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It is crucial to treat Bisphosphonate-related osteonecrosis of the jaw (BRONJ), a serious problem; however, its pathogenesis is unclear. We have previously reported in vivo that bacterial infection or administration of lipopolysaccharide (LPS) aggravates BRONJ in rats. To elucidate the detailed mechanism of BRONJ, we investigated whether co-administration of LPS and zoledronate (ZOL) exacerbated the differentiation of pre-osteoblastic MC3T3-E1 cells in vitro. MC3T3-E1 cells were treated with ZOL, LPS, or both agents at a concentration gradient for the indicated times, after which cell viability was measured using the WST-8 assay. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) staining, ALP activity assay, and Alizarin Red S staining. The expression of relevant genes was detected by quantitative real-time reverse-transcriptase polymerase chain reaction. Western blotting was performed to examine the phosphorylation levels of Smad1/5/8. Co-administration of LPS and ZOL inhibited ALP activity and ALP staining more than a single administration of LPS or ZOL from the early stage of osteoblast differentiation. Co-administration of LPS and ZOL also inhibited the expression of osteoblast differentiation markers, Runx2 , Col I, ALP , and BSP , as well as phosphorylation of Smad1/5/8 more than a single administration of LPS or ZOL. Our data suggest that ZOL and LPS have an inhibitory effect on osteoblast differentiation through BMP2-Smad signaling. This effect may have led to a decrease in mineralization in vitro , suggesting that in patients treated with ZOL, ZOL causes osteonecrosis in their oral cavities, where LPS is universally produced by bacteria. [ABSTRACT FROM AUTHOR]

Osteoclasts, which resorb the bone, and osteoblasts, which form the bone, are the key cells regulating bone homeostasis. Osteoporosis and other metabolic bone diseases occur when osteoclast-mediated bone resorption is increased and bone formation by osteoblasts is decreased. Analyses of tyrosine kinase Src-knockout mice revealed that Src is essential for bone resorption by osteoclasts and suppresses bone formation by osteoblasts. Src-knockout mice exhibit osteopetrosis. Therefore, Src is a potential target for osteoporosis therapy. However, Src is ubiquitously expressed in many tissues and is involved in various biological processes, such as cell proliferation, growth, and migration. Thus, it is challenging to develop effective osteoporosis therapies targeting Src. To solve this problem, it is necessary to understand the molecular mechanism of Src function in the bone. Src expression and catalytic activity are maintained at high levels in osteoclasts. The high activity of Src is essential for the attachment of osteoclasts to the bone matrix and to resorb the bone by regulating actin-related molecules. Src also inhibits the activity of Runx2, a master regulator of osteoblast differentiation, suppressing bone formation in osteoblasts. In this paper, we introduce the molecular mechanisms of Src in osteoclasts and osteoblasts to explore its potential for bone metabolic disease therapy. [ABSTRACT FROM AUTHOR]

Osteoblasts release adenosine triphosphate (ATP) out of the cell following mechanical stress. Although it is well established that extracellular ATP affects bone metabolism via P2 receptors [such as purinergic receptor P2X7 (P2X7R) and purinergic receptor P2Y2 (P2Y2R)], the mechanism of ATP release from osteoblasts remains unknown. Recently, a vesicular nucleotide transporter [VNUT, solute carrier family 17 member 9 (SLC17A9)] that preserves ATP in vesicles has been identified. The purpose of this study was to elucidate the role of VNUT in osteoblast bone metabolism. mRNA and protein expression of VNUT were confirmed in mouse bone and in osteoblasts by quantitative real‐time PCR (qPCR) and immunohistochemistry. Next, when compressive force was applied to MC3T3‐E1 cells by centrifugation, the expression of Slc17a9, P2x7r, and P2y2r was increased concomitant with an increase in extracellular ATP levels. Furthermore, compressive force decreased the osteoblast differentiation capacity of MC3T3‐E1 cells. shRNA knockdown of Slc17a9 in MC3T3‐E1 cells reduced levels of extracellular ATP and also led to increased osteoblast differentiation after the application of compressive force as assessed by qPCR analysis of osteoblast markers such as Runx2, Osterix, and alkaline phosphatase (ALP) as well as ALP activity. Consistent with these observations, knockdown of P2x7r or P2y2r by siRNA partially rescued the downregulation of osteoblast differentiation markers, caused by mechanical loading. In conclusion, our results demonstrate that VNUT is expressed in osteoblasts and that VNUT inhibits osteoblast differentiation in response to compressive force by mechanisms related to ATP release and P2X7R and/or P2Y2R activity. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein (BMP) and canonical Wnt (cWnt) signaling factors are both known to regulate bone mass, fracture risk, fracture repair, and osteoblastogenesis. BMP3 is the most abundant BMP and negatively regulates osteoblastogenesis and bone mass. Thus, identifying the mechanism by which BMP3 acts to depress bone formation may allow for the development of new therapeutics useful in the treatment for osteopenia and osteoporosis. Here, we report that cWnt signaling stimulates BMP3 expression in osteoblast (OB) lineage cells. The expression of BMP3 increases with OB differentiation. Treatment of cells with various cWnt proteins stimulated BMP3 expression. Mice with enhanced cWnt signaling had high expression levels of BMP3. Our data suggest that reduction in BMP3 levels may contribute beneficially to the positive effect of cWnt agonists on bone mass. [ABSTRACT FROM AUTHOR]

Background Bone morphogenetic proteins (BMPs) enhance bone formation and osteoblast differentiation in vivo and in vitro via a Smad signaling pathway. Recent findings have revealed that inhibition of nuclear factor-κB (NF-κB) enhances BMP-induced osteoblast differentiation both in vitro and in vivo , suggesting that NF-κB negatively regulates osteoblastic bone formation. However, the molecular mechanism whereby NF-κB inhibits BMP-induced osteoblastic bone formation is still unclear. Highlight A specific NF-κB inhibitor, BAY11-7082, together with BMP2, enhanced BMP2-induced ectopic bone formation in mice. Furthermore, BMP2 increased alkaline phosphatase (ALP) activity, a typical marker of osteoblast differentiation in mouse embryonic fibroblasts (MEFs) from p65-deficient (p65−/−) mice compared with MEFs from wild type (WT) or p50-deficient (p50−/−) mice. The Smad complex activated by BMP2 bound more stably to the target element, without affecting Smad1/5 phosphorylation, in p65−/− MEFs than in WT MEFs, while p65 overexpression inhibited BMP2 activity by decreasing DNA binding to the Smad complex. Moreover, the C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not of Smad1, and inhibited the BMP-Smad pathway. Conclusion p65 inhibits BMP2-induced osteoblastic bone formation by interfering with DNA binding of the Smad complex via an interaction with Smad4. Thus, targeting the association between p65 and Smad4 may help promote bone regeneration in the treatment of bone diseases. [ABSTRACT FROM AUTHOR]

Summary Background/purpose Adrenocorticotropic hormone (ACTH) plays a vital role in maintaining the function of the hypothalamic–pituitary–adrenal axis. Recent studies have demonstrated that ACTH directly affects the proliferation and differentiation of bone cells. However, the ACTH concentrations used in these studies appear to be markedly higher than the physiological concentrations. Here, we investigated whether ACTH at pathophysiological concentration affects the proliferation and differentiation of osteoblasts and osteoclasts. Materials and methods We evaluated the effect of ACTH at pathophysiological concentration on osteoclasts using tartrate-resistant acid phosphatase staining and on osteoblasts using alkaline phosphatase activity assay. Additionally, we conducted reverse transcriptase-polymerase chain reaction analysis. Results We found that at pathophysiological concentration, ACTH does not affect osteoblast proliferation and inhibits osteoblast differentiation. Moreover, we showed that at pathophysiological concentration, ACTH does not affect the proliferation of bone marrow macrophages, but promotes differentiation of osteoclasts and induces expression of genes involved in bone resorption. Conclusion Taken together, our findings suggest that ACTH modulates the proliferation and differentiation of bone cells in vitro at pathophysiological concentration. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have indicated that acetylcholine (ACh) plays a vital role in various tissues, while the role of ACh in bone metabolism remains unclear. Here we demonstrated that ACh induced cell proliferation and reduced alkaline phosphatase (ALP) activity via nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) in osteoblasts. We detected mRNA expression of several nAChRs and mAChRs. Furthermore, we showed that cholinergic components were up-regulated and subunits/subtypes of acetylcholine receptors altered during osteoblast differentiation. To our knowledge, this is the first report demonstrating that osteoblasts express specific acetylcholine receptors and cholinergic components and that ACh plays a possible role in regulating the proliferation and differentiation of osteoblasts. [Copyright &y& Elsevier]

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten‐eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET‐mediated DNA hydroxymethylation during BMP‐induced C2C12 osteoblast differentiation using a novel cytosine‐based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG‐rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7‐promoter‐reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET‐mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblastic cells. [Copyright &y& Elsevier]