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Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF-kappaB ligand (RANKL) to enhance in vitro differentiation of osteoclast-like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow-derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p-SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p-SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL-stimulated formation of multinuclear TRAP-positive cells. The BMP antagonist noggin inhibited RANKL-mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts-like cells., ((c) 2009 Wiley-Liss, Inc.)

As part of its catabolic action in bone, parathyroid hormone (PTH) inhibits extracellular matrix mineralization. We previously showed that PTH dose-dependently induces matrix gla protein (MGP) expression in osteoblasts and this induction is at least partially responsible for PTH-mediated inhibition of mineralization. Recently, we identified PKA and ERK/MAPK as the key signaling pathways involved in PTH regulation of MGP expression. The goal of this study was to further characterize the mechanism by which PTH stimulates expression of MGP. Deletion analysis of the murine Mgp gene promoter identified a PTH-responsive region between -173 bp and-49 bp. Using gel-mobility shift assays we found that Sp1/Sp3, and Runx2 bind to distinct sites within this region. Mutation of either the Sp or the Runx2 site reduced MGP induction by PTH, while mutation of both sites completely abolished PTH responsiveness. Overexpression of Runx2 or Sp1 activated the Mgp reporter, while Sp3 was a dose-dependent repressor of Sp1 and PTH-induced MGP expression. Collectively, these data show that PTH regulates MGP gene transcription in osteoblasts through altered activities of Sp and Runx2 transcription factors., (2009 Wiley-Liss, Inc.)

Inhibition of osteoblast-mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix gamma-carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH-mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3-E1 cells. Following transient transfection of these cells with a -748 bp murine MGP promoter-luciferase construct (pMGP-luc), PTH (10 (-7) M) induced promoter activity in a time-dependent manner with a maximal four- to six fold induction seen 6 h after PTH treatment. Both H-89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co-transfection of MC3T3-E1 cells with pMGP-luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose-dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12-myristate 13-acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal-regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA- and ERK-dependent pathways.