Positive Regulator Of Osteoblast Differentiation Research Articles

YY2 Promotes Osteoblast Differentiation by Upregulating Osterix Transcriptional Activity

Yin Yang 2 (YY2) is a paralog of YY1, a well-known multifunctional transcription factor containing a C-terminal zinc finger domain. Although the role of YY1 in various biological processes, such as the cell cycle, cell differentiation and tissue development, is well established, the function of YY2 has not been fully determined. In this study, we investigated the functional role of YY2 during osteoblast differentiation. YY2 overexpression and knockdown increased and decreased osteoblast differentiation, respectively, in BMP4-induced C2C12 cells. Mechanistically, YY2 overexpression increased the mRNA and protein levels of Osterix (Osx), whereas YY2 knockdown had the opposite effect. To investigate whether YY2 regulates Osx transcription, the effect of YY2 overexpression and knockdown on Osx promoter activity was evaluated. YY2 overexpression significantly increased Osx promoter activity in a dose-dependent manner, whereas YY2 knockdown had the opposite effect. Furthermore, vectors containing deletion and point mutations were constructed to specify the regulation site. Both the Y1 and Y2 sites were responsible for YY2-mediated Osx promoter activation. These results indicate that YY2 is a positive regulator of osteoblast differentiation that functions by upregulating the promoter activity of Osx, a representative osteogenic transcription factor in C2C12 cells.

Inhibition of miR-29-3p isoforms via tough decoy suppresses osteoblast function in homeostasis but promotes intermittent parathyroid hormone-induced bone anabolism.

miRNAs play a vital role in post-transcriptional regulation of gene expression in osteoblasts and osteoclasts, and the miR-29 family is expressed in both lineages. Using mice globally expressing a miR-29-3p tough decoy, we demonstrated a modest 30-60% decrease all three miR-29-3p isoforms: miR-29a, miR-29b, and miR-29c. While the miR-29-3p decoy did not impact osteoclast number or function, the tough decoy decreased bone formation in growing mice, which led to decreased trabecular bone volume in mature animals. These data support previous in vitro studies suggesting that miR-29-3p is a positive regulator of osteoblast differentiation. In contrast, when mice were treated with intermittent parathyroid hormone (PTH1-34), inhibition of miR-29-3p augmented the effect of PTH on cortical bone anabolism, increased bone formation rate and osteoblast surface, and increased levels of Ctnnb1/βcatenin mRNA, which is a miR-29 target. These findings highlight differences in the mechanisms controlling basal level bone formation and bone formation induced by intermittent PTH. Overall, the global miR-29-3p tough decoy model represents a modest loss-of-function, which could be a relevant tool for assessing the possible impact of systemically administered miR-29-3p inhibitors. Our studies provide a potential rationale for co-administration of PTH1-34 and miR-29-3p inhibitors, to boost bone formation in severely affected osteoporosis patients, particularly in the cortical compartment.

Role of Special AT-Rich Sequence-Binding Protein 2 in the Osteogenesis of Human Dental Mesenchymal Stem Cells.

Dental mesenchymal stem cells (MSCs) are recognized as a critical factor in repair of defective craniofacial bone owing to the multiple differentiation potential, the ability to regenerate distinct tissues, and the advantage that they can be easily obtained by relatively noninvasive procedures. Special AT-rich sequence-binding protein 2 (SATB2) is a nuclear matrix protein, involved in chromatin remodeling and transcriptional regulation, and has been reported to be as a positive regulator of osteoblast differentiation, bone formation, and bone regeneration in MSCs. In this study, we systematically investigated the capability of SATB2 to promote the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and stem cells from human exfoliated deciduous teeth (SHED). RNA-seq analysis and quantitative real-time PCR (RT-PCR) revealed that genes regulating osteogenic differentiation were differentially expressed among three cell types and SATB2 was found to be expressed at a relatively high level. When the three cell types overexpressed SATB2 with AdSATB2 infection, alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red S staining, and quantification tended to increase with an increasing infection rate. It showed opposite results after infection with AdsiSATB2. RNA-seq analysis indicated that the expression of downstream osteogenic genes was affected by AdSATB2 infection and quantitative RT-PCR confirmed that nine osteogenic genes (Spp1, Sema7a, Atf4, Ibsp, Col1a1, Sp7, Igfbp3, Dlx3, and Alpl) were upregulated, to various extents, following SATB2 overexpression. In addition, quantitative PCR results indicated that SATB2 affected the expression of MSC markers. These results suggested an important role of SATB2 in the osteogenesis of PDLSCs, DPSCs, and SHED. Further research is warranted to investigate SATB2-mediated regulation of osteogenic differentiation and to evaluate the therapeutic use of SATB2 for the regeneration of defective craniofacial bone tissue.

Amyloid β peptide promotes bone formation by regulating Wnt/β-catenin signaling and the OPG/RANKL/RANK system.

Amyloid β peptide (Aβ) is involved in osteoporosis, but the effects of Aβ on osteoblast and bone formation remain unclear. In this study, we investigated the effect of Aβ on bone formation. An animal model of osteoporosis was established by ovariectomy in C57BL/6 mice. The mice received intraperitoneal injection of Aβ. The effect of Aβ on the osteogenic differentiation of human bone marrow stromal stem cells (hBMSCs) and differentiation of both pre-osteoblasts and pre-osteoclasts in a co-culture system were investigated. In the animal study, intraperitoneal injection of Aβ for 8 weeks promoted early and late osteogenic differentiation of hBMSCs. Aβ treatment significantly elevated osterix+ (osteoblastic) cells but decreased TRAP+ cells (osteoclasts) in the distal femur bone. In vitro study showed that Aβ treatment significantly enhanced matrix mineralization and osteogenic markers (Runx2 and osteocalcin). Aβ treatment activated Wnt/β-catenin signaling in hBMSCs. The effect of Aβ was blocked by DKK1 (a Wnt/β-catenin inhibitor) treatment. In the co-culture system, Aβ treatment significantly increased the ALP activities of MC3T3-E1 cells (pre-osteoblasts) but reduced the TRAP+ RAW264.7 cells (pre-osteoclasts). Aβ treatment upregulated TCF1 and OPG proteins in MC3T3-E1 cells. Aβ treatment upregulated IκB-α but downregulated NFATc1protein in RAW264.7 cells. These effects were blocked by XAV-939 (a Wnt signaling antagonist), and then rescued by additional Wnt3a (a Wnt agonist). Aβ treatment simultaneously promoted osteogenic differentiation via Wnt/β-catenin signaling, and inhibited osteoclasts differentiation via the OPG/RANKL/RANK system, suggesting Aβ is a positive regulator of osteoblast differentiation and bone formation.

Thy-1 Deficiency Augments Bone Loss in Obesity by Affecting Bone Formation and Resorption

Healthy bone remodeling results from a balanced bone formation and bone resorption realized by bone-forming osteoblasts and bone-resorbing osteoclasts, respectively. Recently, Thy-1 (CD90) was identified as positive regulator of osteoblast differentiation and activation, thus, promoting bone formation while concurrently inhibiting adipogenesis and obesity in mice. Additionally, Thy-1 did not affect bone resorption. An obesity-related co-morbidity that is increasing in prevalence is a disturbed bone formation resulting in an increased fracture risk. The underlying mechanisms of obesity-induced bone alterations are not yet fully elucidated and therefore therapy options for efficient bone-anabolic treatments are limited. Therefore, we investigated the impact of Thy-1 on bone metabolism under obese conditions. Indeed, high fat diet (HFD) induced obese mice lacking Thy-1 (Thy-1−/−) showed increased body fat mass compared to wildtype (WT) mice while bone mass (−38%) and formation (−57%) were decreased as shown by micro-computed tomography (μCT) measurement, histological analysis, and fourier-transform infrared spectroscopy (FTIR). Interestingly, under obese conditions, lack of Thy-1 affected both osteoblast and osteoclast function. Number (−30%) and activity of osteoblasts were decreased in obese Thy-1−/− mice while osteoclast number (+39%) and activity were increased. Facilitated bone marrow fat accumulation (+56%) in obese Thy-1−/− mice compared to obese WT mice was associated with upregulated tumor necrosis factor α (Tnfα, +46%) and colony stimulating factor 1 receptor (Csf1r) expression, strong promoters of osteoclast differentiation. Moreover, lack of Thy-1 was accompanied by a reduction of osteoprotegerin (Tnfrsf11b) expression (−36%), an inhibitor of osteoclast differentiation. Altered Tnfα, Csf1r, and Tnfrsf11b expression might be responsible for elevated osteoclast activity in obese Thy-1-deficient mice. In summary, our findings show that lack of Thy-1 promotes obesity under HFD conditions while concurrently decreasing bone mass and formation. Mechanistic studies revealed that under obese conditions lack of Thy-1 impairs both bone formation and bone resorption.

Monocarboxylate transporter-1 promotes osteoblast differentiation via suppression of p53, a negative regulator of osteoblast differentiation

Monocarboxylate transporter-1 (MCT-1) is a transmembrane transporter for monocarboxylates including lactate and pyruvate. Silencing Mct1 by its small interfering RNA (siRNA) suppressed the expression of marker genes for osteoblast differentiation, namely, Tnap, Runx2, and Sp7, induced by BMP-2 in mouse myoblastic C2C12 cells. Mct1 siRNA also suppressed alkaline phosphatase activity, as well as expressions of Tnap and Bglap mRNAs in mouse primary osteoblasts. On the other hand, Mct1 siRNA did not have effects on the Smad1/5 or ERK/JNK pathways in BMP-2-stimulated C2C12 cells, while it up-regulated the mRNA expression of p53 (Trp53) as well as nuclear accumulation of p53 in C2C12 cells in a BMP-2-independent manner. Suppression of osteoblastic differentiation by Mct1 siRNA in C2C12 cells was abolished by co-transfection of Trp53 siRNA. Together, these results suggest that MCT-1 functions as a positive regulator of osteoblast differentiation via suppression of p53.

2,3,7,8-Tetrachlorodibenzo-p-dioxin dose-dependently increases bone mass and decreases marrow adiposity in juvenile mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) agonists have been shown to regulate bone development and remodeling in a species-, ligand-, and age-specific manner, however the underlying mechanisms remain poorly understood. In this study, we characterized the effect of 0.01–30 μg/kg TCDD on the femoral morphology of male and female juvenile mice orally gavaged every 4 days for 28 days and used RNA-Seq to investigate gene expression changes associated with the resultant phenotype. Micro-computed tomography revealed that TCDD dose-dependently increased trabecular bone volume fraction (BVF) 2.9- and 3.3-fold in male and female femurs, respectively. Decreased serum tartrate-resistant acid phosphatase (TRAP) levels, combined with a reduced osteoclast surface to bone surface ratio and repression of femoral proteases (cathepsin K, matrix metallopeptidase 13), suggests that TCDD impaired bone resorption. Increased osteoblast counts at the trabecular bone surface were consistent with a reciprocal reduction in the number of bone marrow adipocytes, suggesting AhR activation may direct mesenchymal stem cell differentiation towards osteoblasts rather than adipocytes. Notably, femoral expression of transmembrane glycoprotein NMB (Gpnmb; osteoactivin), a positive regulator of osteoblast differentiation and mineralization, was dose-dependently induced up to 18.8-fold by TCDD. Moreover, increased serum levels of 1,25-dihydroxyvitamin D3 were in accordance with the renal induction of 1α-hydroxylase Cyp27b1 and may contribute to impaired bone resorption. Collectively, the data suggest AhR activation tipped the bone remodeling balance towards bone formation, resulting in increased bone mass with reduced marrow adiposity.

Thy1 is a positive regulator of osteoblast differentiation and modulates bone homeostasis in obese mice.

Thy1 (CD90), a glycosylated, glycophosphatidylinositol-anchored membrane protein highly expressed by subsets of mesenchymal stem cells and fibroblasts, inhibits adipogenesis. The role of Thy1 on bone structure and function has been poorly studied and represents a major knowledge gap. Therefore, we analyzed the long bones of wild-type (WT) and Thy1 knockout (KO) mice with micro-computed tomography (micro-CT) and histomorphometry to compare changes in bone architecture and overall bone structure. micro-CT analysis of long bones revealed Thy1 KO and WT mice fed a high-fat diet demonstrated bone structural parameters at 4 mo that differed significantly between WT and KO mice. A significant reduction in trabecular bone volume was noted in Thy1 KO mice. The most prominent differences were observed in trabecular bone volume ratio and trabecular bone connectivity density. Consistent with micro-CT measurements, histomorphometric analysis also showed decreased bone volume in the obese Thy1 KO mice compared to obese WT mice. In vitro assays revealed that osteogenic conditions increased Thy1 expression during OB differentiation and absence of Thy1 attenuated osteoblastogenesis. Together, these findings support the concept that Thy1 serves as a major mechanistic link to regulate bone formation and negatively regulate adipogenesis.-Paine, A., Woeller, C. F., Zhang, H., Garcia-Hernandez, M. L., Huertas, N., Xing, L., Phipps, R. P., Ritchlin, C. T. Thy1 is a positive regulator of osteoblast differentiation and modulates bone homeostasis in obese mice.

MC3T3-E1 세포에서 BMP2에 의한 조골세포의 분화에 일주기 유전자 Per1이 미치는 영향

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that play important roles in a variety of cellular functions. Among BMP family members, BMP2 efficiently promotes osteoblast differentiation through Smad-mediated runt-related transcription factor 2 (Runx2) expression. Several recent studies suggest that BMPs are associated with clock genes, in particular Bmal1. Bmal1 protein heterodimerizes with Clock protein and then induces period 1 (Per1) expression. However, the role of Per1 on osteoblast differentiation remains unclear. In this study, we investigated whether Per1 is involved in osteoblast differentiation. MC3T3-E1 cells were treated with BMP2 for induction of osteoblastic differentiation. Osteogenic maker gene and Per1 mRNA expression were measured using real-time PCR. Interestingly, BMP2 treatment induced Per1 mRNA expression in MC3T3-E1 cells. To further investigate the function of Per1 on osteoblast differentiation, MC3T3-E1 cells were transiently transfected with pCMV-Per1. Per1 overexpression increased Runx2 mRNA and protein levels. Also, mRNA expression and promoter activity of osteocalcin were upregulated by Per1 overexpression. To investigate the effect of interaction between Per1 and osteogenic condition, MC3T3-E1 cells were cultured in osteogenic medium containing ascorbic acid and β-glycerophosphate. Osteogenic medium-induced ALP staining level and mineralization were synergistically increased by overexpression of Per1. Taken together, these results demonstrate that Per1 is a positive regulator of osteoblast differentiation.

TRIM33 is essential for osteoblast proliferation and differentiation via BMP pathway.

Tripartite motif containing 33 (TRIM33) functions both as a positive and negative regulator of the TGF-β/BMP pathway in tumors; however, its effect and mechanism during osteoblast proliferation and differentiation, which involves the TGF-β/BMP pathway is not defined. In this study, we used mouse C3H10T1/2 mesenchymal stem cell line and MC3T3-E1 preosteoblasts to investigate the role of TRIM33 during this process. The results demonstrated that the expression of TRIM33 increased during the differentiation. Moreover, the overexpression or knockdown of TRIM33 resulted in both an augmentation or decrease in osteoblast differentiation, which were measured by the expression of alkaline phosphatase (ALP) at the mRNA level, both Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) at the protein level, and the formation of mineral modules. To further demonstrate the mechanism of TRIM33 in this process, we found that TRIM33 could positively mediate the BMP pathway by forming TRIM33-Smad1/5 complex. This interaction between TRIM33 and Smad1/5 triggered the phosphorylation of Smad1/5. In addition, the essential role of TRIM33 in osteoblast proliferation was determined in this study by CellCounting Kit (CCK) -8 and cell cycle assays. In summary, we establish the function of TRIM33 as a positive regulator of osteoblast differentiation in BMP pathway, which mediates its effect through its interaction with and activation of Smad1/5. In addition, the results clearly demonstrate that TRIM33 is necessary for osteoblast proliferation by regulating cell cycle. These results suggest that TRIM33 can be a positive target of osteoblast proliferation and differentiation through BMP pathway.

Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins.

ObjectivesUbiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown.MethodsWe examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-κB on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay.ResultsThe expression levels of WW Domain Containing E3 Ubiquitin Protein Ligase 1 (Wwp1), SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1), SMAD Specific E3 Ubiquitin Protein Ligase 2 (Smurf2) and Itch were all significantly increased in the fracture callus of WT mice, which was associated with elevated expression of NF-κB members and total ubiquitinated proteins. Callus tissue isolated from Itch KO mice expressed higher levels of osteoblast-associated genes, including Runx2, a positive regulator of osteoblast differentiation, but osteoclast-associated genes were not increased. Both NF-κB RelA and RelB proteins were found to bind to the NF-κB binding site in the mouse Itch promoter.ConclusionsOur findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus. Thus, ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing.Cite this article: J. Liu, X. Li, H. Zhang, R. Gu, Z. Wang, Z. Gao, L. Xing. Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins. Bone Joint Res 2017;6:154–161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1.

Angiopoietin-like protein 2 is a positive regulator of osteoblast differentiation

Introduction and AimsSeveral studies have reported that angiopoietin-like protein 2 (Angptl2) is expressed abundantly in adipocytes and is associated with adipose tissue inflammation. In the present study, we found that osteoblasts and mesenchymal stem cells also expressed Angptl2 at high levels. The aim of this study was to understand the role of Angptl2 in osteoblastic cell differentiation. MethodsAngptl2 expression was examined during osteoblast and adipocyte differentiation. The role of Angptl2 on cell differentiation and associated signaling was analyzed by gene knockdown using Angptl2 small interfering ribonucleic acid (siRNA). ResultsAngptl2 was highly expressed in MC3T3-E1 cells, ST2 cells and primary osteoblasts, but not in RAW264 cells. Inhibition of Angptl2 expression using siRNA markedly inhibited alkaline phosphatase (ALP) expression and osteoblastic differentiation in MC3T3-E1, ST2 cells and primary osteoblasts. Angptl2 siRNA also inhibited adipocyte differentiation in ST2 cells. Treatment of MC3T3-E1 cells with Angptl2 siRNA led to the down-regulation of the activities of several cell signaling pathways, including extracellular signal-regulated kinase (ERK), Jun amino-terminal kinase (JNK), Akt, and nuclear factor kappa B (NF-κB) signals. It also down-regulated the expression of Osterix, but not that of runt-related transcription factor 2 (Runx2), suggesting that Angptl2 is a positive activator of Osterix and its down-stream signals. Treatment of MC3T3-E1 cells with anti-Angptl2 antibodies suppressed ALP gene expression. In addition, treatment of Angptl2 siRNA-treated cells with culture supernatants of normal MC3T3-E1 cells restored ALP gene expression, indicating that Angptl2 acts in an autocrine manner. ConclusionsThe results suggest that Angptl2 is an autocrine positive regulator of cell differentiation. Thus, it is suggested that Angptl2 regulates not only adipose tissue metabolism but also bone metabolism.

Protein tyrosine phosphatase SHP-1 modulates osteoblast differentiation through direct association with and dephosphorylation of GSK3β

SHP-1, the Src homology-2 (SH2) domain-containing phosphatase 1, is a cytosolic protein-tyrosine phosphatase (PTP) predominantly expressed in hematopoietic-derived cells. Previous studies have focused on the involvement of SHP-1 in osteoclastogenesis. Using primary cultured mouse fetal calvaria-derived osteoblasts as a model, this study aims to investigate the effects of SHP-1 on differentiation and mineralization of osteoblasts and elucidate the signaling pathways responsible for these effects. We found that osteoblasts treated by osteogenic media showed significant increase in SHP-1 expression, which contributed to osteoblastic differentiation and mineralization. Using immunoprecipitation assay, we found that a direct association between SHP-1 and glycogen synthase kinase (GSK)-3β could be detected in differentiated osteoblasts and was significantly inhibited by SHP-1 inhibitor NSC87877. Inhibition of SHP-1 activated GSK3β, thereby leading to suppression of osteoblast differentiation and mineralization, which could be rescued by the inhibitor of GSK3β. In addition, we found that rosiglitazone (RSG) treatment led to significant decrease in SHP-1 expression. Overexpression of SHP-1 reversed RSG-induced GSK3β activation, thus rescuing the inhibitory effect of RSG on osteoblast differentiation and mineralization. These findings suggest that protein tyrosine phosphatase SHP-1 may act as a positive regulator of osteoblast differentiation through direct association with and dephosphorylation of GSK3β. Downregulation of SHP-1 may contribute to RSG-induced inhibition of mouse calvaria osteoblast differentiation by activating GSK3β-dependent pathway.

BMP-2 Induced Expression of PLCβ1 That is a Positive Regulator of Osteoblast Differentiation.

Bone morphogenetic protein 2 (BMP-2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide-phospholipase Cβ 1 (PLCβ1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCβ1. Here, the function of PLCβ1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP-2 we assist to a remarkable increase in PLCβ1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCβ1 promotes osteogenic differentiation by up-regulating alkaline phosphatase (ALP). Moreover, PLCβ1 is present in the nuclear compartment of these cells and overexpression of a cytosolic-PLCβ1mutant (cyt-PLCβ1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR-214 is a regulator of Osterix (Osx) which is an osteoblast-specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCβ1 could be a potential target of miR-214 in the control of osteogenic differentiation by gain- and loss- of function experiment. The results indicated that inhibition of miR-214 in C2C12 cells significantly enhances the protein level of PLCβ1 and promotes C2C12 BMP-2-induced osteogenesis by targeting PLCβ1.

Regulation of proliferation and apoptosis in human osteoblastic cells by microRNA-15b

MicroRNAs (miRNAs) play an important role in proliferation and differentiation of osteoblasts. We recently reported that miR-15b acts as a positive regulator of osteoblast differentiation, whereas its functional role in osteoblastic proliferation remains not known. In this study, we found that there was increased proliferation of human osteoblastic cells (MG63) when they were transiently transfected with miR-15b inhibitor. A significant level of cell population was found to be decreased at G0/G1 phase, and increased at S and G2/M phase by miR-15b inhibitor treatment. Cyclin E1 was found to be one of the putative target genes of miR-15b, and miR-15b mimic and miR-15b inhibitor treatments in cells decreased and increased cyclin E1 expression, respectively. We further identified that the cyclin E1 3′UTR is directly targeted by miR-15b using the luciferase reporter gene system. No significant effect was found on apoptosis of MG63 cells with miR-15b inhibitor. Thus, these findings provide new insights in understanding the role of miR-15b expression as negative regulator of osteoblast proliferation.

LIM mineralization protein-1 inhibits the malignant phenotypes of human osteosarcoma cells.

Osteosarcoma (OS), also known as osteogenic sarcoma, is the most common primary malignancy of bone tumor in children and adolescents. However, its underlying molecular pathogenesis is still only vaguely understood. Recently, LIM mineralization protein-1 (LMP-1) was reported to be an essential positive regulator of osteoblast differentiation. In the present study, we found that the expression of LMP-1 is downregulated in OS tissues compared with adjacent normal tissues. Moreover, we restored the expression of LMP-1 through a recombinant adenovirus. Overexpression of LMP-1 inhibited cell proliferation and invasion, arrested cell cycle progression, and induced apoptosis in vitro. Finally, ectopic LMP-1 expression suppressed the expression of Runx2 and BMP-2 in OS cells. These data demonstrate that LMP-1 is an essential tumor suppressor in the OS pathological process, which will provide a new opportunity for discovering and identifying novel effective treatment strategies.

BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation

Bone morphogenetic proteins (BMPs) regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3) which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophila aristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp), Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

Distal‐less homeobox 5 inhibits adipogenic differentiation through the down‐regulation of peroxisome proliferator‐activated receptor γ expression

Distal-less homeobox 5 (Dlx5) is a positive regulator of osteoblast differentiation that contains a homeobox domain. Because there are possible reciprocal relationships between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (MSCs), we examined the regulatory role of Dlx5 in adipogenic differentiation in this study. Adipogenic stimuli suppressed the expression levels of Dlx5 mRNA in mouse bone marrow stromal cells. Over-expression of Dlx5 inhibited adipogenic differentiation in human bone marrow MSCs and 3T3-L1 preadipocytic cells whereas knockdown of Dlx5 enhanced adipogenic differentiation in 3T3-L1 cells. Over-expression of Dlx5 suppressed the expression of adipogenic marker genes, including CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Dlx5-mediated suppression of adipogenic differentiation was overcome by over-expression of PPARγ but not by that of cAMP response element binding protein (CREB) or C/EBPα. Dlx5 decreased the transcriptional activity of CREB and C/EBPα in a dose-dependent manner. Dlx5 directly bound to CREB and C/EBPα and prevented them from binding to and subsequently transactivating the PPARγ promoter. These results suggest that Dlx5 plays an important regulatory role in fate determination of bone marrow MSCs toward the osteoblast lineage through the inhibition of adipocyte differentiation as well as the direct stimulation of osteoblast differentiation.

Tunicamycin negatively regulates BMP2-induced osteoblast differentiation through CREBH expression in MC3T3E1 cells

Tunicamycin, an endoplasmic reticulum (ER) stress inducer, specifically inhibits N-glycosylation. The cyclic AMP (cAMP) response element-binding protein H (CREBH) was previously shown to be regulated by UPR-dependent proteolytic cleavage in the liver. On the other hand, the role of CREBH in other tissues is unknown. In the present study, tunicamycin increased the level of CREBH activation (cleavage) as well as mRNA expression in osteoblast cells. Adenoviral (Ad) overexpression of CREBH suppressed BMP2-induced expression of alkaline phosphatase (ALP) and osteocalcin (OC). Interestingly, the BMP2-induced OASIS (structurally similar to CREBH, a positive regulator of osteoblast differentiation) expression was also inhibited by CREBH overexpression. In addition, inhibition of CREBH expression using siRNA reversed the tunicamycin-suppressed ALP and OC expression. These results suggest that CREBH inhibited osteoblast differentiation via suppressing BMP2-induced ALP, OC and OASIS expression in mouse calvarial derived osteoblasts.

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α

POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.