The cells of the 10T1/2 line are pluripotent and differentiate into myoblasts, adipocytes and chondrocytes following treatment with 5-aza-cytidine [33]. The expression of MyoD, a master gene for skeletal muscle differentiation, was identified as a gene Y-27632 datasheet expressed in the 5-aza-cytidine-treated 10T1/2 cells but not in the parental cells [34]. BMP-2 stimulated osteoblastic differentiation in cells of the ROB-C26 line, which are pluripotent cells that were established from rat calvaria [35]. C2C12 cells are murine myoblasts established from cells of the regenerating thigh muscles of C3H mice [36]. C2C12 cells are widely used to study myogenesis in vitro because they grow while
expressing MyoD, differentiate into myocytes and form multinucleated myotubes that express contractile proteins, such as myosin heavy chain and troponin T. Treatment with BMP-2 inhibits the expression of the myogenic phenotype in C2C12 cells and maintains them as mononuclear cells [37].
Moreover, BMP-2 induces osteoblastic features in C2C12 cells, including high levels of ALP activity and the expression of PTHR and osteocalcin [37]. TGF-β1 does not induce osteoblastic characteristics in these cells, although it inhibits myogenic differentiation [37]. Therefore, the induction Crizotinib in vivo of osteoblastic characteristics in C2C12 cells correlates with the heterotopic bone-inducing activity of the TGF-β members that is observed in vivo. The in vitro activities of 14 types of BMPs (BMP-2 through BMP-15) were directly compared using
an adenoviral-expression system in vitro and in vivo [23]. Among these factors, BMP-2, BMP-6 and BMP-9 induced high levels of ALP activity in C2C12 cells [23]. Moreover, C2C12 cells transduced with BMP-2, BMP-6, BMP-7 or BMP-9 constructs induced bone formation in the quadriceps muscle of nude mice in vivo [24]. BMPs bind to transmembrane serine/threonine kinase receptors, which are classified into type I and type II receptors based on their amino acid sequences (Fig. 2) [38]. Type I receptors, which include ALK1, ALK2, ALK3/BMPR-IA and ALK6/BMPR-IB, contain a conserved glycine/serine-rich domain (GS domain) inside of the transmembrane domains. The kinase activity of type II receptors, which include BMPR-II, ActR-IIA and ActR-IIB, is constitutively activated regardless of whether they are bound to BMPs. In contrast, the kinase Depsipeptide price activity of type I receptors is inactive in the absence of ligands but is activated by type II receptor kinases via phosphorylation of the GS domain in response to ligand binding. The signal transduction role of phosphorylation in the GS domain has been elucidated by identifying constitutively active type I receptors in which the conserved residues of the GS domain have been replaced with acidic amino acids to introduce a negative charge that mimics the phosphorylated state [39]. The constitutively activated type I receptors activate intracellular signaling in the absence of ligands [39].