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Bone morphogenetic protein 3b (BMP3b), also known as growth differentiation factor 10 (GDF10), is a non-osteogenic BMP highly expressed in the skeleton. Although in vitro studies have shown that BMP3b suppresses osteoblast differentiation, the physiological role of BMP3b in regulating bone mass in vivo remains unknown. Here, we show that BMP3b deletion in mice leads to a high bone mass phenotype via an unexpected novel mechanism involving de-repression of canonical BMP/Smad signaling. BMP3b null mice were viable, and exhibited no significant difference in body size compared to wildtype control. Trabecular bone parameters assessed by histomorphometry and μCT, revealed a significant increase in bone volume and bone mineral density. Expression of osteoblast-differentiation genes were elevated in bone tissue of BMP3b null mice, whereas expression of osteoclast-related genes remained unchanged. Consistent with this, Bmp3b was highly expressed in osteoblasts relative to osteoclast cells. Ex-vivo culture of primary bone marrow mesenchymal stem cells (BMSCs) and primary bone marrow-derived osteoclasts revealed that inactivation of BMP3b enhances osteogenesis without affecting osteoclastogenesis. Mechanistically, we found that BMP3b suppressed BMP4-induced Smad1/5 phosphorylation and inhibited the activity of a BMP4-driven Id-1 luciferase reporter. Protein-protein interaction assays revealed that BMP3b competitively interfered with the association of BMP4 and BMP type I receptors. These findings suggest that BMP3b regulates bone mass by acting as a BMP receptor antagonist. Thus, maintenance of bone mass involves antagonism of canonical BMP/Smad signaling by a member of the BMP family., Competing Interests: Declaration of competing interest The authors declare that this article and related all data are original, is not under consideration by any other journals. The authors also declare that we will not submit this manuscript to any other journals during the review period of Bone. The authors also confirm that data from previously published papers have not been used in this manuscript and agree to provide information about any relevant publications., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022 Federation of American Societies for Experimental Biology.)

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., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 -/- ) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 -/- mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 -/- mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 -/- mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 -/- mice. Finally, bone marrow cells from Bif-1 -/- mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 -/- mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words)., (© 2019 Wiley Periodicals, Inc.)

Background/aim: While netrin-4 plays a vital role in the vascular system, the role of netrin-1 in osteoblast differentiation is not well understood. In this study we explored whether netrin-4 has functional roles in osteoblasts., Materials and Methods: Quantitative reverse-transcriptase polymerase chain reaction (PCR), RNA interference, the generation of plasmids, transfections, measurement of alkaline phosphatase activity, a mineralization assay, a migration assay and a cell proliferation assay were performed., Results: Netrin-4 expression was up-regulated during osteoblast differentiation and an RNA interference experiment showed that small interfering RNA used to silence netrin-4 inhibited osteoblast differentiation. Recombinant mouse netrin-4 promoted alkaline phosphatase (ALP) activity of osteoblasts and enhancement of calcium deposits. Moreover, we constructed a vector containing the netrin-4 gene on the basis of the plasmid pcDNA3.1/V5-His. Overexpression of netrin-4 enhanced differentiation of osteoblasts. Finally, recombinant mouse netrin-4 promoted cell migration of osteoblasts., Conclusion: Netrin-4 promotes differentiation and migration of osteoblasts., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aim: Recent studies have demonstrated that netrin-1 plays a vital role in bone metabolism. Previous studies have shown that osteoblasts produce netrin-1 which affects osteoclast differentiation. However, the role of netrin-1 in osteoblast differentiation is not well understood. In this study, we explored the roles of netrin-1 in osteoblasts., Materials and Methods: Quantitative reverse-transcriptase polymerase chain reaction (qPCR), RNA interference for netrin receptors, the generation of netrin-1 plasmid, transfection of plasmids, and cell proliferation assay were performed., Results: During osteoblast differentiation by ascorbic acid, netrin-1 expression was significantly decreased. Gene expression related with osteoblast differentiation was down-regulated by netrin-1 treatment. We also found that osteoblast differentiation by bone morphogenetic protein-4 (BMP-4) was inhibited in the presence of recombinant netrin-1. Forced expression of both BMP-4 and netrin-1 significantly decreased alkaline phosphatase expression. On the other hand, Unc5b, neogenin, and A2b which belong to netrin receptors were expressed by osteoblasts. Moreover, alkaline phosphatase expression was significantly decreased by knockdown for the combination of two receptors among these receptors., Conclusion: Netrin-1 is involved in the regulation of osteoblast differentiation., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

In senile osteoporosis the balance of adipogenesis and osteoblastogenesis in bone marrow stromal cells (BMSCs) is disrupted so that adipogenesis is increased with respect to osteoblastogenesis, and as a result, bone mass is decreased. While the molecular mechanisms controlling the balance between osteoblastogenesis and adipogenesis are of great interest, the exact nature of the signals regulating this process remains to be determined. In general, adipogenesis is a reciprocal relationship with osteoblastogenesis in BMSCs. Recently transducin-like enhancer of split 3 (TLE3), was reported to enhance adipogenesis in pre adipocytes. However, the effect of TLE3 on osteoblast differentiation of BMSCs is completely unknown. Here we report that TLE3 not only enhances adipocyte differentiation in BMSCs but also suppresses osteoblast differentiation. Firstly we examined the expression and localization of TLE3. We found that TLE3 is expressed in the nucleus of bone marrow stromal cells and that over-expression of TLE3 induced adipocyte differentiation and suppressed ALP activity induced by treatment with BMP2 in these cells. In contrast, adipocyte differentiation was decreased and ALP activity increased when endogenous TLE3 was knocked down by shRNA in BMSCs. To examine the mechanism by which TLE3 is able to suppress osteoblast differentiation, we focused on Runx2, a transcription factor essential for osteoblast differentiation. We found that TLE3 strongly suppressed ALP activity and OSE2-luciferase activity induced by Runx2 and this repression of Runx2 by TLE3 occurs via HDACs because treatment with TSA, a class I and II HDAC inhibitor, rescued this repression. In conclusion, we identify TLE3 as a suppressor of BMSC differentiation in osteoblast lineage cells in vitro. Our data suggest that TLE3 activity may be a key in balancing adipocyte and osteoblast differentiation in the adult bone marrow microenvironment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Enhancing bone morphogenetic protein (BMP) signaling increases bone formation in a variety of settings that target bone repair. However, the role of BMP in the maintenance of adult bone mass is not well understood. Targeted disruption of BMP3 in mice results in increased trabecular bone formation, whereas transgenic overexpression of BMP3 in skeletal cells leads to spontaneous fracture, consistent with BMP3 having a negative role in bone mass regulation. Here we investigate the importance of BMP3 as a mediator of BMP signaling in the adult skeleton. We find that osteoblasts (OBL) and osteocytes are the source of BMP3 in adult bone. Using in vitro cultures of primary bone marrow stromal cells, we show that overexpression of BMP3 suppresses OBL differentiation, whereas loss of BMP3 increases colony-forming unit fibroblasts and colony-forming unit OBL. The ability of BMP3 to affect OBL differentiation is due to its interaction with activin receptor type 2b (Acvr2b) because knockdown of endogenous Acvr2b in bone marrow stromal cells reduces the suppressive effect of BMP3 on OBL differentiation. These findings best fit a model in which BMP3, produced by mature bone cells, acts to reduce BMP signaling through Acvr2b in skeletal progenitor cells, limiting their differentiation to mature OBL. Our data further support the idea that endogenous BMPs have a physiological role in regulating adult bone mass.

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation in myogenic cells via the phosphorylation of Smads. Two types of Smad phosphatases--small C-terminal domain phosphatase 1 (SCP1) and protein phosphatase magnesium-dependent 1A--have been shown to inhibit BMP activity. Here, we report that SCP1 inhibits the osteoblastic differentiation induced by BMP-4, a constitutively active BMP receptor, and a constitutively active form of Smad1. The phosphatase activity of SCP1 was required for this suppression, and the knockdown of SCP1 in myoblasts stimulated the osteoblastic differentiation induced by BMP signaling. In contrast to protein phosphatase magnesium-dependent 1A, SCP1 did not reduce the protein levels of Smad1 and failed to suppress expression of the Id1, Id2, and Id3 genes. Runx2-induced osteoblastic differentiation was suppressed by SCP1 without affecting the transcriptional activity or phosphorylation levels of Runx2. Taken together, these findings suggest that SCP1 may inhibit the osteoblastic differentiation induced by the BMP-Smad axis via Runx2 by suppressing downstream effector(s).

Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of beta-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3beta activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3beta-dependent but beta-catenin-independent mechanism., (Copyright 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Bone morphogenetic proteins (BMPs) induce ectopic bone formation in muscle tissue in vivo and convert myoblasts such that they differentiate into osteoblastic cells in vitro. We report here that constitutively active Smad1 induced osteoblastic differentiation of C2C12 myoblasts in cooperation with Smad4 or Runx2. In floxed Smad4 mice-derived cells, Smad4 ablation partially suppressed BMP-4-induced osteoblast differentiation. In contrast, the BMP-4-induced inhibition of myogenesis was lost by Smad4 ablation and restored by Smad4 overexpression. A nuclear zinc finger protein, E4F1, was identified as a possible component of the Smad4 complex that suppresses myogenic differentiation in response to BMP signaling. In the presence of Smad4, E4F1 stimulated the expression of Ids. Taken together, these findings suggest that the Smad signaling pathway may play a dual role in the BMP-induced conversion of myoblasts to osteoblastic cells.

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., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

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.