Runx2 is a critical transcription factor for osteoblast differentiation. Regulation of Runx2 expression levels and transcriptional activity is important for bone morphogenetic protein (BMP)-induced osteoblast differentiation. Previous studies have shown that extracellular signal-regulated kinase (Erk) activation enhances the transcriptional activity of Runx2 and that BMP-induced Runx2 acetylation increases Runx2 stability and transcriptional activity. Because BMP signaling induces Erk activation in osteoblasts, we sought to investigate whether BMP-induced Erk signaling regulates Runx2 acetylation and stability. Erk activation by overexpression of constitutively active MEK1 increased Runx2 transcriptional activity, whereas U0126, an inhibitor of MEK1/2, suppressed basal Runx2 transcriptional activity and BMP-induced Runx2 acetylation and stabilization. Overexpression of constitutively active MEK1 stabilized Runx2 protein via up-regulation of acetylation and down-regulation of ubiquitination. Erk activation increased p300 protein levels and histone acetyltransferase activity. Knockdown of p300 using siRNA diminished Erk-induced Runx2 stabilization. Overexpression of Smad5 increased Runx2 acetylation and stabilization. Erk activation further increased Smad-induced Runx2 acetylation and stabilization, whereas U0126 suppressed these functions. On the other hand, knockdown of Smad1 and Smad5 by siRNA suppressed both basal and Erk-induced Runx2 protein levels. Erk activation enhanced the association of Runx2 with p300 and Smad1. Taken together these results indicate that Erk signaling increases Runx2 stability and transcriptional activity, partly via increasing p300 protein levels and histone acetyltransferase activity and subsequently increasing Runx2 acetylation by p300. In addition to the canonical Smad pathway, a BMP-induced non-Smad Erk signaling pathway cooperatively regulates osteoblast differentiation partly via increasing the stability and transcriptional activity of Runx2.The bone morphogenetic proteins (BMPs) 3 are members of the transforming growth factor-␤ superfamily and are primary growth factors that induce formation of both cartilage and bone. Receptors for BMP are serine/threonine kinase receptors and consist of type I (BMPR-I) and II (BMPR-II) receptors. After ligand binding, BMPR-I kinases are activated by BMPR-II kinase-induced phosphorylation. R-Smad proteins are then recruited to activated receptors and play a role in transmitting the BMP signal from the receptor to target genes such as alkaline phosphatase (ALP), bone sialoprotein, osteocalcin (OC), Runx2, and Dlx5 (1, 2). In addition to the Smad pathway, diverse intracellular signaling molecules also participate in BMP-induced osteoblast differentiation. These are collectively called the non-Smad pathway of BMP signaling and include extracellular signal-regulated protein kinase (Erk), p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, phosphatidylinositol 3-kinase, and protein kinases C and D (3-7). These cooperate with and/or reg...

Fibrodysplasia ossificans progressiva (FOP), a rare genetic and catastrophic disorder characterized by progressive heterotopic ossification, is caused by a point mutation, c.617G>A; p.R206H, in the activin A receptor type 1 (ACVR1) gene, one of the bone morphogenetic protein type I receptors (BMPR-Is). Although altered BMP signaling has been suggested to explain the pathogenesis, the molecular consequences of this mutation are still elusive. Here we studied the impact of ACVR1 R206H mutation on BMP signaling and its downstream signaling cascades in murine myogenic C2C12 cells and HEK 293 cells. We found that ACVR1 was the most abundant of the BMPR-Is expressed in mesenchymal cells but its contribution to osteogenic BMP signal transduction was minor. The R206H mutant caused weak activation of the BMP signaling pathway, unlike the Q207D mutant, a strong and constitutively active form. The R206H mutant showed a decreased binding affinity for FKBP1A/FKBP12, a known safeguard molecule against the leakage of transforming growth factor (TGF)-␤ or BMP signaling. The decreased binding affinity of FKBP1A to the mutant R206H ACVR1 resulted in leaky activation of the BMP signal, and moreover, it decreased steady-state R206H ACVR1 protein levels. Interestingly, while WT ACVR1 and FKBP1A were broadly distributed in plasma membrane and cytoplasm without BMP-2 stimulation and then localized in plasma membrane on BMP-2 stimulation, R206H ACVR1 and FKBP1A were mainly distributed in plasma membrane regardless of BMP-2 stimulation. The impaired binding to FKBP1A and an altered subcellular distribution by R206H ACVR1 mutation may result in mild activation of osteogenic BMP-signaling in extraskeletal sites such as muscle, which eventually lead to delayed and progressive ectopic bone formation in FOP patients.

Sclerostin decreases bone mass by antagonizing the Wnt signaling pathway. We examined whether obesity-induced bone loss is associated with the expression of sclerostin. Five-week-old male mice were assigned to one of two groups (n = 10 each) and fed either a control diet (10% kcal from fat; CON) or a high-fat diet (60% kcal from fat; HF) for 12 weeks. Thex final body weight and whole body fat mass of the HF mice were higher than those of the CON mice. The distal femur cancellous bone mineral density and bone formation rate was lower in HF mice than in CON mice. The percent erosion surface was higher in the HF mice than the CON mice. The serum levels and femoral osteocytic protein expression levels of tumor necrosis factor-α (TNF-α) were significantly higher in HF mice than in CON mice. Sclerostin mRNA levels and osteocytic sclerostin protein levels in femoral cortex were also higher in HF mice than in CON mice. Sclerostin expression in MLO-Y4 osteocytes increased with TNF-α treatment, and TNF-α-induced sclerostin expression was blocked by the inhibition of NF-κB activation. Chromatin immunoprecipitation and a luciferase reporter assay demonstrated that NF-κB directly binds to the NF-κB binding elements on the mouse sost promoter and stimulates sclerostin expression. These results support a model in which, in the context of obesity or other inflammatory diseases that increase the production of TNF-α, TNF-α upregulates the expression of sclerostin through NF-κB signaling pathway, thus contributing to bone loss.

The epidermal growth factor receptor (EGFR) functions in various cellular physiological processes such as proliferation, differentiation, and motility. Although recent studies have reported that EGFR signaling is involved in osteoclast recruitment and formation, the molecular mechanism of EGFR signaling for the regulation of osteoclastogenesis remains unclear. We investigated the role of the EGFR in osteoclast differentiation and survival and show that the expression of the EGFR was highly up-regulated by receptor activator of nuclear factor-kappaB ligand (RANKL) during osteoclast differentiation. EGFR-specific tyrosine kinase inhibitors and EGFR knockdown blocked RANKL-dependent osteoclast formation, suggesting that EGFR signaling plays an important role in osteoclastogenesis. EGFR inhibition impaired the RANKL-mediated activation of osteoclastogenic signaling pathways, including c-Jun N-terminal kinase (JNK), NF-kappaB, and Akt/protein kinase B (PKB). In addition, EGFR inhibition in differentiated osteoclasts caused apoptosis through caspase activation. Inhibition of the phosphoinositide-3 kinase (PI3K)-Akt/PKB pathway and subsequent activation of BAD and caspases-9 and -3 may be responsible for the EGFR inhibition-induced apoptosis. The EGFR physically associated with receptor activator of nuclear factor-kappaB (RANK) and Grb2-associated binder 2 (Gab2). Moreover, RANKL transactivated EGFR. These data indicate that EGFR regulates RANKL-activated signaling pathways by cross-talking with RANK, suggesting that the EGFR may play a crucial role as a RANK downstream signal and/or a novel type of RANK co-receptor in osteoclast differentiation and survival.

Estrogen deficiency causes osteoporosis via increased generation of reactive oxygen species (ROS), and thus, antioxidants may prove to be the effective therapeutic candidates. We examined the effects of the antioxidant N-acetylcysteine (NAC) on osteoblastic differentiation in mouse calvarial cells. NAC (10-30 mM) enhanced alkaline phosphatase activity, mRNA expression of osteoblast differentiation-associated genes and mineralized nodule formation. It also increased expression of bone morphogenetic proteins-2, -4, and -7. The osteogenic activity of NAC was partially reduced by inhibition of glutathione synthesis. Since caffeic acid phenethyl ester did not stimulate osteoblast differentiation, it is unlikely that ROS scavenging activity of NAC is sufficient for osteogenic activity. We observed that NAC suppressed small GTPase RhoA activity and activation of RhoA by Pasteurella multocida toxin suppressed the osteogenic activity of NAC. These results suggest that NAC might exert its osteogenic activity via increased glutathione synthesis and inhibition of RhoA activation.