Regulation of Myostatin Expression and Myoblast Differentiation by FoxO and SMAD Transcription Factors

Abstract

Myostatin, a member of the TGF-β family, plays an important role in regulating skeletal muscle growth and differentiation. The purpose of this work was to determine the role of FoxO and SMAD transcription factors in regulating myostatin gene expression and myoblast differentiation in C2C12 myotubes in vitro. We used RT-PCR to examine FoxO1 and myostatin mRNA expression levels in fast and slow muscle fibers, and myostatin mRNA levels in control and FoxO 1-expressing myotubes. In addition, 1100 bp of the upstream promoter region of the mouse myostatin gene was cloned into a luciferase reporter plasmid and transfected into C2C12 muscle cells. Both myostatin and FoxO1 mRNA expression was greater in fast vs. slow skeletal muscles in vivo. Moreover, expression of a constitutively active form of FoxO1 increased myostatin mRNA and increased activity of a myostatin promoter reporter construct in differentiated C2C12 myotubes. Mutagenesis of highly conserved FoxO or SMAD binding sites significantly decreased myostatin promoter activity, and binding assays showed that both FoxO1 and SMADs 2/3 bind to their respective sites in the myostatin promoter. Treatment with TGF-β and/or over-expression of SMADs 2, 3, or 4 also resulted in a significant increase in myostatin promoter activity. Treatment with TGF-β along with over-expression of SMAD2 and FoxO 1 resulted in the largest increase in myostatin promoter activity. Finally, TGF-β treatment and SMAD2 over-expression greatly potentiated FoxO 1-mediated suppression of myoblast differentiation. Together these data demonstrate that FoxO and SMAD transcription factors regulate the expression of myostatin and contribute to the control of muscle cell growth and differentiation. These transcription factors may also regulate expression of myostatin during periods atrophy associated with decreased muscle use in vivo.