Myostatin auto‐regulates its expression by feedback loop through Smad7 dependent mechanism

Abstract

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.

Myostatin promotes a fibrotic phenotypic switch in multipotent C3H 10T1/2 cells without affecting their differentiation into myofibroblasts

“…The upregulation of Smad7 by myostatin was already observed in an elegant study where myostatin induced the expression of Smad7 in C2C12 myoblasts. The latter in turn inhibited myostatin promoter activity, thus suggesting that myostatin autoregulates its expression by feedback loop through Smad7 (Forbes et al 2006), which involves the interaction of Smad2/3 with the Smad7 promoter (Zhu et al 2004). The fact that Smad7 abrogates myostatin -but not TGF-b1-mediated repression of myogenesis (Kollias et al 2007) -raises the question of why myofibroblast differentiation was not triggered by the myostatin-induced TGF-b1 expression in our experiments.…”

“…An antifibrotic process was simultaneously elicited, as evidenced by the stimulation of the expression of a) the myostatin activity inhibitor, follistatin (Hill et al 2002, Amthor et al 2004, Kocamis et al 2004, b) a Smad signaling inhibitor, Smad7 (Forbes et al 2006), and c) a collagen breakdown inducer, matrix metalloproteinase 8 (MMP-8;Siller-Lopez et al 2004). Follistatin did block the upregulation of TGF-b1 expression by myostatin, as shRNA against myostatin (Magee et al 2006) inhibited the expression of PAI-1, which in turn was stimulated autocrinely by the forced over-expression of myostatin.…”

“…This is likely to occur via the Smad pathway, since myostatin signals through Smad2-4, and phosphorylates Smad3 (Zhu et al 2004). In addition, it triggers a feedback compensatory mechanism through the inhibitory Smad7 that inactivates myostatin promoter, and counteracts myostatin and TGF-b action (Zhu et al 2004, Forbes et al 2006, Kollias et al 2006. In turn, Smad2-4 upregulate myostatin expression by activating its promoter (Zhu et al 2004).…”

Smad3 signaling is required for satellite cell function and myogenic differentiation of myoblasts

“…Among the TGF-β superfamily members, TGF-β and myostatin rely on canonical Smad signaling, specifically Smad2/3, to elicit biological functions during myogenesis [11,12,26]. Since myostatin is a negative regulator of myogenesis, one might expect that loss of Smad3 would enhance myogenesis due to interference with the myostatin-signaling pathway.…”

“…Myostatin is widely known to signal through canonical Smad signaling (mainly Smad3) to inhibit myoblast proliferation, differentiation, SC activation and selfrenewal [12,26,38]. However, other signaling pathways including Smad2 are also shown to be involved in myostatin signaling [9,13,[39][40][41][42].…”