Myostatin, a secreted growth factor, is a member of the TGF-beta superfamily and an inhibitor of myogenesis. Previously, we have shown that myostatin gene expression is regulated at the level of transcription and that myostatin is a downstream target gene of MyoD. Here we show that myostatin gene expression is auto-regulated by a negative feedback mechanism. Northern blot analysis indicated that there are relatively higher levels of myostatin mRNA in the biceps femoris muscle of cattle that express a non- functional myostatin allele (Belgian Blue) as compared to normal cattle. In contrast, addition of exogenous myostatin decreases endogenous myostatin mRNA. Consistent with this result, wild type myostatin protein is able to repress myostatin promoter activity via Activin type IIb receptor (ActRIIB) and ALK5 (P < 0.001). However, non-functional myostatin (Piedmontese) failed to repress the myostatin promoter suggesting that myostatin auto-regulates its promoter by negative feedback inhibition. Auto-regulation by myostatin appears to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by myostatin and the over-expression of Smad7 in turn inhibits the myostatin promoter activity (P < 0.001). In contrast down regulation of Smad7 by siRNA results in increased myostatin mRNA indicating that Smad7 is a negative regulator of myostatin gene expression. Consistent with these results, a decrease in Smad7 mRNA and concomitant increase in myostatin expression is seen in myotubes that express non functional myostatin. In addition, interference with myostatin signaling prevents the induction of Smad7 promoter activity by myostatin. Based on these results, we propose that myostatin auto-regulates its gene expression through a Smad7 dependent mechanism in myogenic cells.

. Molecular analysis of fiber type-specific expression of murine myostatin promoter. Am J Physiol Cell Physiol 287: C1031-C1040, 2004. First published June 9, 2004; 10.1152/ajpcell.00492.2003.-Myostatin is a negative regulator of muscle growth, and absence of the functional myostatin protein leads to the heavy muscle phenotype in both mouse and cattle. Although the role of myostatin in controlling muscle mass is established, little is known of the mechanisms regulating the expression of the myostatin gene. In this study, we have characterized the murine myostatin promoter in vivo. Various constructs of the murine myostatin promoter were injected into the quadriceps muscle of mice, and the reporter luciferase activity was analyzed. The results indicate that of the seven E-boxes present in the 2.5-kb fragment of the murine myostatin promoter, the E5 E-box plays an important role in the regulation of promoter activity in vivo. Furthermore, the in vitro studies demonstrated that MyoD preferentially binds and upregulates the murine myostatin promoter activity. We also analyzed the activity of the bovine and murine promoters in murine skeletal muscle and showed that, despite displaying comparable levels of activity in murine myoblast cultures, bovine myostatin promoter activity is much weaker than murine myostatin promoter in mice. Finally, we demonstrate that in vivo, the 2.5-kb region of the murine myostatin promoter is sufficient to drive the activity of the reporter gene in a fiber type-specific manner. myogenic regulatory factor; E-box; naked DNA MYOSTATIN, A NEW MEMBER of the transforming growth factor-␤ superfamily, is predominantly expressed in developing and adult skeletal muscle. Myostatin-null mice display a two-to threefold increase in skeletal muscle mass that is due to both hyperplasia (i.e., increase in the number of fibers) and hypertrophy (i.e., increase in fiber thickness) (26). Naturally occurring mutations in the myostatin gene coding sequence in Belgian Blue and Piedmontese cattle breeds result in the heavy muscle phenotype (14,22,27). Hence, myostatin functions as a negative regulator of muscle growth.Myostatin expression is detected in myogenic precursors during early embryogenesis, and the expression continues in postnatal skeletal muscle (22,26). Changes in muscle mass have been shown to be related to changes in myostatin expression. Recently, Roth et al. (32) reported that myostatin mRNA levels are reduced in response to heavy-resistance strength training in humans. On the other hand, higher levels of circulatory and muscle myostatin have been observed in humans with acquired immunodeficiency syndrome-related muscle wasting or age-associated sarcopenia (13,25). Furthermore, chronic underfeeding in sheep and hindlimb suspension in rats resulted in increased levels of myostatin (6, 21, 42). Collectively, these results and those described in other reports indicate that myostatin expression is regulated at the transcription level.Although the functional role of myostatin in controlling muscle ma...

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