We found elevated levels and activation of the p38 activator MKK6 in the C2p38α(-/-) cells, leading to activation of p38β and p38γ, which are not active in differentiating parental C2C12 cells. Thus, p38α is an inhibitor of p38β+p38γ, which perhaps replace it in promoting differentiation. To test this notion, we generated C2p38α/γ(-/-) and C2p38α/β(-/-) cells and found that in both clones, the myogenic program was induced. C2p38β/γ(-/-) cells also formed myotubes. These observations could be interpreted in two ways: either each p38 isoform can promote, by itself, the myogenic program, or p38 activity is not required at all for the process. Generating C2p38α/β/γ(-/-) cells in which the myogenic program was shut-off altogether, showed that p38 activity is critical for differentiation. Notably, C2p38α/β/γ(-/-) cells proliferate uncontrollably and give rise to foci, reminiscence of oncogenically transformed cells. In summary, our study shows that a crosstalk between p38 isoforms functions in C2C12 cells as a safeguard mechanism that ensures resilience of the p38 activity in promoting the myogenic program and enforcing cell cycle arrest.