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Bone homeostasis is regulated by bone morphogenic proteins (BMPs), among which BMP9 is one of the most osteogenic. Here, we have found that BMP9 rapidly increases the protein expression of hypoxia-inducible factor-1α (HIF-1α) in osteoblasts under normoxic conditions more efficiently than BMP2 or BMP4. A combination of BMP9 and hypoxia further increased HIF-1α protein expression. HIF-1α protein induction by BMP9 is not accompanied by messenger RNA (mRNA) increase and is inhibited by the activation of prolyl hydroxylase domain (PHD)-containing protein, indicating that BMP9 induces HIF-1α protein expression by inhibiting PHD-mediated protein degradation. BMP9-induced HIF-1α protein increase was abrogated by inhibitors of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) kinase, indicating that it is mediated by PI3K-AKT signaling pathway. BMP9 increased mRNA expression of pyruvate dehydrogenase kinase 1 (PDK1), a glycolytic enzyme, and vascular endothelial growth factor-A (VEGF-A), an angiogenic factor, in osteoblasts. Notably, BMP9-induced mRNA expression of PDK1, but not that of VEGF-A, was significantly inhibited by small interference RNA-mediated knockdown of Hif-1α. BMP9-induced matrix mineralization and osteogenic marker gene expressions were significantly inhibited by chemical inhibition and gene knockdown of either Hif-1α or Pdk-1, respectively. Since increased glycolysis is an essential feature of differentiated osteoblasts, our findings indicate that HIF-1α expression is important in BMP9-mediated osteoblast differentiation through the induction of PDK1.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are cell wall components of Gram-positive and Gram-negative bacteria, respectively. Notably, oral microflora consists of a variety of bacterial species, and osteomyelitis of the jaw caused by dental infection presents with symptoms of bone resorption and osteosclerosis. However, the effects of LTA and LPS on osteogenic differentiation have not yet been clarified. We examined the effects of LTA and LPS on osteoblasts and found that LTA alone promoted alizarin red staining at low concentrations and inhibited it at high concentrations. Additionally, gene expression of osteogenic markers (ALP, OCN, and OPG) were enhanced at low concentrations of LTA. High concentrations of LPS suppressed calcification potential, and the addition of low concentrations of LTA inhibited calcification suppression, restoring the gene expression levels of suppressed bone differentiation markers (ALP, BSP, and OCN). Moreover, the suppression of p38, a signaling pathway associated with bone differentiation, had opposing effects on gene-level expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), suggesting that mixed LTA and LPS infections have opposite effects on bone differentiation through concentration gradients, involving inflammatory markers (TNF-α and IL-6) and the p38 pathway.

Bone morphogenetic proteins (BMPs) are essential regulators of skeletal homeostasis, and BMP9 is the most potently osteogenic among them. Here, we found that BMP9 and BMP2 rapidly induced early growth response 1 (EGR1) protein expression in osteoblasts through MEK/ERK pathway-dependent transcriptional activation. Knock-down of EGR1 using siRNA significantly inhibited BMP9-induced matrix mineralization and osteogenic marker gene expression in osteoblasts. Knock-down of EGR1 significantly reduced SMAD1/5 phosphorylation and inhibited the expression of their transcriptional targets in osteoblasts stimulated by BMP9. In contrast, forced EGR1 overexpression in osteoblasts enhanced BMP9-mediated osteoblast differentiation and SMAD1/5 phosphorylation. An intracellular association between EGR1 and SMAD1/5 was identified using immunoprecipitation assays. These results indicated that EGR1 plays an important role in BMP9-stimulated osteoblast differentiation by enhancing SMAD1/5 phosphorylation.

Bone morphogenetic protein (BMP) 9 is one of the most osteogenic BMPs, but its mechanism of action has not been fully elucidated. Hes1, a transcriptional regulator with a basic helix-loop-helix domain, is a well-known effector of Notch signaling. Here, we find that BMP9 induces periodic increases of Hes1 mRNA and protein expression in osteoblasts, presumably through an autocrine negative feedback mechanism. BMP9-mediated Hes1 induction is significantly inhibited by an ALK inhibitor and overexpression of Smad7, an inhibitory Smad. Luciferase and ChIP assays revealed that two Smad-binding sites in the 5' upstream region of the mouse Hes1 gene are essential for transcriptional activation by BMP9. Thus, our data indicate that BMP9 induces Hes1 expression in osteoblasts via the Smad signaling pathway.

Mesenchymal Stem Cells (MSCs) are pluripotent cells that can be differentiated as osteoblasts, adipocytes, myocytes or chondrocytes depending on the culture condition. However, MSCs are known to lose their differentiation potency after long-term culture. Development of a new cell culture method to maintain their stemness is required for successful application of MSCs. Here, we revealed that low-intensity pulsed ultrasound (LIPUS) stimulation was useful for maintaining the MSC stemness as LIPUS inhibited the loss of osteogenic differentiation potency of osteo-progenitor cells induced by serial subculture. LIPUS also increased the transcriptional and phosphorylation level of Nanog, a crucial stem cell marker gene in a MSC cell line. We also found that LIPUS induced the secretion of extracellular adenosine triphosphate (ATP) in MSC. The treatments of the conditioned medium from LIPUS-stimulated MSC and exogenous ATP promoted Nanog expression. Thus, LIPUS may maintain the long-term differentiation potency of MSCs and osteo-progenitor cells by induction in Nanog transcript level and phosphorylation.

Mesenchymal Stem Cells (MSCs) are pluripotent cells with the capability of self-renewal. It has been established that MSCs can transform into osteoblasts, adipocytes or chondrocytes depending on the culture condition. Regenerative medicine is considered to become an essential therapy in the field of