Myoblasts, including C2C12

cells, are maintained in vitro in the presence of highly concentrated serum to sustain their self-renewal ability and to inhibit their differentiation. Over-expression of Smad7 in C2C12 cells markedly enhanced their myogenic differentiation even in the presence of concentrated serum, suggesting that the TGF-β family is involved in the inhibition of myogenesis by serum [55]. This result also indicated that serum contains a BMP-like activity because serum induced the expression of the Id1 gene and ALP activity in C2C12 cells, which were inhibited by adding a soluble form of ALK3/BMPR-IA learn more or noggin [55]. Bovine BMP-4 was identified in the highly purified BMP-like fraction from calf serum [55]. BMP-9 is also found in the circulating blood of humans, suggesting that BMPs are not only local factors but also systemic factors [56]. The phosphorylation of Smad1/5/8 is induced within 1 h after BMP stimulation, and the transcription of several genes is also induced by BMPs within the same period. Id1 was identified as a dominant-negative regulator of MyoD activity and an inhibitor of myogenesis [57] and [58]. The expression of Id1 mRNA

is up-regulated within 1 h in C2C12 cells following BMP-2 stimulation, suggesting that the induction Src inhibitor of Id1 expression would inhibit myogenesis [37]. A GC-rich 29-bp region was identified as a BMP-responsive element (BRE) in the Id1 gene, and this BRE is conserved 100% in mice and humans [59]. Smad1 and Smad4 form a DNA–protein complex with the BRE in response to BMP-2 stimulation [59]. Smad4 is an essential co-activator of Id1 expression because deletion of the Smad4 gene prevented its transcription in response to BMP stimulation [48] and [59]. In addition to Id1, Id2 and Id3 are also early-responsive genes of BMP signaling, and GC-rich

Smad-binding elements were identified in these genes [60], [61] and [62]. A novel non-coding RNA, called BMP-inducible transcript-1 (BIT-1), was identified by searching databases for the conserved Montelukast Sodium BRE sequence [62]. The expression of BIT-1 mRNA is increased in C2C12 cells in response to BMP-4 stimulation [62]. Although BIT-1 mRNA is highly expressed in cartilage, the physiological role of BIT-1 remains unknown. Among the genes induced by BMP stimulation, osterix was shown to play an important role in bone formation [63]. Osterix (also called SP7) was identified as a transcription factor that is expressed in C2C12 cells treated with BMP-2 but not in the parental C2C12 cells [63]. The level of Osterix mRNA is increased up to 24 h after the BMP-2-stimulation of C2C12 cells and is specifically expressed in bone tissue in vivo [63].