SMAD2 promotes myogenin expression and terminal myogenic differentiation.

SMAD2 is a transcription factor, the activity of which is regulated by members of the transforming growth factor ß (TGFß) superfamily. Although activation of SMAD2 and SMAD3 downstream of TGFß or myostatin signaling is known to inhibit myogenesis, we found that SMAD2 in the absence of TGFß signaling promotes terminal myogenic differentiation. We found that, during myogenic differentiation, SMAD2 expression is induced. Knockout of SMAD2 expression in primary myoblasts did not affect the efficiency of myogenic differentiation but produced smaller myotubes with reduced expression of the terminal differentiation marker myogenin. Conversely, overexpression of SMAD2 stimulated myogenin expression, and enhanced both differentiation and fusion, and these effects were independent of classical activation by the TGFß receptor complex. Loss of Smad2 in muscle satellite cells in vivo resulted in decreased muscle fiber caliber and impaired regeneration after acute injury. Taken together, we demonstrate that SMAD2 is an important positive regulator of myogenic differentiation, in part through the regulation of Myog.

SOX7 Is Required for Muscle Satellite Cell Development and Maintenance.

Satellite cells are skeletal-muscle-specific stem cells that are activated by injury to proliferate, differentiate, and fuse to enable repair. SOX7, a member of the SRY-related HMG-box family of transcription factors is expressed in quiescent satellite cells. To elucidate SOX7 function in skeletal muscle, we knocked down Sox7 expression in embryonic stem cells and primary myoblasts and generated a conditional knockout mouse in which Sox7 is excised in PAX3+ cells. Loss of Sox7 in embryonic stem cells reduced Pax3 and Pax7 expression. In vivo, conditional knockdown of Sox7 reduced the satellite cell population from birth, reduced myofiber caliber, and impaired regeneration after acute injury. Although Sox7-deficient primary myoblasts differentiated normally, impaired myoblast fusion and increased sensitivity to apoptosis in culture and in vivo were observed. Taken together, these results indicate that SOX7 is dispensable for myogenesis but is necessary to promote satellite cell development and survival.

Expression of CCAAT/Enhancer Binding Protein Beta in Muscle Satellite Cells Inhibits Myogenesis in Cancer Cachexia.

Cancer cachexia is a paraneoplastic syndrome that causes profound weight loss and muscle mass atrophy and is estimated to be the cause of up to 30% of cancer deaths. Though the exact cause is unknown, patients with cancer cachexia have increased muscle protein catabolism. In healthy muscle, injury activates skeletal muscle stem cells, called satellite cells, to differentiate and promote regeneration. Here, we provide evidence that this mechanism is inhibited in cancer cachexia due to persistent expression of CCAAT/Enhancer Binding Protein beta (C/EBPß) in muscle myoblasts. C/EBPß is a bzip transcription factor that is expressed in muscle satellite cells and is normally downregulated upon differentiation. However, in myoblasts exposed to a cachectic milieu, C/EBPß expression remains elevated, despite activation to differentiate, resulting in the inhibition of myogenin expression and myogenesis. In vivo, cancer cachexia results in increased number of Pax7+ cells that also express C/EBPß and the inhibition of normal repair mechanisms. Loss of C/EBPß expression in primary myoblasts rescues differentiation under cachectic conditions without restoring myotube size, indicating that C/EBPß is an important inhibitor of myogenesis in cancer cachexia.

Retinoic acid promotes myogenesis in myoblasts by antagonizing transforming growth factor-beta signaling via C/EBPß.

BACKGROUND: The effects of transforming growth factor-beta (TGFß) are mediated by the transcription factors Smad2 and Smad3. During adult skeletal myogenesis, TGFß signaling inhibits the differentiation of myoblasts, and this can be reversed by treatment with retinoic acid (RA). In mesenchymal stem cells and preadipocytes, RA treatment can function in a non-classical manner by stimulating the expression of Smad3. Smad3 can bind to and prevent the bzip transcription factor CCAAT/enhancer-binding protein beta (C/EBPß) from binding DNA response elements in target promoters, thereby affecting cell differentiation. In skeletal muscle, C/EBPß is highly expressed in satellite cells and myoblasts and is downregulated during differentiation. Persistent expression of C/EBPß in myoblasts inhibits their differentiation. METHODS: Using both C2C12 myoblasts and primary myoblasts, we examined the regulation of C/EBPß expression and activity following treatment with TGFß and RA. RESULTS: We demonstrate that treatment with RA upregulates Smad3, but not Smad2 expression in myoblasts, and can partially rescue the block of differentiation induced by TGFß. RA treatment reduces C/EBPß occupancy of the Pax7 and Smad2 promoters and decreased their expression. RA also inhibits the TGFß-mediated phosphorylation of Smad2, which may also contribute to its pro-myogenic activities. TGFß treatment of C2C12 myoblasts stimulates C/EBPß expression, which in turn can stimulate Pax7 and Smad2 expression, and inhibits myogenesis. Loss of C/EBPß expression in myoblasts partially restores differentiation in the presence of TGFß. CONCLUSIONS: TGFß acts, at least in part, to inhibit myogenesis by upregulating the expression of C/EBPß, as treatment with RA or loss of C/EBPß can partially rescue differentiation in TGFß-treated cells. This work identifies a pro-myogenic role for Smad3, through the inhibition of C/EBPß's actions in myoblasts, and reveals mechanisms of crosstalk between RA and TGFß signaling pathways.