Skeletal muscle mass is maintained as a consequence of two main molecular mechanisms: protein synthesis and protein degradation. Despite the profound knowledge of the role of myostatin in the regulation of muscle growth, many details of the molecular mechanisms of its action are poorly understood. It has been suggested that binding of myostatin to the ActRIIB results in the phosphorylation of two serine residues of Smad2 or Smad3 at COOH domains. This leads to the assembly of Smad2/3 with Smad4 to the heterodimer that is able to translocate to the nucleus and activate transcription of target genes [
51‐
53]. One of the known downstream targets of Smad signalling is MyoD, a transcriptional factor that is involved in skeletal muscle development and takes part in the repair of damaged skeletal muscle [
54‐
57]. Downregulation of
myoD expression was shown in vitro and in vivo during cachexia, possibly via TNFα through the induction of the NF-κB pathway. Interestingly, myostatin downregulates
myoD in an NF-κB-independent way [
58,
59]. Myostatin also inhibits Pax3 expression, which is possibly an upstream target of MyoD [
58,
60,
61]. Moreover, Smad signalling targets other genes such as
myf5 and
myogenin, known to be important for myogenesis [
62].
Interestingly, other Smad proteins (Smad6 and Smad7) work as agonists. They compete for the binding to the activin type I receptor and thereby inhibit signalling of TGF-β family members [
63,
64]. Myostatin was also shown to activate Smad7 transcription. Smad7, in turn, is able to inhibit the association of Smad2/3 with Smad4 [
65,
66]. Therefore, the Smad pathway is regulated by feedback control [
67].
Myostatin signalling via Smad has been intensively investigated, but it is not the only feasible signalling pathway. TGF-β family members were shown to activate mitogen-activated protein kinases (MAPKs), particularly p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) [
68‐
71]. p38 MAPK is responsible for the cell response to stress factors and was shown to be activated by myostatin via TGF-β activated kinase 1 (TAK-1)/MAPK kinase (MAPKK) cascade. Its signalling results in the downregulation of myogenesis-related genes, but it does not target Smads [
72]. The role of ERK1/2 in the regulation of muscle mass is controversial. On the one hand, there are studies confirming that ERK1/2 takes part in the process of satellite cell proliferation that is necessary for the maintenance of skeletal muscle weight [
73] and induces protein synthesis under physiological conditions [
74,
75]. On the other hand, there are experiments showing an opposite effect of ERK1/2 activation [
76‐
79]. Thus, the data suggest that increased activity of ERK1/2 leads to the differentiation inhibition in several cell types [
76,
77]. At the same time, myostatin significantly activated ERK1/2 in C2C12 cells. Similar effects were observed in mice during systematic administration of myostatin [
78]. Taken together, it seems likely that myostatin mediates its signal at least partially through ERK1/2 activation. Therefore, different responses through ERK1/2 could be caused by different levels of myostatin corresponding to normal and pathological conditions. MAPK cascade normally involves the activation of Ras/Raf/MEK1. To check whether myostatin uses the same pathways to activate ERK1/2, some experiments were done. Using an inhibitor of MEK1 in C2C12 cells, Yang et al. showed that this kinase is involved in the myostatin-induced activation of ERK1/2 [
78]. Moreover, such inhibition of MEK1 leads to the rescue of cell differentiation, which means that MEK-1/ERK1/2 play a role in differentiation suppression by myostatin. The presence of dominant negative form of Ras was shown to positively influence MEK1/ERK1/2 through the downstream activation of Raf. Therefore, myostatin activates ERK1/2 via Ras/Raf/MEK1 pathway [
78].