Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin.

Muscle growth results from a set of complex processes including myogenic transcription factor's expression and activity, cell cycle withdrawal, myoblast fusion in myotubes, and acquisition of an apoptosis-resistant phenotype. Myostatin, a member of the TGFbeta family, described as a strong regulator of myogenesis in vivo Nature 387 (1997), 83; FEBS Lett. 474 (2000), 71 is upregulated during in vitro differentiation Biochem. Biophys. Res. Commun. 280 (2001), 561. To improve characterization of myostatin's myogenic influence, we stably transfected vectors expressing myostatin and myostatin antisense in C2C12 myoblasts. Here, we found that myostatin inhibits cell proliferation and differentiation. Our results also indicate that myogenin is an important target of myostatin. In addition, overexpressed but not endogenous myostatin decreases MyoD protein levels and induces changes in its phosphorylation pattern. We also established that myostatin overexpression reduces the frequency of G0/G1-arrested cells during differentiation. Conversely, inhibition of myostatin synthesis leads to enhanced cell cycle withdrawal and consequently stimulates myoblast differentiation. We examined the expression patterns of the pRb, E2F1, p53, and p21 proteins involved in cell cycle withdrawal. We found that myostatin overexpression increases p21 and p53 expression, as it does accumulation of hypophosphorylated Rb. Interestingly, myostatin overexpression strongly reduced low-mitogen-induced apoptosis, whereas antisense expression induced contrary changes. In conclusion, these data show the influence of overexpressed myostatin on myoblast proliferation, differentiation, and apoptosis is extended to endogenous myostatin. Though some differences in overexpression or inhibition of endogenous myostatin were observed, it appears that myogenin and p21 are essential targets of this growth factor.