The critical PD0325901 ic50 roles of BMP type I receptors in bone-inducing activity were examined in C2C12 cells. Over-expression of a kinase

domain-truncated ALK3/BMPR-IA or ALK6/BMPR-IB induced resistance to BMP-2 [40]. C2C12 cells expressing dominant negative BMP receptors differentiated into mature myocytes and formed myotubes without the induction of ALP activity, even in the presence of high concentrations of BMP-2 [40]. BAMBI is a natural dominant negative receptor of BMPs because it contains the ligand-binding domain but lacks the kinase domain [41]. Over-expression of a constitutively active form of ALK2, ALK3/BMPR-IA or ALK6/BMPR-IB in C2C12 cells induced an osteoblastic phenotype without the addition of exogenous BMPs [42], [43] and [44]. These results indicate that type I receptors directly control the bone-inducing activity of BMPs. Smad proteins (identified as homologs of Sma and Mad) were found in Caenorhabditis elegans and Drosophila, respectively; SB203580 supplier they are critical transcription factors for the intracellular signal transduction of TGF-β family members [45] and [46]. Smad1, Smad5 and Smad8 are phosphorylated by activated BMP type I receptors at the carboxyl serine–valine–serine (SVS) motif [1], [38] and [47]. A substitution mutation in this SVS

motif that changes it into a DVD (aspartic acid–valine–aspartic acid) sequence constitutively activates the transcriptional activity of Smad1 not only in C2C12 cells in vitro but also in Xenopus embryos in vivo [48]. The mutant Smad1 protein was recognized by a specific antibody directed against phosphorylated Smad1/5/8, suggesting that the three-dimensional structure of the mutant mimics that of phosphorylated Smad proteins [48]. Over-expression of the constitutively active

form of Smad1 induces osteoblastic phenotypes in C2C12 cells without activating BMP receptors, confirming that Smad proteins are substrates and downstream effectors of BMP type I receptors in heterotopic bone induction [48]. Constructing a constitutively active form of Smad1 allowed us to examine the roles of Smad phosphatases, ROS1 which de-phosphorylate the carboxyl terminal that is phosphorylated at the SVS motif [49], [50], [51] and [52]. Co-expression of protein phosphatase, magnesium-dependent 1A (PPM1A) with the constitutively active Smad1 inhibited transcriptional activity and osteoblastic differentiation of C2C12 cells [53]. PPM1A induced the degradation of Smad proteins via a proteasomal pathway, suggesting that another substrate(s) de-phosphorylated by PPM1A increases ubiquitin-ligase activity [53]. Small C-terminal domain phosphatase 1 (SCP1) also inhibits the osteoblastic differentiation of C2C12 cells induced by the constitutively active Smad1 by suppressing transcriptional activity of downstream effectors within the type I receptor-Smad axis [54].