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Highlights: [•] BMP2 induces Y1 receptor expression in C2C12 cells. [•] Smad1/4 induces Y1 receptor expression in C2C12 cells. [•] Y1 gene promoter activity is regulated by BMP2 signaling. [•] Y1 receptor suppression by siRNA increases osteoblast-related gene expression. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, degradation of most intracellular proteins is carried out by the ubiquitin-proteasome pathway. Recent investigations suggest that bone metabolism is also regulated by this pathway. The clinical efficacy of bortezomib, a 26 S proteasome inhibitor used as an anticancer drug, has been linked to an increase in bone formation. In this study, we show that proteasome inhibitors induce expression of osteoblastic differentiation-related genes such as osteocalcin and alkaline phosphatase in C2 C12 cells. In contrast, myogenic differentiation is inhibited. Among the proteasome inhibitors tested, bortezomib induced the greatest increase in osteocalcin expression. Although these effects were similar to that of bone morphogenetic protein ( BMP) 2, proteasome inhibitors did not induce transcriptional activity of Smad1/4-dependent reporter or BMP2 signaling target gene expression. Transient transfection of osteocalcin promoter-luciferase constructs with bortezomib resulted in an increase in luciferase activity. Mutation of OSE2, but not OSE1, sites of the osteocalcin promoter diminished the bortezomib-induced activity. Also, Runx2 binding activity and protein levels were induced by bortezomib treatment. These results suggest that the bortezomib induces osteoblastic differentiation by modifying the activity of Runx2 and that the function of the proteasome in controlling degradation of differentiation-related transcription factors plays an important role in osteoblast differentiation. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that are emerging as important post-transcriptional gene regulators. miR-206 is unique in that it is expressed only in skeletal muscle, including the myoblastic C2C12 cell line. In C2C12 cells, miR-206 expression was reduced dramatically after bone morphogenetic protein (BMP)-2 treatment. The down-regulation of miR-206 expression was also observed after co-transfection with constitutively-active Smad1 and Smad4, which are the intracellular signaling molecules of the BMP pathway. BMP-2 also reduced miR-206 expression in the presence of α-amanitin in a similar manner to that in the absence of α-amanitin. Moreover, the expression of pri-miR-206 was increased upon BMP-2 treatment for 6h compared to that in the absence of BMP-2. These results suggested that BMP-2 down-regulates miR-206 expression at the post-transcriptional level, by inhibiting the processing of pri-miR-206 into mature miR-206, and that BMP-2 could regulate miRNA biogenesis by a novel mechanism. [Copyright &y& Elsevier]

Wnt/β-catenin signaling plays an important role in the developing skeletal system. Our previous studies demonstrated that Wnt/β-catenin signaling inhibits the ability of bone morphogenetic protein (BMP)-2 to suppress myotube formation in the multipotent mesenchymal cell line C2C12 and that this inhibition is mediated by Id1. In this study, we examined the role of intracellular signaling by Wnt/β-catenin and BMP-2 in regulating the expression of osteoprotegerin (OPG) and of the receptor activator of NFκB ligand (RANKL). OPG expression was induced by Wnt/β-catenin signaling in C2C12 cells and osteoblastic MC3T3-E1 cells. Silencing of glycogen synthase kinase-3β also increased OPG expression. In contrast, R expression was suppressed by Wnt/β-catenin signaling. In a transfection assay, β-catenin induced the activity of a reporter gene, a 1.5 kb fragment of the 5′-flanking region of the OPG gene. Deletion and mutation analysis revealed that Wnt/β-catenin signaling regulates transcription of OPG via a promoter region containing two Wnt/β-catenin responsive sites. BMP-2 enhanced Wnt/β-catenin-dependent transcriptional activation of the OPG promoter. In response to BMP-2 stimulation, Smad 1 and 4 interacted with Wnt/β-catenin responsive sites. These results show that the regulation of OPG expression is mediated through two transcription pathways that involve the OPG promoter. [ABSTRACT FROM AUTHOR]