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Myostatin is subject to regulation at all levels, ensuring coordinated and tightly controlled activity. A number of mechanisms are apparent including post-translational modification, competition for receptor binding and feedback control.

Transcriptional regulation

The myostatin promoter contains a number of transcription factor and hormone binding sites. Eight putative E-boxes corresponding to MyoD binding sites have been identified (Du, Chen et al. 2005). Stimulation of promoter activity by MyoD suggests that direct up-regulation of myostatin expression is involved in the ability of MyoD to control differentiation (Joulia-Ekaza and Cabello 2006). The down-regulation of MyoD expression by myostatin signaling (Langley, Thomas et al. 2002) suggests that feedback mechanisms are in place.

The androgen, progesterone and glucocorticoid response elements were also observed (Du, Chen et al. 2005). Glucocorticoid-induced muscle atrophy is a well-known condition observed during treatment of inflammatory disorders in humans (Joulia- Ekaza and Cabello 2006); an increase in myostatin gene expression by glucocorticoids may contribute to muscle atrophy, with a dose-dependent induction of endogenous myostatin transcription in response to dexamethasone reported (Joulia- Ekaza and Cabello 2006). FoxO1 regulatory sites are present in the myostatin promoter region (Allen and Unterman 2007). FoxO1 overexpression leads to an upregulation of myostatin activity and FoxO transcription factors have been suggested to play a central role in the regulation of gene expression during skeletal muscle atrophy.

Post-translational processing and modification

As introduced previously, production of myostatin as a precursor protein constitutes a significant means of inhibition, as the precursor must be cleaved either intra- or extracellularly by furin convertases to generate a latent complex. This complex can be purified from the serum of mammalian cells (Lee and McPherron 2001) with myostatin growth factor activity only detected following acid treatment to disrupt the latent complex (Hill, Davies et al. 2002; Zimmers, Davies et al. 2002). Transgenic mice expressing high levels of the myostatin propeptide exhibit dramatic increases in skeletal muscle mass comparable to those seen in the myostatin-knockout mouse, indicating that the propeptide inhibits myostatin function in vivo (Lee and McPherron 2001). The propeptide domain may compete for receptor-binding; studies of the BMP-7 latent complex suggest that type II receptors displace binding of TGF-β family propeptide domains to the growth factor (Sengle, Ono et al. 2008).

Intracellular binding partners

In skeletal muscle, myostatin can be found complexed with hSGT (human small glutamine-rich TPR-containing protein), a protein required for progress through cell division (Winnefeld, Grewinig et al. 2006). hSGT interacts with the N-terminal signal peptide region of myostatin, suggesting an inhibition of myostatin translation and/or translocation (Joulia-Ekaza and Cabello 2006).

The sarcomeric protein titin cap binds to mature myostatin, appearing to prevent latent complex formation and consequently inhibit myostatin secretion; overexpression of the titin cap inhibits the antiproliferative influence of myostatin without alteration in myosatin production (Nicholas, Thomas et al. 2002; Joulia- Ekaza and Cabello 2006). However, the relationship between the sarcomeric localization of the titin cap protein and the secreted nature of myostatin is unclear.

Extracellular binding partners

Follistatin is a major TGF-β family antagonist (Lee and McPherron 2001); transgenic mice over-expressing follistatin show increased muscling (Lee and McPherron 2001). The crystal structure of myostatin in complex with follistatin provides key information regarding this inhibition (Cash, Rejon et al. 2009). Two follistatin molecules wrap around the myostatin growth factor dimer and completely block all four receptor binding sites, as seen in the activin:follistatin complex (Thompson, Lerch et al. 2005). The myostatin:follistatin interaction is more intimate than the corresponding activin complex, with a larger amount of buried surface area (Cash, Rejon et al. 2009). Comparison of the published structures suggests that follistatin adopts alternate conformations to adapt to the shape of the different ligands (Thompson, Lerch et al. 2005; Cash, Rejon et al. 2009). The key interaction between myostatin and follistatin involves hydrogen bonding between the ‘fingertip’ motif of myostatin and the N-terminal domain helix of follistatin (Cash, Rejon et al. 2009); the latter is suggested to confer specificity to the antagonist. In addition to a blockage of receptor binding, complexed follistatin is known to bind heparin on the cell surface causing cellular uptake and ligand degradation. Follistatin complexed with myostatin has increased affinity for heparin (Cash, Rejon et al. 2009), suggesting two modes of myostatin regulation by follistatin.

Two other myostatin-binding proteins have been detected complexed with myostatin in serum; the follistatin-related gene (FLRG) and the growth and differentiation factor-associated serum protein-1 (GASP-1) (Hill, Qiu et al. 2003). FLRG is known to bind and inhibit the action of other TGF-β family members in vitro and functional studies show that FLRG inhibits myostatin activity in a reporter gene assay (Hill, Davies et al. 2002). GASP-1 binds directly to both mature myostatin and its propeptide and contains multiple protease domains, suggesting function is via inhibition of propeptide cleavage, preventing activation of mature myostatin (Hill, Qiu et al. 2003).

Myostatin activity is also modulated by decorin, a small leucine-rich ECM-localised proteoglycan previously shown to bind and regulate the activity of TGF-β1 (Miura, Kishioka et al. 2006); decorin binding prevented the growth inhibitory effect of myostatin on C2C12 myoblasts.

Autoregulation by myostatin

Myostatin signaling has been shown to negatively regulate the expression of furin convertase (McFarlane, Langley et al. 2005). Myostatin exhibits reduced processing during differentiation; it is possible that this is due to a down-regulation of furin protease activity by an increase in myostatin expression prior to differentiation.

Myostatin upregulates the expression of Smad7, an inhibitory Smad (Forbes, Jackman

et al. 2006). Smad7 overexpression leads to an inhibition of myostatin gene

expression and the inhibition of Smad7 expression by RNA interference increases myostatin promoter activity. These mechanisms may autonomously control myostatin expression in response to extracellular myostatin levels.