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Carbohydrate responsive element binding protein (ChREBP) is a major transcription factor of lipogenesis regulated by glucose status in the liver. However, the function of ChREBP in osteogenic differentiation is unclear. The present study examined the role of ChREBP in osteoblast differentiation in MC3T3-E1 preosteoblast cell line. The mRNA expression of ChREBP, protein phosphatase 2A catalytic subunit-α (PP2A Cα) and the osteogenic genes such as, DNA-binding protein inhibitor (Id1), runt-related transcription factor-2 (Runx2), and alkaline phosphatase (ALP) was measured by qPCR and RT-PCR. Runx2, ChREBP, and PP2A Cα, protein levels were evaluated by Western blotting. ALP staining experiment was carried out to evaluate ALP enzyme activity, and a luciferase reporter assay was performed to analyze Runx2 transcriptional activity. Expression of ChREBP and PP2A Cα did not change during bone morphogenetic protein-2 (BMP2)-induced osteoblast differentiation. Overexpression of ChREBP reduced the osteogenic genes (Runx2 and ALP) expression and ALP activity, while knockdown of ChREBP had the opposite effects. Overexpression of PP2A Cα increased ChREBP expression, while inhibition of PP2A Cα using okadaic acid not only inhibited the expression of ChREBP, but also restored the mRNA and protein expression of Runx2 and activity of ALP enzyme. These results demonstrate that ChREBP inhibits BMP2-induced osteoblast differentiation in a PP2A Cα- dependent manner., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

OVO homologue-like 1 (OVOL1) encodes a C2H2 zinc finger protein and is an evolutionarily conserved gene in mammals. The OVOL1 expression is required for development. However, the function of OVOL1 in bone metabolism remains unreported. Here, we show for the first time the role of OVOL1 in osteoblast differentiation. To determine the role of OVOL1 in osteogenic differentiation, we analyzed OVOL1 expression in the preosteoblastic cell line. OVOL1 messenger RNA expression was induced during osteoblast differentiation. In addition, OVOL1 overexpression enhanced the expression of osteogenic genes including bone morphogenetic protein 2 (BMP2), the inhibitor of DNA binding 1 (Id1), distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), osteocalcin (OC), and alkaline phosphatase (ALP). Moreover, mineralization of the extracellular matrix was increased by OVOL1 overexpression in MC3T3-E1 cells. Furthermore, knockdown of the OVOL1 experiment demonstrated that OVOL1 is required for osteoblast differentiation. Collectively, these results suggest that OVOL1 function as an important regulator of osteoblast differentiation by inducing BMP2 expression in MC3T3-E1 cells., (© 2018 Wiley Periodicals, Inc.)

Peroxiredoxin II (Prx II), an antioxidant enzyme in the Prx family, reduces oxidative stress by decreasing the intracellular ROS levels. Osteoblast differentiation is promoted by bone morphogenetic protein 2 (BMP2), which upregulates the expression of osteoblast differentiation marker genes, through Smad1/5/9 phosphorylation. We found that Prx II expression was increased by a high dose of lipopolysaccharide (LPS) but was not increased by a low dose of LPS. Prx II itself caused a decrease in the osteogenic gene expression, alkaline phosphatase (ALP) activity, and Smad1/5/9 phosphorylation induced by BMP2. In addition, BMP2-induced osteogenic gene expression and ALP activity were higher in Prx II knockout (KO) cells than they were in wild-type (WT) cells. These inhibitory effects were mediated by protein phosphatase 2A Cα (PP2A Cα), which was increased and is known to induce the dephosphorylation of Smad1/5/9. The overexpression of Prx II increased the expression of PP2A Cα, and PP2A Cα was not expressed in Prx II KO cells. Moreover, PP2A Cα reduced the level of BMP2-induced osteogenic gene expression and Smad1/5/9 phosphorylation. LPS inhibited BMP2-induced Smad1/5/9 phosphorylation and the suppressed phosphorylation was restored by the PP2A inhibitor okadaic acid (OA). Bone phenotype analyses using microcomputed tomography (μCT) revealed that the Prx II KO mice had higher levels of bone mass than the levels of the WT mice. We hypothesize that Prx II has a negative role in osteoblast differentiation through the PP2A-dependent dephosphorylation of Smad1/5/9.

Aims: CREB (cAMP response element-binding protein)-regulated transcription coactivator (CRTC2) has been reported to act as a coactivator of CREB during gluconeogenesis. The role of CRTC2 in osteoblastic differentiation has not yet been elucidated. The aim of this study is to identify the mechanism of CRTC2 in osteoblast differentiation., Main Methods: The mRNA expression was determined by RT-PCR and qPCR. Protein levels were measured using Western blot assay. Alkaline phosphatase (ALP) staining was performed to evaluate ALP activity. Alizarin red S (ARS) staining was performed to measure extracellular mineralization. Transcriptional activity was detected using a luciferase assay., Key Findings: In the present study, TNF-α was found to stimulate CRTC2 expression. However, TNF-α did not increase the gene expression of osteoblast differentiation markers and inhibited BMP2-induced osteoblastic differentiation. Overexpression of CRTC2 decreased the expression of osteogenic genes, ALP activity and extracellular matrix mineralization. Knockdown of CRTC2 restored BMP2-induced osteogenic gene expression and ALP activity. CRTC2 increased Smurf1 mRNA expression, Smurf 1 promoter activity, and protein level. Furthermore, Smurf 1 decreased Smad 1/5/9 protein levels. These results suggest that CRTC2 decreased BMP2-induced osteoblastic differentiation via Smurf 1 expression., Significance: Our results indicate that CRTC2 regulates the expression of Smurf1 in osteoblast differentiation., (Copyright © 2018. Published by Elsevier Inc.)

The RNA-binding motif protein 3 (RBM3) belongs to a small group of proteins whose synthesis increases during hypothermia while global protein production is slowed down. Bone homeostasis is maintained by a balance between bone resorption and bone formation. Osteoblasts are key components of the bone and have an important role in bone remodeling cycle. However, hypothermia-induced RBM3 between osteoblasts remains unclear. At 32°C, expression of RBM3 and Runx2 was increased in a time-dependent manner and mineralization was also increased. RBM3 was also increased in a time-dependent manner under osteogenic conditions. Overexpression of RBM3 increased the expression of osteogenic genes such as Runx2 and OC. The osteogenic condition-induced expressions of RBM3, Runx2 and OC gene were decreased by RBM3 siRNA. Moreover, RBM3 promoted ERK and p38 phosphorylation. The inhibitor of ERK decreased the expression of Runx2 but did not affect the expression of RBM3. Taken together, these results demonstrate that RBM3 stimulates osteoblast differentiation via the ERK signaling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Kisspeptin-10 (KP-10) acts as a tumor metastasis suppressor via its receptor, G-protein-coupled receptor 54 (GPR54). The KP-10-GPR54 system plays an important role in embryonic kidney development. However, its function in osteoblast differentiation is unknown. Osteoblast differentiation is controlled by a range of hormones and cytokines, such as bone morphogenetic protein (BMPs), and multiple transcription factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and Distal-less homeobox 5 (Dlx5). In the present study, KP-10-treatment significantly increased the expression of osteogenic genes, including mRNA and protein levels of BMP2, in C3H10T1/2 cells. Moreover, KP-10 induced BMP2-luc activity and increased phosphorylation of Smad1/5/9. In addition, NFATc4 specifically mediated KP-10-induced BMP2 gene expression. However, KP-10 treatment did not induce expression of the BMP2 and Runx2 genes in GPR54 -/- cells. To examine whether KP-10 induced secretion of BMP2 to the culture medium, we used the conditioned-medium (C.M) of KP-10 treated medium on C3H10T1/2 cells. Dlx5 and Runx2 expressions were higher in GPR54 -/- cells treated with C.M than in those treated with KP-10. These results demonstrate that BMP2 protein has an autocrine effect upon KP-10 treatment. Taken together, these findings suggest that KP-10/GPR54 signaling induces osteoblast differentiation via NFATc4-mediated BMP2 expression.

Aims: Curcumin (diferuloylmethane or [1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6heptadiene-3,5-dione) is a phenolic natural product derived from the rhizomes of the turmeric plant, Curcuma longa. It is reported to have various biological actions such as anti-oxidative, anti-inflammatory, and anti-cancer effects. However, the molecular mechanism of osteoblast differentiation by curcumin has not yet been reported., Main Methods: The cytotoxicity of curcumin was identified using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of osteogenic markers and endoplasmic reticulum (ER) stress markers in C3H1-T1/2 cells were measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity in C3H10T1/2 cells. Transcriptional activity was detected using a luciferase reporter assay., Key Findings: Curcumin increased the expression of genes such as distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC), which subsequently induced osteoblast differentiation in C3H10T1/2 cells. In addition, ALP activity and mineralization was found to be increased by curcumin treatment. Curcumin also induced mild ER stress similar to bone morphogenetic protein 2 (BMP2) function in osteoblast cells. Next, we confirmed that curcumin increased mild ER stress and osteoblast differentiation similar to BMP2 in C3H10T1/2 mesenchymal stem cells. Transient transfection studies also showed that curcumin increased ATF6-Luc activity, while decreasing the activities of CREBH-Luc and SMILE-Luc. In addition, similar to BMP2, curcumin induced the phosphorylation of Smad 1/5/9., Significance: Overall, these results demonstrate that curcumin-induced mild ER stress increases osteoblast differentiation via ATF6 expression in C3H10T1/2 cells., (Copyright © 2017. Published by Elsevier Inc.)

Piperine is an alkaloid responsible for the pungency of black pepper and long pepper. It is reported to have various biological actions such as anti-oxidative and anti-inflammatory, and aids cancer prevention. Antioxidants have been shown to promote osteoblast differentiation. However, osteoblast differentiation by piperine has not yet been elucidated. Piperine-induced expression of the osteogenic genes such as distal-less homeobox 5 (Dlx5), inhibitor of DNA binding-1 (Id1), and runt-related transcription factor 2 (Runx2) was investigated using RT-PCR. In addition, alkaline phosphatase (ALP) activity and mineralization was found to be increased by piperine treatment. Finally, we confirmed that piperine induced phosphorylation of AMPK in MC3T3-E1 cells. Taken together, these results demonstrate that piperine enhance osteoblast differentiation through AMPK phosphorylation in MC3T3-E1 cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aims: Costunolide is a sesquiterpene lactones used in many herbal medicines, with well-established anti-inflammatory and anti-oxidant functions modulating endoplasmic reticulum (ER) stress pathways, and which promotes the expression of anti-oxidant genes. The aim of this study is to investigate whether costunolide is involved in osteoblast differentiation and, determine the mechanisms of differentiation in mesenchymal stem cells., Main Methods: The cytotoxicity of costunolide was identified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression levels of osteogenic genes were determined by RT-PCR and Western blot analysis. Alkaline phosphate (ALP) staining and Alizarin red S (ARS) staining were performed to evaluate ALP activity and matrix mineralization. Transcriptional activity was detected using a luciferase reporter assay., Key Findings: In this study, we determined that costunolide increased the expression of distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC) in C3H10T 1/2 cells. Furthermore, costunolide increased ALP activity and matrix mineralization. Interestingly, costunolide increased ER stress by Bip, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). However, it did not exert effects on expression of activating transcription factor 6 (ATF6). ATF4 activation has a protective role in oxidative stress, and its transcription induces anti-oxidant genes in cells. Heme oxygenase-1 (HO-1) is a major anti-oxidant enzyme, and is regulated by ATF4. We showed that costunolide treatment increased HO-1 expression. Furthermore, the HO-1 inhibitor, Sn(IV) Protoporphyrin IX dichloride (SnPP) was blocked costunolide-induced Runx2 expression., Significance: Our results revealed that costunolide-induced osteoblast differentiation is regulated by ATF4-dependent HO-1 expression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aims: Mammalian circadian rhythms regulate many metabolic processes. Recent studies suggest that brain and muscle Arnt-like 1 (BMAL1), an important component of mammalian circadian rhythm, is associated with insulin signaling. Several studies have shown that insulin is associated with bone metabolism; however, the relationship between BMAL1 and osteoblasts remains unclear., Main Methods: Expression of osteogenic markers and Bmal1 in MC3T3-E1 cells was measured by RT-PCR and Western blotting. Alizarin red S staining was performed to assess matrix mineralization in MC3T3-E1 cells., Key Findings: mRNA levels of osteogenic genes and Bmal1 were up-regulated in MC3T3-E1 cells upon insulin treatment. In addition, Bmal1 overexpression increased the expression of osteogenic genes including inhibitor of DNA binding (Id1), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC). Interestingly, expression of Bone morphogenetic protein-2 (BMP2), an important upstream factor of Id1, Runx2, and OC, was markedly increased by Bmal1. Finally, we confirmed that insulin-induced BMP2 expression was attenuated in Bmal1 knockout (KO) cells. PCR analysis and alizarin red S staining showed that insulin-mediated increases gene expression and calcium deposition were reduced in Bmal1 KO cells compared to wild-type cells., Significance: Taken together, these results demonstrate that Bmal1 promotes osteoblast differentiation by regulating BMP2 expression in MC3T3-E1 cells., (Copyright © 2016. Published by Elsevier Inc.)

Endoplasmic reticulum (ER) stress transducers, such as old astrocyte specifically induced substance (OASIS) and activating transcription factor 6 (ATF6), which are induced by bone morphogenetic protein 2 (BMP2), regulate bone formation and osteoblast differentiation. Here, we examined the role of cAMP response element-binding protein H (CREBH), a member of the same family of ER membrane-bound basic leucine zipper (bZIP) transcription factors as OASIS and ATF6, in osteoblast differentiation and bone formation. Proinflammatory cytokine TNFα increased CREBH expression by up-regulating the nuclear factor-κB (NF-κB) signaling pathway in osteoblasts, increased the level of N-terminal fragment of CREBH in the nucleus, and inhibited BMP2 induction of osteoblast specific gene expression. Overexpression of CREBH suppressed BMP2-induced up-regulation of the osteogenic markers runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OC) in MC3T3-E1 cells and primary osteoblasts, as well as BMP2-induced ALP activity and OC protein production. In contrast, knockdown of CREBH attenuated the inhibitory effect of TNFα on BMP2-induced osteoblast differentiation. Mechanistic studies revealed that CREBH increased the expression of Smad ubiquitination regulatory factor 1 (Smurf1), leading to ubiquitin-dependent degradation of Smad1, whereas knockdown of CREBH inhibited TNFα-mediated degradation of Smad1 by Smurf1. Consistent with these in vitro findings, administration of Ad-CREBH inhibited BMP2-induced ectopic and orthotopic bone formation in vivo. Taken together, these results suggest that CREBH is a novel negative regulator of osteoblast differentiation and bone formation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Small heterodimer partner interacting leucine zipper protein (SMILE) is an orphan nuclear receptor and a member of the bZIP family of proteins. Several recent studies have suggested that SMILE is a novel co-repressor that is involved in nuclear receptor signaling; however, the role of SMILE in osteoblast differentiation has not yet been elucidated. This study demonstrates that SMILE inhibits osteoblast differentiation by regulating the activity of Runt-related transcription factor-2 (RUNX2). Tunicamycin, an inducer of endoplasmic reticulum stress, stimulated SMILE expression. Bone morphogenetic protein-2-induced expression of alkaline phosphatase and osteocalcin, both of which are osteogenic genes, was suppressed by SMILE. The molecular mechanism by which SMILE affects osteocalcin expression was also determined. An immunoprecipitation assay revealed a physical interaction between SMILE and RUNX2 that significantly impaired the RUNX2-dependent activation of the osteocalcin gene. A ChIP assay revealed that SMILE repressed the ability of RUNX2 to bind to the osteocalcin gene promoter. Taken together, these findings demonstrate that SMILE negatively regulates osteocalcin via a direct interaction with RUNX2., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: MicroRNAs (miRNA) are involved in various biological processes including cellular differentiation. However, the role of miR-433 in osteoblast differentiation remains poorly understood. The objective of this study was to investigate the effect of miR-433 on BMP2-induced osteoblast differentiation., Main Methods: The expression of mature miR-433 in cells was detected by real-time PCR. RT-PCR or real-time PCR was used to confirm the expression of osteogenic genes. For the activation or inhibition of miR-433 expression, we used a precursor form of miR-433 or anti-miR-433. Functional activity of miR-433 and Runx2 was evaluated by promoter study. Osteoblast differentiation was evaluated by analyzing alkaline phosphatase (ALP) activity., Key Finding: ERRγ increased miR-433 expression in the mesenchymal stem cell lineage C3H10T1/2. During the BMP2-induction of osteoblastic differentiation of C3H10T1/2, ERRγ and miR433 expression decreased. In addition, during the osteoblastic differentiation, overexpression of ERRγ or miR-433 inhibited the expression of osteogenic marker genes such as Runx2 and ALP. A computer-based prediction algorithm led to the identification of three miR-433 binding sites [S1 (114-145 bp), S2 (3735-3766 bp) and S3 (3828-3860 bp)] on the 3'-UTR of Runx2 mRNA. Furthermore, miR-433 directly targeted S1 and S2, and decreased the level of Runx2 transcript. In addition, miR-433 inhibited BMP2-induced 6×OSE-Luc activities. Anti-miR-433 recovered ERRγ-suppressed Runx2 expression and ALP activity., Significance: These results demonstrated that miR-433 suppressed BMP2-indcued osteoblast differentiation by decreasing the level of Runx2 transcript., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

Bone morphogenetic protein 2 (BMP2) activates unfolded protein response (UPR) transducers, such as PERK and OASIS, in osteoblast cells. ATF6, a bZIP transcription factor, is also a UPR transducer. However, the involvement of ATF6 in BMP2-induced osteoblast differentiation has not yet been elucidated. In the present study, BMP2 treatment was shown to markedly induce the expression and activation of ATF6 with an increase in alkaline phosphatase (ALP) and OC expression in MC3T3E1 cells. In contrast, ATF6 activation by BMP2 was not observed in the Runx2(-/-) primary calvarial osteoblasts, and Runx2 overexpression recovered BMP2 action. BMP2 stimulated ATF6 transcription by enhancing the direct binding of Runx2 to the osteoblast-specific cis-acting element 2 (OSE2, ACCACA, -205 to -200 bp) motif of the Atf6 promoter region. In addition, the overexpression of ATF6 increased the Oc promoter activity by enhancing the direct binding to a putative ATF6 binding motif (TGACGT, -1126 to -1121 bp). The inhibition of ATF6 function with the dominant negative form of ATF6 (DN-ATF6) blocked BMP2- or Runx2-induced OC expression. Interestingly, OASIS, which is structurally similar to ATF6, did not induce Oc expression. ALP and Alizarin red staining results confirmed that BMP2-induced matrix mineralization was also dependent on ATF6 in vitro. Overall, these results suggest that BMP2 induces osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates Oc transcription.

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.

Metformin is an oral anti-diabetic drug of the biguanide class that is commonly used to treat type 2 diabetes mellitus. This study examined the molecular mechanism for the action of metformin on osteoblast differentiation. Metformin-induced mRNA expression of the osteogenic genes and small heterodimer partner (SHP) in MC3T3E1 cells were determined by RT-PCR and real-time PCR. Metformin increased significantly the expression of the key osteogenic genes, such as alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP) as well as SHP. Transient transfection assays were performed in MC3T3E1 cells to confirm the effects of metformin on SHP, OC and Runx2 promoter activities. Metformin increased the transcription of the SHP and OC genes, and the metformin effect was inhibited by dominant negative form of AMPK (DN-AMPK) or compound C (an inhibitor of AMPK). The adenoviral overexpression of SHP increased significantly the level of ALP staining and OC production. However, metformin did not have any significant effect on osteogenic gene expression, ALP staining and activity, and OC production in SHP null (SHP-/-) primary calvarial cells. Moreover, upstream stimulatory factor-1 (USF-1) specifically mediated metformin-induced SHP gene expression. In addition, metformin-induced AMPK activation increased the level of Runx2 mRNA and protein. However, USF-1 and SHP were not involved in metformin-induced Runx2 expression. Transient transfection and chromatin immunoprecipitation assays confirmed that metformin-induced SHP interacts physically and forms a complex with Runx2 on the osteocalcin gene promoter in MC3T3E1 cells. These results suggest that metformin may stimulate osteoblast differentiation through the transactivation of Runx2 via AMPK/USF-1/SHP regulatory cascade in mouse calvaria-derived cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

This study examined the role of AMPK activation in osteoblast differentiation and the underlining mechanism. An AMPK activator (AICAR or metformin) stimulated osteoblast differentiation with increases in ALP and OC protein production as well as the induction of AMPK phosphorylation in MC3T3E1 cells. In addition, metformin induced the phosphorylation of Smad1/5/8 and expression of Dlx5 and Runx2, whereas compound C or dominant negative AMPK inhibited these effects. Transient transfection studies also showed that metformin increased the BRE-Luc and Runx2-Luc activities, which were inhibited by DN-AMPK or compound C. Down-regulation of Dlx5 expression by siRNA suppressed metformin-induced Runx2 expression. These results suggest that the activation of AMPK stimulates osteoblast differentiation via the regulation of Smad1/5/8-Dlx5-Runx2 signaling pathway., (Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.)

In this study, we propose a new bioactive bone cement (BBC), composed of natural bone powder (hydroxyapatite; HA), chitosan powder, and the currently available polymethylmethacrylate (PMMA) bone cement, for use in orthopedic surgeries such as vertebroplasty or as bone filler. Three types of BBCs (BBC I, BBC II, and BBC III) were prepared with different composition ratios. In vitro tests and animal studies were performed with the new BBCs, and with a currently available commercial PMMA bone cement. Surface morphology, chemical composition, changes in pH over time, exothermic temperatures, intrusion, and cellular responses were investigated in vitro. Scanning electron microscopy (SEM) and radiological and histological examinations were performed in animal studies. The results showed that the major components of the BBCs were C, O, Ca, P, Cl, Si, S, Ba, and Mg. The pH values of the BBCs decreased after 1 day, but eventually recovered to 7.2-7.4. The water absorbency, weight loss, and porosity of the BBCs were higher than those of pure PMMA, but the compressive Young's modulus and the ultimate compressive strength (UCS) of the BBCs were lower than those of pure PMMA. The exothermic temperatures of the BBCs were considerably lower than that of pure PMMA. BBC II and III required longer times to solidify than did pure PMMA. Intrusion tests showed that the BBCs were more intrusive than was pure PMMA. Cell proliferation tests demonstrated that BBC II was preferable to pure PMMA for cell attachment and proliferation. No cytotoxic characteristics were found associated with any of the BBCs. In animal tests, BBC II was more biocompatible and osteoconductible than was pure PMMA. The results of in vitro and animal studies indicated that the proposed BBCs have potential clinical application as replacements for the pure PMMA bone cements currently in use.