Regulation of osteoblastic differentiation by the proteasome inhibitor bortezomib.

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 26S 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 C2C12 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.

Bone morphogenetic protein-2 enhances Wnt/beta-catenin signaling-induced osteoprotegerin expression.

Wnt/beta-catenin signaling plays an important role in the developing skeletal system. Our previous studies demonstrated that Wnt/beta-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/beta-catenin and BMP-2 in regulating the expression of osteoprotegerin (OPG) and of the receptor activator of NFkappaB ligand (RANKL). OPG expression was induced by Wnt/beta-catenin signaling in C2C12 cells and osteoblastic MC3T3-E1 cells. Silencing of glycogen synthase kinase-3beta also increased OPG expression. In contrast, R expression was suppressed by Wnt/beta-catenin signaling. In a transfection assay, beta-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/beta-catenin signaling regulates transcription of OPG via a promoter region containing two Wnt/beta-catenin responsive sites. BMP-2 enhanced Wnt/beta-catenin-dependent transcriptional activation of the OPG promoter. In response to BMP-2 stimulation, Smad 1 and 4 interacted with Wnt/beta-catenin responsive sites. These results show that the regulation of OPG expression is mediated through two transcription pathways that involve the OPG promoter.

Bone morphogenetic protein-2 down-regulates miR-206 expression by blocking its maturation process.

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 alpha-amanitin in a similar manner to that in the absence of alpha-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.

Regulation of CXCL12 expression by canonical Wnt signaling in bone marrow stromal cells.

CXCL12 (stromal cell-derived factor-1, SDF-1), produced by stromal and endothelial cells including cells of the bone marrow, binds to its receptor CXCR4 and this axis regulates hematopoietic cell trafficking. Recently, osteoclast precursor cells were found to express CXCR4 and a potential role for the CXCL12-CXCR4 axis during osteoclast precursor cell recruitment/retention and development was proposed as a regulator of bone resorption. We examined the role of canonical Wnt signaling in regulating the expression of CXCL12 in bone marrow stromal cells. In mouse stromal ST2 cells, CXCL12 mRNA was expressed, while its expression was reduced in Wnt3a over-expressing ST2 (Wnt3a-ST2) cells or by treatment with lithium chloride (LiCl). Wnt3a decreased CXCL12 levels in culture supernatants from mouse bone marrow stromal cells. The culture supernatant from Wnt3a-ST2 cells also reduced migratory activity of bone marrow-derived cells in a Transwell migration assay. Silencing of glycogen synthase kinase-3ß decreased CXCL12 expression, suggesting that the canonical Wnt signaling pathway regulates CXCL12 expression. In a transfection assay, LiCl down-regulated the activity of a reporter gene, a 1.8kb fragment of the 5'-flanking region of the CXCL12 gene. These results show that canonical Wnt signaling regulates CXCL12 gene expression at the transcriptional level, and this is the first study linking chemokine expression to canonical Wnt signaling.

Role of the Wnt signaling pathway in bone and tooth.

Signaling by the Wnt plays a central role in many processes during embryonic development and adult homeostasis. At least 19 types of Wnts, several families of secreted antagonists and multiple receptors have been identified. Two distinct Wnt signaling pathways, the canonical pathway and the noncanonical pathway have been described. Functional studies and experimental analysis of relevant animal models confirmed the effects of Wnt on regulation of developing mineralized tissue formation and adult homeostasis. In osteoblasts, the canonical Wnt pathway modulates differentiation, proliferation and mineralization, while it blocks apoptosis and osteoclastogenesis by increasing osteoprotegerin. Functional crosstalk between Wnt and bone morphogenetic protein signaling during osteoblastic differentiation has been reported. Recently, non-canonical Wnt signaling was shown to play a role in bone formation. The Wnt signaling pathway also plays an important role not only in tooth formation but also in differentiation and proliferation of cementoblasts and odontoblasts in the tooth. This present review provides an overview of progress in elucidating the role of Wnt signaling pathways in bone and tooth and the resulting possibilities for therapeutic potential.