Search

Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1-3 regulation by Runx2. To investigate the physiological role of Runx2 in the regulation of Fgfr1-3, we compared osteoblast progenitors in Sp7 -/- and Runx2 -/- mice. Osteoblast progenitors accumulated and actively proliferated in calvariae and mandibles of Sp7 -/- but not of Runx2 -/- mice, and the number of osteoblast progenitors and their proliferation were dependent on the gene dosage of Runx2 in Sp7 -/- background. The expression of Fgfr2 and Fgfr3, which were responsible for the proliferation of osteoblast progenitors, was severely reduced in Runx2 -/- but not in Sp7 -/- calvariae. Runx2 directly regulated Fgfr2 and Fgfr3, increased the proliferation of osteoblast progenitors, and augmented the FGF2-induced proliferation. The proliferation of Sp7 -/- osteoblast progenitors was enhanced and strongly augmented by FGF2, and Runx2 knockdown reduced the FGF2-induced proliferation. Fgfr inhibitor AZD4547 abrogated all of the enhanced proliferation. These results indicate that Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation, at least partly, by regulating Fgfr2 and Fgfr3 expression.

Long noncoding RNAs (lncRNAs) were proposed to be important regulators influencing various differentiation processes. Yet, the molecular mechanisms of lncRNAs governing osteogenic differentiation of Periodontal Ligament Stem Cells (PDLSCs) remain unclear. Here, PDLSCs were isolated from normal periodontal ligament of human (PDL) whereas P-PDLSCs were isolated from periodontitis affected PDL. Quantitative real-time PCR (qRT-PCR) was performed to examine the relative expression level of lncRNA-ANCR and of Osterix (OSX), Alkaline Phosphatase (ALP) as well as Runt-related transcription factor 2 (RUNX2) in PDLSCs. Gain- and loss-of- function experiments was performed to study the role of lncRNA-ANCR. Alizarin Red staining was used to evaluate the function of lncRNA-ANCR and miRNA-758 on osteogenic differentiation. In addition, via dual luciferase reporter assay and RNA immunoprecipitation the microRNA sponge potential of lncRNA-ANCR was assessed. A luciferase reporter assay identified the correlation between miR-758 and Notch2. Our results showed that the expression of ALP, RUNX2 and OSX were increased whereas lncRNA-ANCR was decreased during the process of differentiation in PDLSCs. Overexpression of lncRNA-ANCR decreased the expression of ALP, RUNX2 and OSX as confirmed by Alizarin red staining. Overexpression of lncRNA-ANCR resulted in reduction of the miR-758 expression level. Furthermore, RNA immunoprecipitation proved that lncRNA-ANCR targets miR-758 directly. The results of dual luciferase reporter assay also demonstrated that miR-758 regulated Notch2 expression by targeting 3'-UTR of Notch2. In conclusion, the novel pathway lncRNA-ANCR/miR-758/Notch2 plays an important role in the process of regulating osteogenic differentiation of PDLSCs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study investigated the effects of conditional deletion of ephrinB1 in osteoprogenitor cells driven by the Osterix (Osx) promoter, on skeletal integrity in a murine model of ovariectomy-induced (OVX) osteoporosis. Histomorphometric and μCT analyses revealed that loss of ephrinB1 in sham Osx:cre-ephrinB1 fl/fl mice caused a reduction in trabecular bone comparable to OVX Osx:Cre mice, which was associated with a significant reduction in bone formation rates and decrease in osteoblast numbers. Interestingly, these observations were not exacerbated in OVX Osx:cre-ephrinB1 fl/fl mice. Furthermore, sham Osx:cre-ephrinB1 fl/fl mice displayed significantly higher osteoclast numbers and circulating degraded collagen type 1 compared to OVX Osx:Cre mice. Confirmation studies found that cultured monocytes expressing EphB2 formed fewer TRAP + multinucleated osteoclasts and exhibited lower resorption activity in the presence of soluble ephrinB1-Fc compared to IgG control. This inhibition of osteoclast formation and function induced by ephrinB1-Fc was reversed in the presence of an EphB2 chemical inhibitor. Collectively, these observations suggest that ephrinB1, expressed by osteoprogenitors, influences bone loss during the development of osteoporosis, by regulating both osteoblast and osteoclast formation and function, leading to a loss of skeletal integrity.

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for the human bone formation, and emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in hBMSC osteogenic differentiation. MALAT1 is often regarded as a tumor-related lncRNA, but its function in mesenchymal stem cell differentiation remains to be defined. In this study, we aimed to investigate whether MALAT1 regulates Osterix (Osx) expression by sponging miR-143 to promote hBMSC osteogenic differentiation. Firstly, we found that the expression of MALAT1 was much lower in hBMSCs from osteoporosis patients and miR-143 was contrarily higher. In addition, MALAT1 expression increased, and miR-143 decreased when hBMSCs were treated with osteogenic induction. Then, we used short hairpin RNAs to knockdown MALAT1, and the results showed that hBMSC osteogenic differentiation decreased significantly, indicating that MALAT1 is a positive regulator of osteogenic differentiation in hBMSCs. Furthermore, by luciferase assays, we found that MALAT1 could directly bind to miR-143 and negatively regulate its expression. Similarly, miR-143 could directly bind to the target site on the Osx 3'-UTR and then inhibit Osx expression. Knockdown of MALAT1 decreased Osx expression, and co-transfection of miR-143 inhibitor could rescue Osx mRNA expression. While Osx expression was increased in MALAT1-overexpressing hBMSCs, it was reversed by the miR-143 mimics. Moreover, Osx silencing decreased ALP, OCN, and OPN mRNA expression induced by the miR-143 inhibitor. Altogether, our findings suggest that MALAT1 acts to regulate Osx expression through targeting miR-143; thus, it is considered as a positive regulator in hBMSC osteogenic differentiation., (© 2018 Wiley Periodicals, Inc.)

Objective: To determine whether quercetin inhibits oxidized low-density lipoprotein (Ox-LDL)-induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) and understand the underlying mechanism., Methods: The calcification of human VSMCs following Ox-LDL treatment was assessed using alizarin red staining and by detecting ALP activity. The mRNA expressions of the bone-related genes including Msx2, BMP2 and Osterix, and the contractile proteins including SMA and SM22a were analyzed using qPCR. The effects of quercetin were investigated on OxLDL-induced VSMC calcification and changes in ALP activity, expressions of Msx2, BMP2, Osterix, SMA and SM22a, ROS levels and SOD activity. The effect of Toll like receptor 4 (TLR4) silencing mediated by siRNA transfection on cell calcification, ALP activity, gene expressions and ROS levels were investigated., Results: Ox-LDL treatment promoted VSMC calcification and up-regulated TLR4 expression. Quercetin treatment significantly attenuated Ox-LDL-induced VSMC calcification, reduced ALP activity, down-regulated the expression levels of Msx2, BMP2 and Osterix, and up-regulated the expressions of vascular smooth muscle contractile proteins SMA and SM22a. In addition, Quercetin treatment markedly increased SOD activity, reduced ROS levels and TLR4 expression in VSMCs. Silencing TLR4 expression using TLR4 siRNA also significantly decreased calcification of the VSMCs., Conclusions: Quercetin inhibits Ox-LDL-induced VSMC calcification in VSMCs possibly by targeting the ROS/TLR4 signaling pathway.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the main source of extracellular pyrophosphate. Along with tissue-nonspecific alkaline phosphatase (TNAP), ENPP1 plays an important role in balancing bone mineralisation. Although well established in pre-osteoblasts, the regulating mechanisms of ENPP1 in osteoblasts and osteocytes remain largely unknown. Using bioinformatic methods, osterix (Osx), an essential transcription factor in osteoblast differentiation and osteocyte function, was found to have five predicted binding sites on the ENPP1 promoter. ENPP1 and Osx showed a similar expression profile both in vitro and in vivo. Over-expression of Osx in MC3T3-E1 and MLO-Y4 cells significantly up-regulated the expression of ENPP1 (p < 0.05). The consensus Sp1 sequences, located in the proximal ENPP1 promoter, were identified as Osx-regulating sites using promoter truncation experiments and chromatin immunoprecipitation (ChIP) assays. The p38-mitogen-activated protein kinase (MAPK) signalling pathway was demonstrated to be responsible for ENPP1 promoter activation by Osx. Runt-related transcription factor 2 (Runx2) was confirmed to have synergistic effects with Osx in activating ENPP1 promoter. Taken together, these results provided evidence of the regulating mechanisms of ENPP1 transcription in osteoblasts and osteocytes.

SP7/Osterix (OSX) is a master regulatory transcription factor that activates a variety of genes during differentiation of osteoblasts. However, the influence of post-translational modifications on the regulation of its transactivation activity is largely unknown. Here, we report that sirtuins, which are NAD(+)-dependent deacylases, regulate lysine deacylation-mediated transactivation of OSX. Germline Sirt7 knockout mice develop severe osteopenia characterized by decreased bone formation and an increase of osteoclasts. Similarly, osteoblast-specific Sirt7 knockout mice showed attenuated bone formation. Interaction of SIRT7 with OSX leads to the activation of transactivation by OSX without altering its protein expression. Deacylation of lysine (K) 368 in the C-terminal region of OSX by SIRT7 promote its N-terminal transactivation activity. In addition, SIRT7-mediated deacylation of K368 also facilitates depropionylation of OSX by SIRT1, thereby increasing OSX transactivation activity. In conclusion, our findings suggest that SIRT7 has a critical role in bone formation by regulating acylation of OSX.

Corticalization, coalescence of trabecular bone into the metaphyseal cortex, is important for the longitudinal growth of long bones. However, little is known about the molecular mechanisms controlling corticalization. To understand the molecular mechanisms underlying corticalization, we analyzed osteoblast-specific Osterix-knockout mice (Col-OMT). In control mice, corticalization was initiated after 7 postnatal days, and the number of osteoblasts in the peripheral spongiosa was increased compared to the number in the central spongiosa. In contrast, in Col-OMT mice, corticalization was delayed, and the number of osteoblasts in peripheral zones was unchanged compared to the central zone. Furthermore, femoral length was decreased in Col-OMT mice at 1 month. Because Col-OMT mice exhibited impaired matrix coalescence and osteoblast migration, we evaluated integrin signaling in Col-OMT mice. Osterix bound to the Itgb3 promoter and increased transcription of the Itgb3 gene in osteoblast cells. Interestingly, the inner and outer cortical bones were separated in Itgb3-null mice at postnatal day 7. In Itgb3-null mice, the number of osteoblasts in peripheral zones was not changed, and the femoral length was decreased. Taken together, these results indicate that Osterix regulates corticalization for longitudinal bone growth via the control of integrin β3 expression in osteoblasts. Our findings imply that the ability to control osteoblast function during corticalization may help in the treatment of short stature.

Objective: To investigate the effects and related mechanisms of miR-204 on fracture healing., Materials and Methods: Mouse osteoblastic cell line MC3T3-E1 was used in our experiment. Three groups were established to investigate the potential function between miR-204 and osteoblastic cells: miR-NC group (negative control), miR-204 mimics group (MC3T3-E1 cells transfected with miR-204 mimics) and miR-204 mimics + inhibitor group (MC3T3-E1 cells transfected with miR-204 mimics and inhibitor). After incubation, cell viability, activity of caspase-3, and migration ability of MC3T3-E1 cells, were measured. Further, the expression levels of Runt-related transcription factor 2 (RUNX2) and Osterix (OSX) were detected and analyzed., Results: Compared with miR-NC group, the cell viability and migration ability of MC3T3-E1 cells were enhanced while the activity of caspase-3 was respectively mitigated. Besides, the expression level of RUNX2 and OSX was increased by treatment of miR-204 mimics. However, these variations of the indicators were reversed by the intervention using miR-204 inhibitor., Conclusions: We revealed the promotion effect of miR-204 on fracture healing, indicating that miR-204 could be a potential therapeutic target for the treatment of a fracture.

Background: With the impaired regenerative potential in patients with diabetes mellitus (DM), Periodontal ligament stem cells (PDLSCs) are regarded as an attractive source of stem cells for periodontal cytotherapy. Recent studies have shown that Exendin-4 (Ex-4) exerts cell-protective effects and bone remodeling ability on many types of cells. The aim of this study was to investigate whether Ex-4 alleviates the inhibition of high glucose on the proliferation and osteogenic differentiation of PDLSCs., Methods: PDLSCs were incubated in medium supplemented with 5.5 mM d-glucose (NG), 30 mM d-glucose (HG), NG plus Ex-4, and HG plus different concentration (1, 10, 20, 100 nM) of Ex-4 respectively. Cell proliferation was detected by CCK-8 assay and cell cycle analysis. Osteogenesis was assessed by Alizarin Red S staining and evaluation of the mRNA expression of Runx2, ALP and Osx at day 7, 14 and 21. Intracellular level of reactive oxygen species (ROS) was detected using 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate (CMH2DCF-DA)., Results: The proliferation ability, mineralized nodules forming capacity and the mRNA expression of Runx2, ALP and Osx of PDLSCs in HG group were decreased, the ROS level was increased compared to NG group. With the treatment of Ex-4, the HG-inhibited proliferation ability and osteogenic differentiation ability of PDLSCs were significantly reversed, the HG-increased ROS level could be down-regulated. Moreover, Ex-4 enhanced the osteogenic differentiation of normal PDLSCs., Conclusions: Ex-4 alleviates the inhibitory effect of HG on the proliferation and osteoblastic differentiation of PDLSCs, and has a significant enhance in the osteoblastic differentiation of normal PDLSCs, giving new insights into the possible therapeutic method of diabetic periodontitis., (Copyright © 2018. Published by Elsevier Ltd.)

Human adipose mesenchymal stem/stromal cells (Ad-MSCs) have been proposed as a suitable option for bone tissue engineering. However, donor age, weight, and gender might affect the outcome. There is still a lack of knowledge of the effects the donor tissue site might have on Ad-MSCs function. Thus, this study investigated proliferation, stem cell, and osteogenic differentiation capacity of human Ad-MSCs obtained from subcutaneous fat tissue acquired from different locations (abdomen, hip, thigh, knee, and limb). Ad-MSCs from limb and knee showed strong proliferation despite the presence of osteogenic stimuli, resulting in limited osteogenic characteristics. The less proliferative Ad-MSCs from hip and thigh showed the highest alkaline phosphatase (AP) activity and matrix mineralization. Ad-MSCs from the abdomen showed good proliferation and osteogenic characteristics. Interestingly, the observed differences were not dependent on donor age, weight, or gender, but correlated with the expression of Sox2 , Lin28A , Oct4α , and Nanog . Especially, low basal Sox2 levels seemed to be pivotal for osteogenic differentiation. Our data clearly show that the donor tissue site affects the proliferation and osteogenic differentiation of Ad-MSCs significantly. Thus, for bone tissue engineering, the donor site of the adipose tissue from which the Ad-MSCs are derived should be adapted depending on the requirements, e.g., cell number and differentiation state.

Purpose: Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis. However, detailed studies of the effects of AnTT on preosteoblastic cells were limited. This study was conducted to investigate the osteogenic effect of AnTT on preosteoblast MC3T3-E1 cells in a time-dependent manner., Materials and Methods: Murine MC3T3-E1 preosteoblastic cells were cultured in the different concentrations of AnTT (0.001-1 µg/mL) up to 24 days. Expression of osteoblastic differentiation markers was measured by qPCR (osterix [OSX], collagen 1 alpha 1 [COL1α1], alkaline phosphatase [ALP], and osteocalcin [OCN]) and by fluorometric assay for ALP activity. Detection of collagen and mineralized nodules was done via Direct Red staining and Alizarin Red staining, respectively., Results: The results showed that osteoblastic differentiation-related genes, such as OSX, COL1α1, ALP, and OCN, were significantly increased in the AnTT-treated groups compared to the vehicle group in a time-dependent manner ( P <0.05). Type 1 collagen level was increased from day 3 to day 15 in the AnTT-treated groups, while ALP activity was increased from day 9 to day 21 in the AnTT-treated groups ( P <0.05). Enhanced mineralization was observed in the AnTT-treated groups via increasing Alizarin Red staining from day 3 to day 21 ( P <0.05)., Conclusion: Our results suggest that AnTT enhances the osteogenic activity by promoting the bone formation-related genes and proteins in a temporal and sequential manner., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7 , whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a , evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1 , pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Objective: To investigate the role of microRNAs (miRNAs) and its mechanism in osteoblast mineralization., Materials and Methods: Real-time polymerase chain reaction (PCR), Northern Blot, and Western Blot were used to identify the expression mode of regulators. Overexpression and down-regulation experiments were carried out to study the role of miR-98 and interactions between regulators. Bioinformatics calculation and luciferase reporter assay were used to prove the target gene. Electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (CHIP), and promoter luciferase reporter assay confirmed the relationship between the regulator and the promoter of miR-98., Results: MiR-98 was up-regulated during osteoblast mineralization. Overexpression of miR-98 promoted osteoblast mineralization. Factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT), a negative regulator of osteoblast differentiation, was confirmed to be a target of miR-98. As a motivator in osteoblast mineralization, Sp7 transcription factor 7 (Sp7) promoted miR-98 transcription by a combination on the promoter region., Conclusions: Our study showed that miR-98 was an important regulator in osteoblast mineralization and miR-98 carried out its function through a novel miR-98-FIAT/Sp7 regulatory loop. It provides new insights into the roles of miRNAs in osteoblast mineralization.

Previous studies have indicated that bone morphogenetic protein 9 (BMP9) can promote the osteogenic differentiation of mesenchymal stem cells (MSCs) and increase bone formation in bone diseases. However, the mechanisms involved remained poorly understood. It is necessary to investigate the specific regulatory mechanisms of osteogenic differentiation that were induced by BMP9. During the process of osteogenic differentiation induced by BMP9, the expression of microRNA-155 (miR-155) exhibited a tendency of increasing at first and then decreasing, which made us consider that miR-155 may have a modulatory role in this process, but the roles of this process have not been elucidated. This study aimed to uncover miR-155 capable of concomitant regulation of this process. mmu-miR-155 mimic (miR-155) was transfected into MSCs and osteogenesis was induction by using recombinant adenovirus expressing BMP9. Overexpressed miR-155 in MSCs led to a decrease in alkaline phosphatase (ALP) staining and Alizarin red S staining during osteogenic differentiation, and reduced the expression of osteogenesis-related genes, such as runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OCN) and osteopontin (OPN). On protein levels, overexpressed miR-155 markedly decreased the expression of phosphorylated Smad1/5/8 (p-Smad1/5/8), Runx2, OCN and OPN. Luciferase reporter assay revealed Runx2 and bone morphogenetic protein receptor 9 (BMPR2) are two direct target genes of miR-155. Downregulation of the expression of Runx2 and BMPR2, respectively could offset the inhibitory effect of miR-155 in the osteogenesis of MSCs. In vivo, subcutaneous ectopic osteogenesis of MSCs in nude mice showed miR-155 inhibited osteogenic differentiation. In conclusion, our results demonstrated that miR-155 can inhibit the osteogenic differentiation induced by BMP9 in MSCs.

MicroRNAs (miRNAs) play important roles in bone metabolism and aging. Here we show that miR-96 was markedly up-regulated in serum of elderly patients with osteoporosis by miRNA microarray analysis and qRT-PCR. Moreover miR-96 was also up-regulated in bone marrow mesenchymal stem cells (BMSCs) of aged humans and mice. Our results show that the over-expression of miR-96 reduced osteogenic differentiation of BMSCs, whereas the inhibition of miR-96 increased osteogenic differentiation of BMSCs. At the molecular level, miR-96 regulated osteogenesis by targeting osterix. Interestingly, over-expression of miR-96 in young mice by intravenous injection of agomiR-96 developed a low bone mass due to impaired osteogenesis. However, inhibition of miR-96 in aged mice attenuated the age-related bone loss. Thus, our data suggest that miR-96 regulates osteogenesis and may represent a potential diagnostic marker or therapeutic target for age-related bone loss., (© 2017 John Wiley & Sons Australia, Ltd.)

The Sp7/Osterix transcription factor is essential for bone development. Mutations of the Sp7 gene in humans are associated with craniofacial anomalies and osteogenesis imperfecta. However, the role of Sp7 in embryonic tooth development remains unknown. Here we identified the functional requirement of Sp7 for dentin synthesis and tooth development. Sp7-null mice exhibit craniofacial dysmorphogenesis and are completely void of alveolar bone. Surprisingly, initial tooth morphogenesis progressed normally in Sp7-null mice. Thus the formation of alveolar bone is not a prerequisite for tooth morphogenesis. Sp7 is required for mineralization of palatal tissue but is not essential for palatal fusion. The reduced proliferative capacity of Sp7-deficient ectomesenchyme results in small and misshapen teeth with randomly arranged cuboidal preodontoblasts and preameloblasts. Sp7 promotes functional maturation and polarization of odontoblasts. Markers of mature odontoblast (Col1a, Oc, Dspp, Dmp1) and ameloblast (Enam, Amelx, Mmp20, Amtn, Klk4) are barely expressed in incisors and molar tissues of Sp7-null mice. Consequently, dentin and enamel matrix are absent in the Sp7-null littermates. Interestingly, the Sp7 expression is restricted to cells of the dental mesenchyme indicating the effect on oral epithelium-derived ameloblasts is cell-nonautonomous. Abundant expression of Fgf3 and Fgf8 ligand was noted in the developing tooth of wild-type mice. Both ligands were remarkably absent in the Sp7-null incisor and molar, suggesting cross-signaling between mesenchyme and epithelium is disrupted. Finally, promoter-reporter assays revealed that Sp7 directly controls the expression of Fgf-ligands. Together, our data demonstrate that Sp7 is obligatory for the differentiation of both ameloblasts and odontoblasts but not for the initial tooth morphogenesis. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

Osteogenesis imperfecta (OI) is a connective tissue disorder characterized by low bone density and recurrent fractures with a wide genotypic and phenotypic spectrum. Common features include short stature, opalescent teeth, blue sclerae and hearing impairment. The majority (>90%) of patients with OI have autosomal dominant variants in COL1A1/COL1A2, which lead to defects in type 1 collagen. More recently, numerous recessive variants involving other genes have also been identified. Sp7/Osx gene, is a protein coding gene that encodes a zinc finger transcription factor, osterix, which is a member of the Sp subfamily of sequence-specific DNA-binding proteins. Osterix is expressed primarily by osteoblasts and has been shown to be vital for bone formation and bone homeostasis by promoting osteoblast differentiation and maturation. In animal models, Sp7/Osx has also been shown to regulate biomineralization of otoliths, calcium carbonate structures found in the inner ear of vertebrates. Until recently, only one report of a boy with an Sp7/Osx pathogenic variant presenting with bone fragility, limb deformities and normal hearing has been described in the literature. We have identified a novel Sp7/Osx variant in another sibship that presented with osteoporosis, low-trauma fractures and short stature. Progressive moderate-to-severe and severe-to-profound hearing loss secondary to otospongiosis and poor mineralization of ossicles and petrous temporal bone was also noted in two of the siblings. A homozygous pathogenic variant in exon 2 of the Sp7/Osx gene was found in all affected relatives; c.946C>T (p.Arg316Cys). Bone biopsies in the proband and his male sibling revealed significant cortical porosity and high trabecular bone turnover. This is the second report to describe children with OI associated with an Sp7/Osx variant. However, it is the first to describe the bone histomorphometry associated with this disorder and identifies a significant hearing loss as a potential feature in this OI subtype. Early audiology screening in these children is therefore warranted., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bone and bone marrow serve as an imperative ecosystem to various types of cells participating in critical body functions. The chemokine Cxcl12, also known as stromal cell-derived factor 1 (Sdf1), is one of the communication factors in the marrow microenvironment that regulates hematopoietic stem/progenitor cell homeostasis. However, the function of Cxcl12 in other bone marrow cells in vivo is yet to be discovered. Here we report a novel function of Cxcl12 in postnatal bone development and homeostasis. Targeted deletion of Cxcl12 in Paired related homeobox 1 (Prx1)-expressing or osterix (Osx)-expressing mesenchymal stem/progenitor cells (MSPCs), but not in mature osteoblasts, resulted in marrow adiposity and reduced trabecular bone content. In vivo lineage tracing analysis revealed biased differentiation of MSPCs toward adipocytes. In contrast, adult-stage deletion of Cxcl12 in Osx-expressing cells led to reduced bone content but not adiposity. Targeting the receptor Cxcr4 in the Prx1-expressing cells also resulted in reduced trabecular bone content but not adiposity. Our study reveals a previously unidentified role of the MSPC-secreting Cxcl12 that regulates its osteogenesis and adipogenesis through the cell-autonomous and non-autonomous mechanism, respectively; which could further influence the homeostatic control of the hematopoietic system. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

Background and Objective: Induced pluripotent stem cells (iPSCs) are a candidate cell source in periodontal regenerative therapy. Enamel matrix derivative (EMD) has been shown to regenerate periodontal tissues, and atelocollagen sponge (ACS) is considered a suitable scaffold or carrier for growth factors. This study aimed to investigate the effect of combined use of EMD and an ACS scaffold on cell behaviors and differentiation of mouse iPSCs (miPSCs) in vitro., Material and Methods: Following embryonic body formation from miPSCs, dissociated cells (miPS-EB-derived cells) were seeded onto ACS with or without EMD, and cultured in osteoblast differentiation medium. Scanning electron microscopy and histological analyses were used to assess cell morphology and infiltration within the ACS. Cell viability (metabolism) was determined using an MTS assay, and expression of mRNA of osteoblastic differentiation markers was assessed by quantitative RT -PCR. Alkaline phosphatase (ALP) staining intensity and activity were evaluated. Mineralization was assessed by von Kossa staining, and calcium content was quantitated using the methylxylenol blue method., Results: By 24 hours after seeding, miPS-EB-derived cells in both the EMD and control groups had attached to and infiltrated the ACS scaffold. Scanning electron microscopy images revealed that by day 14, many cytoplasmic protrusions and extracellular deposits, suggestive of calcified matrix, were present in the EMD group. There was a time-dependent increase in cell viability up to day 3, but no difference between groups was observed at any time point. The levels expressed of ALP and osterix genes were significantly higher in the EMD group than in the control group. Expression of runt-related transcription factor 2 was increased in the EMD group compared with the control group on day 7. EMD upregulated the expression of bone sialoprotein and osteopontin on day 14, whereas expression of osteocalcin was lower at all time points. The staining intensity and activity of ALP were higher in the EMD group than in the control group. Mineralization levels and calcium contents were significantly higher in the EMD group throughout the observation period., Conclusion: These data suggest that combining ACS with EMD increases levels of osteoblastic differentiation and mineralization in miPS-EB-derived cells, compared with ACS used alone., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Serine/threonine protein phosphatase 2A (PP2A) is involved in regulating various physiological processes including cell cycle, growth, apoptosis, and signal transduction. Osteoblast differentiation is controlled by main bone specific transcription factors including Osterix, distal-less homeobox 5 (Dlx5), and Runt-related transcription factor 2 (Runx2). We previously reported that knockdown of PP2A Cα increases the expression of Osterix, leading to the accelerated osteoblast differentiation through the upregulation of bone-related genes. In this study, we examined whether Dlx5 and Runx2 are involved in the upregulated Osterix expression in PP2A Cα-knockdown osteoblasts (shPP2A cells). The expression of Dlx5 as well as Osterix was significantly higher in shPP2A cells in the initial stage of osteoblast differentiation compared with the control cells (shCont). The expression of Runx2 protein was also higher in shPP2A cells compared with shCont cells although its mRNA level was comparable. Reduction of Dlx5 or Runx2 decreased Osterix expression and alkaline phosphatase activity in shPP2A cells. Luciferase assay showed that Osterix promoter activity was drastically elevated in shPP2A cells compared with that in shCont cells. The deletion or mutation of the Dlx5 and Runx2 binding sites significantly suppressed Osterix promoter activity in shPP2A cells. These results indicate that Dlx5 and Runx2 are critical factors for the upregulated Osterix expression in shPP2A cells, which is considered to be important for the accelerated osteoblast differentiation in these cells., (© 2017 International Federation for Cell Biology.)

Osteoblasts provide a microenvironmental niche for B-cell commitment and maturation in the bone marrow (BM). Any abnormity of osteoblasts function may result in the defect of B lymphopoiesis. Signaling from mechanistic target of rapamycin complex 1 (mTORC1) has been implicated in regulating the expansion and differentiation of osteoblasts. Thus, we raise a hypothesis that mTORC1 signaling in osteoblasts plays a vital role in B-cell development. Inactivation of mTORC1 in osterix-expressing cells (mainly osteoblast lineage) through Osx-Cre-directed deletion of Raptor (an mTORC1-specific component) resulted in a reduction in the total B-cell population in the BM, which was due to a block in early B-cell development from the pro-B to pre-B cell stage. Further mechanistic studies revealed that this defect was the result of reduction of interleukin-7 (IL-7) expression in osterix-expressing immature osteoblasts, which caused the abnormality of IL-7/Stat5 signaling in early B lymphocytes, leading to an increased apoptosis of pre-B plus immature B cells. In vitro and in vivo studies demonstrated that the addition of exogenous IL-7 partially restored B lymphopoiesis in the BM of Raptor mutant mice. Furthermore, total BM cells cultured in conditioned media from Raptor null immature osteoblasts or media with anti-IL-7 neutralizing antibody failed to differentiate into pre-B and immature B cells, indicating that inactivation of mTORC1 in immature osteoblast cannot fully support normal B-cell development. Taken together, these findings demonstrate a novel role for mTORC1 in the regulation of bone marrow environments that support B-cell differentiation via regulating IL-7 expression. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

The capacity to fully replace teeth continuously makes zebrafish an attractive model to explore regeneration and tooth development. The requirement of attachment bone for the appearance of replacement teeth has been hypothesized but not yet investigated. The transcription factor sp7 (osterix) is known in mammals to play an important role during odontoblast differentiation and root formation. Here we study tooth replacement in the absence of attachment bone using sp7 zebrafish mutants. We analysed the pattern of tooth replacement at different stages of development and demonstrated that in zebrafish lacking sp7, attachment bone is never present, independent of the stage of tooth development or fish age, yet replacement is not interrupted. Without bone of attachment we observed abnormal orientation of teeth, and abnormal connection of pulp cavities of predecessor and replacement teeth. Mutants lacking sp7 show arrested dentinogenesis, with non-polarization of odontoblasts and only a thin layer of dentin deposited. Osteoclast activity was observed in sp7 mutants; due to the lack of bone of attachment, remodelling was diminished but nevertheless present along the pharyngeal bone. We conclude that tooth replacement is ongoing in the sp7 mutant despite poor differentiation and defective attachment. Without bone of attachment tooth orientation and pulp organization are compromised., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Our previous study showed that wedelolactone, a compound isolated from Ecliptae herba , has the potential to enhance osteoblastogenesis. However, the molecular mechanisms by which wedelolactone promoted osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs) remain largely unknown. In this study, treatment with wedelolactone (2 μg/mL) for 3, 6, and 9 days resulted in an increase in phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N -terminal protein kinase (JNK), and p38. Phosphorylation of mitogen-activated protein kinases (MAPKs), ERK and JNK started to increase on day 3 of treatment, and p38 phosphorylation was increased by day 6 of treatment. Expression of bone morphogenetic protein (BMP2) mRNA and phosphorylation of Smad1/5/8 was enhanced after treatment of cells with wedelolactone for 6 and 9 days. The addition of the JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580 suppressed wedelolactone-induced alkaline-phosphatase activity, bone mineralization, and osteoblastogenesis-related marker genes including Runx2, Bglap, and Sp7. Increased expression of BMP2 mRNA and Smad1/5/8 phosphorylation was blocked by SP600125 and PD98059, but not by SB203580. These results suggested that wedelolactone enhanced osteoblastogenesis through induction of JNK- and ERK-mediated BMP2 expression and Smad1/5/8 phosphorylation., Competing Interests: The authors declare no conflict of interest.

Purpose: This study evaluated the expression pattern of micro RNAs (miRNAs) on a surface with nanotopography compared with a smooth surface (control)., Materials and Methods: Human mesenchymal stem cells (hMSCs) were plated on different surfaces and compared at 3, 7, and 14 days for alkaline phosphatase (ALP) activity, expression of genes (osterix [OSX], runt-related transcription factor 2 [RUNX2], bone morphogenetic protein 2 [BMP2], and ALP), and expression of miRNAs. Western blot was also used to detect osteogenic proteins (BMP2, OSX, and osteocalcin [OCN]). Scanning electron microscopy of cells plated onto the surfaces was obtained., Results: ALP activity on different surfaces was significantly greater in the nanotopography surface. At day 14, there was a 3.5-fold and a 9-fold increase for the RUNX2 and OSX genes, respectively. BMP2 and ALP also increased by fourfold and sevenfold compared with the control. Protein levels for OSX and BMP2 were also upregulated compared with the control group. Using RNA sequencing technology (RNA-Seq), a total of 117 miRNAs were found to be differentially expressed comparing the control (day 7) with the nanosurface (day 14). Forty-five miRNAs were upregulated, and 72 were downregulated. Several of the miRNAs that were differently expressed regulate osteogenic genes. For example, hsa-miR-135b-5p targets OCN, BSP, RUNX2, COL15A1, and OSX; hsa-miR-122-5p targets OPN; hsa-miR-196a-5p targets BMP4; hsa-miR-26b-5p targets BMP2; and hsa-miR-148b-3p targets OPN., Conclusion: Surfaces with nanotopography have the potential to improve the osseointegration response in order to reduce the osseointegration time and also increase bone formation around the implants, improving areas with low bone quality. Within the limitation of this study, nanotopography surfaces affected MSC differentiation to osteoblasts. Several miRNAs were differentially regulated by surface topography. These miRNAs could be related to the differentiation response to help control the osseointegration process.

Studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in bone development. Thus, to investigate in vivo roles of osteoblast AMPK, we conditionally inactivated Ampk in osterix (Osx)-expressing cells by crossing Osx-Cre mice with floxed AMPKα1 to generate mice lacking AMPKα1 in osteoblasts (Ampk-/- mice). Compared with wild-type and Ampk+/- mice, Ampk-/- mice displayed retardation of postnatal bone development, although bone deformity was not observed at birth. Microcomputed tomography showed significant reductions in trabecular bone volume, cortical bone length, and density, as well as increased cortical porosity in femur as well as development defects of skull in 8-week-old Ampk-/- mice. Surprisingly, histomorphometric analysis demonstrated that the number of osteoclasts was significantly increased, although bone formation rate was not altered. Loss of trabecular network connections and mass, as well as shortened growth plates and reduced thickness of cartilage adjacent to the growth plate, was observed in Ampk-/- mice. In primary cultured osteoblasts from calvaria, the expressions of alkaline phosphatase, type 1 collagen, osteocalcin, bone morphogenetic protein 2, Runx2, and osterix were significantly inhibited in Ampk-/- osteoblasts, whereas the expression of receptor activator of nuclear κB ligand (RANKL) and the RANKL/osteoprotegerin ratio were significantly increased. These findings indicate that osteoblastic AMPK plays important roles in bone development in vivo and that deletion of AMPK in osteoblasts decreases osteoblastic differentiation and enhances bone turnover by increasing RANKL expression., (Copyright © 2018 Endocrine Society.)

The timing of gene transfection greatly influences stem cell differentiation. Sequential transfection is crucial for regulation of cell behavior. When transfected several days after differentiation initiation, genes expressed at the late stage of differentiation can regulate cell behaviors and functions. To determine the optimal timing of key gene delivery, we sequentially transfected human mesenchymal stem cells (hMSCs). This method can easily control osteogenesis of stem cells. hMSCs were first transfected with RUNX2 and SP7 using poly(lactic-co-glycolic acid) nanoparticles to induce osteogenesis, and then with ATF4 after 5, 7, and 14 days. Prior to transfecting hMSCs with all three genes, each gene was individually transfected and its expression was monitored. Transfection of these genes was confirmed by RT-PCR, Western blotting, and confocal microscopy. The pDNAs entered the nuclei of hMSCs, and RUNX2 and SP7 proteins were translated and triggered osteogenesis. Second, the ATF4 gene was delivered when cells were at the pre-osteoblasts stage. To induce the osteogenesis of hMSCs, the optimal timing of ATF4 gene delivery was 14 days after RUNX2/SP7 transfection. Experiments in 2- and 3-dimensional culture systems confirmed that transfection of ATF4 at 14 days after RUNX2/SP7 promoted osteogenic differentiation of hMSCs.

Human mesenchymal stem cells (hMSCs) show promise for bone and cartilage regeneration. Our previous studies demonstrated that hMSCs with periodic mild heating had enhanced osteogenic and chondrogenic differentiation with significantly upregulated heat shock protein 70 (HSP70). However, the role of HSP70 in adult tissue regeneration is not well studied. Here, we revealed an essential regulatory mechanism of HSP70 in osteogenesis and chondrogenesis using adult hMSCs stably transfected with specific shRNAs to knockdown HSP70. Periodic heating at 39 °C was applied to hMSCs for up to 26 days. HSP70 knockdown resulted in significant reductions of alkaline phosphatase activity, calcium deposition, and gene expression of Runx2 and Osterix during osteogenesis. In addition, knockdown of HSP70 led to significant decreases of collagens II and X during chondrogenesis. Thus, downregulation of HSP70 impaired hMSC osteogenic and chondrogenic differentiation as well as the enhancement of these processes by thermal treatment. Taken together, these findings suggest a putative mechanism of thermal-enhanced bone and cartilage formation and underscore the importance of HSP70 in adult bone and cartilage differentiation.

Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O -linked-β- N -acetylglucosamine glycosylation ( O -GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O -GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N -acetylglucosamine concentrations or by O -GlcNAc transferase (OGT) overexpression. The effect of O -GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N -acetylglucosamine increased O -GlcNAcylation of Runx2 and the total levels of O -GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O -GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β- N -acetylglucosaminidase. Our findings suggest that excessive protein O -GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation., Competing Interests: The authors declare no conflict of interest.

Postmenopausal osteoporosis affected most elderly women with co-existence of lipid and bone metabolism disorders. However, the cellular and molecular mechanisms underlying the parallel progression and cross-talk of these systems remained unclear. In the present study, low-density lipoprotein receptor knockout (LDLR -/- ) mice were chosen to elucidate the effect of LDLR in regulating the differentiation of osteoblasts, which were responsible for bone formation and modulation of osteoclastogenesis. Primary osteoblasts were isolated from the calvarium of newborn LDLR -/- or wild-type mice followed by osteoblastic differentiation culture in vitro. Alkaline phosphatase activity was significantly decreased in LDLR -/- osteoblasts compared to wild-type controls, combined with calcium deposit formation delay, implying impaired osteoblastogenesis in vitro. Consistent with these findings, the expression of runt-related transcription factor 2 (Runx2) was decreased 3 days after differentiation in LDLR -/- osteoblasts compared to wild-type controls. Moreover, the expression of Osterix was decreased 7 days after differentiation in LDLR -/- osteoblasts compared to wild-type controls, later than Runx2.However, the osteoclastogenesis modulation role of osteoblasts was unaffected by the LDLR deficiency, evidenced by the same level of osteoprotegerin (OPG)/receptor activator of nuclear factor-κ B ligand (RANKL) axis between LDLR -/- and wild-type control osteoblasts. Our results provide a novel insight into the role of LDLR during osteoblastic differentiation and improve understanding of cross-talk between bone and lipid metabolisms.

Background/aims: This study aimed to investigate the effect of Nell-1 on the osteogenic behaviors of pre-osteoblasts on titanium (Ti) surfaces and to identify the underlying signaling pathway., Methods: Nell-1 at different concentrations was added to culture medium to stimulate MC3T3-E1 subclone 14 on Ti surfaces. A CCK-8 colorimetric assay was used to detect cell proliferation. Alkaline phosphatase activity (ALP) assay and enzyme-linked immunosorbent assay (ELISA) were used to evaluate ALP activity and the osteocalcin (OCN) secretion, respectively. Indicators of osteoblastic differentiation were assessed using real-time polymerase chain reaction analysis (RT-PCR). Western blot (WB) assay was used to analyze the expression changes of the osteogenic proteins and the mitogen-activated protein kinase (MAPK) pathway., Results: Nell-1 significantly increased the osteogenic gene and protein expression levels of ALP, OCN, Runx2, osteoprotegerin (OPG), collagen type I (Col-I), and Osterix (Osx) in pre-osteoblasts on Ti surfaces. The optimal concentration of Nell-1 was 100 ng/ ml. In addition, Nell-1 activated ERK and JNK, but not P38, in MC3T3-E1 cells on the Ti surface. Except for ALP and Col-I, the promotive effects of Nell-1 on the expression of osteogenic markers were suppressed by ERK inhibitor U0126., Conclusion: Certain concentrations of Nell-1 can promote the osteogenic differentiation of pre-osteoblasts on Ti surfaces by activating the MAPK/ERK signaling pathway., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Pseudoshikonin I (PSI), a novel biomaterial isolated from Lithospermi radix, has been recognized as an herbal medicine for the treatment of infectious and inflammatory diseases. Bone remodeling maintains a balance through bone resorption (osteoclastogenesis) and bone formation (osteoblastogenesis). Bone formation is generally attributed to osteoblasts. However, the effects of PSI on the bone are not well known. In this study, we found that the ethanol extracts of PSI induced osteoblast differentiation by increasing the expression of bone morphogenic protein 4 (BMP 4). PSI positively regulates the transcriptional expression and osteogenic activity of osteoblast-specific transcription factors such as Runx2 and Osterix. To identify the signaling pathways that mediate PSI-induced osteoblastogenesis, we examined the effects of serine-threonine kinase inhibitors that are known regulators of Osterix and Runx2. PSI-induced upregulation of Osterix and Runx2 was suppressed by treatment with AKT and PKA inhibitors. These results suggest that PSI enhances osteoblast differentiation by stimulating Osterix and Runx2 via the AKT and PKA signaling pathways. Thus, the activation of Runx2 and Osterix is modulated by PSI, thereby demonstrating its potential as a treatment target for bone disease., (© 2017 Wiley Periodicals, Inc.)

Teriparatide is a bone anabolic treatment for osteoporosis, modeled in animals by intermittent PTH (iPTH) administration, but the cellular and molecular mechanisms of action of iPTH are largely unknown. Here, we show that Teriparatide and iPTH cause a ~two-threefold increase in the number of regulatory T cells (Tregs) in humans and mice. Attesting in vivo relevance, blockade of the Treg increase in mice prevents the increase in bone formation and trabecular bone volume and structure induced by iPTH Therefore, increasing the number of Tregs is a pivotal mechanism by which iPTH exerts its bone anabolic activity. Increasing Tregs pharmacologically may represent a novel bone anabolic therapy, while iPTH-induced Treg increase may find applications in inflammatory conditions and transplant medicine., (© 2017 The Authors.)

Methoxsalen (MTS) is a natural bioactive compound found in a variety of plants that has many known biofunctions; however, its effects on osteoporosis and related mechanisms are not clear. This study examined whether MTS exhibited preventive effects against postmenopausal osteoporosis. Female C3H/HeN mice were divided into four groups: Sham, ovariectomy (OVX), OVX with MTS (0.02% in diet), and OVX with estradiol (0.03 μg/day, s.c). After 6 weeks, MTS supplementation significantly increased femur bone mineral density and bone surface along with bone surface/total volume. MTS significantly elevated the levels of serum formation markers (estradiol, osteocalcin and bone-alkaline phosphatase) such as estradiol in OVX mice. Tartrate resistant acid phosphatase staining revealed that MTS suppressed osteoclast numbers and formation in femur tissues compared with the OVX group. Supplementation of MTS slightly up-regulated osteoblastogenesis-related genes (Runx-2, osterix, osteocalcin, and Alp) expression, whereas it significantly down-regulated inflammatory genes (Nfκb and Il6) expression in femur tissue compared with the OVX group. These results indicate that MTS supplementation effectively prevented OVX-induced osteoporosis via enhancement of bone formation and suppression of inflammatory response in OVX mice. Our study provides valid scientific information regarding the development and application of MTS as a food ingredient, a food supplement or an alternative agent for preventing postmenopausal osteoporosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Osteoblast differentiation is achieved by activating a transcriptional network in which Dlx5, Runx2 and Osx/SP7 have fundamental roles. The tumour suppressor p53 exerts a repressive effect on bone development and remodelling through an unknown mechanism that inhibits the osteoblast differentiation programme. Here we report a physical and functional interaction between Osx and p53 gene products. Physical interaction was found between overexpressed proteins and involved a region adjacent to the OSX zinc fingers and the DNA-binding domain of p53. This interaction results in a p53-mediated repression of OSX transcriptional activity leading to a downregulation of the osteogenic programme. Moreover, we show that p53 is also able to repress key osteoblastic genes in Runx2-deficient osteoblasts. The ability of p53 to suppress osteogenesis is independent of its DNA recognition ability but requires a native conformation of p53, as a conformational missense mutant failed to inhibit OSX. Our data further demonstrates that p53 inhibits OSX binding to their responsive Sp1/GC-rich sites in the promoters of their osteogenic target genes, such as IBSP or COL1A1. Moreover, p53 interaction to OSX sequesters OSX from binding to DLX5. This competition blocks the ability of OSX to act as a cofactor of DLX5 to activate homeodomain-containing promoters. Altogether, our data support a model wherein p53 represses OSX-DNA binding and DLX5-OSX interaction, and thereby deregulates the osteogenic transcriptional network. This mechanism might have relevant roles in bone pathologies associated to osteosarcomas and ageing.

Small non-coding RNAs, which are 20-25 nucleotide ribonucleic acids, have emerged as an important transformation in the biological evolution over almost three decades. microRNAs (miRNAs) and short interfering RNAs (siRNAs) are two significant categories of the small RNAs that exert important effects on bone endocrinology and skeletology. Therefore, clarifying the expression and function of these important molecules in bone endocrine physiology and pathology is of great significance for improving their potential therapeutic value for metabolism-associated bone diseases. In the present review, we highlight the recent advances made in understanding the function and molecular mechanism of these small non-coding RNAs in bone metabolism, especially their potentially therapeutic values in bone-related diseases., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

It is assumed that the activity of osteoblasts and osteoclasts is decreased in bone tissue of aged individuals. However, detailed investigation of the molecular signature of human bone from young compared to aged individuals confirming this assumption is lacking. In this study, quantitative expression analysis of genes related to osteogenesis and osteoclastogenesis of human cancellous bone derived from the distal radius of young and aged individuals was performed. Furthermore, we additionally performed immunohistochemical stainings. The young group included 24 individuals with an average age of 23.2 years, which was compared to cancellous bone derived from 11 body donators with an average age of 81.0 years. In cancellous bone of young individuals, the osteogenesis-related genes RUNX-2, OSTERIX, OSTEOPONTIN and OSTEOCALCIN were significantly up-regulated compared to aged individuals. In addition, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in cancellous bone of young individuals, as well as the WNT gene family member WNT5a and the matrix metalloproteinases MMP-9. However, quantitative RT-PCR analysis of BMP-2, ALP, FGF-2, CYCLIN-D1, MMP-13, RANK, OSTEOPROTEGERIN and TGFb1 revealed no significant difference. Furthermore, Tartrate-resistant acid phosphatase (TRAP) staining was performed which indicated an increased osteoclast activity in cancellous bone of young individuals. In addition, pentachrome stainings revealed significantly less mineralized bone matrix, more osteoid and an increased bone density in young individuals. In summary, markers related to osteogenesis as well as osteoclastogenesis were significantly decreased in the aged individuals. Thus, the present data extends the knowledge about reduced bone regeneration and healing capacity observed in aged individuals., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

MicroRNAs have emerged as pivotal regulators in various physiological and pathological processes, including osteogenesis. Here we discuss the contribution of miR-5100 to osteoblast differentiation and mineralization. We found that miR-5100 was upregulated during osteoblast differentiation in ST2 and MC3T3-E1 cells. Next, we verified that miR-5100 can promote osteogenic differentiation with gain-of-function and loss-of-function experiments. Target prediction analysis and experimental validation demonstrated that Tob2, which acts as a negative regulator of osteogenesis, was negatively regulated by miR-5100. Furthermore, we confirmed that the important bone-related transcription factor osterix, which can be degraded by binding to Tob2, was influenced by miR-5100 during osteoblast differentiation. Collectively, our results revealed a new molecular mechanism that fine-tunes osteoblast differentiation through miR-5100/Tob2/osterix networks.

Human mesenchymal stem cells (hMSCs) can differentiate into osteoblasts and are regulated by chemical cues. The recombinant N-terminal (1-34 amino acids) fragment of the parathyroid hormone (PTH (1-34)) is identified to promote osteogenesis. The osteoanabolic effects of intermittent PTH (1-34) treatment are linked to a complex consisting of signaling pathways; additionally, protein kinase C (PKC) act as mediators of multifunctional signaling transduction pathways, but the role of PKC δ (PKCδ), a downstream target in regulating osteoblast differentiation during intermittent administration of PTH (1-34) is less studied and still remains elusive. The purpose of this study is to examine the role of PKCδ during intermittent and continuous PTH (1-34) administration using osteoblast-lineage-committed hMSCs. Relative gene expression of osteoblast-specific genes demonstrated significant upregulation of RUNX2 , type I Collagen , ALP , and Osterix and increased alkaline phosphatase activity in the presence of PTH (1-34). Intermittent PTH (1-34) administration increased PKC activity at day 7 of osteogenic differentiation, whereas inhibition of PKC activity attenuated these effects. In addition, the specific isoform PKCδ was activated upon treatment. These findings demonstrate that intermittent PTH (1-34) treatment enhances the osteogenesis of hMSCs by upregulating osteoblast-specific genes via PKCδ activation., Competing Interests: The authors declare no conflict of interest.

Dentin sialophosphoprotein (Dspp) as a differentiation marker of odontoblasts is regulated by BMP-2. However, the intimate mechanism is still unknown. Transcription factors Dlx3 and Osx are essential for odontoblasts differentiation. We hypothesized that BMP-2 regulation of Dspp transcription was mediated by Dlx3 and/or Osx in odontoblasts. In the present investigation, we found that BMP-2 stimulated expression and nuclear translocation of Dlx3 and Osx in odontoblasts both in vitro and in vivo. Osx was a downstream target of Dlx3 and both of them stimulated Dsp expression. Both Dlx3 and Osx were able to activate Dspp promoter from nucleotides (nt) -318 to +54 by transfections of luciferase reports containing different lengths of mouse Dspp promoters. The binding of Dlx3 and Osx with nt -318 to +54 of Dspp promoter was verified by chromatin immunoprecipitation in vivo. Two Dlx3 binding sites and one Osx binding site on Dspp promoter were found by EMSA. Furthermore, the exact biological function of these binding sites was confirmed by site-directed mutagenesis. At last, the protein-protein interaction between Dlx3 and Osx in odontoblasts was detected by co-immunoprecipitation. In conclusion, in this study we found a novel signaling pathway in which BMP-2 activates Dspp gene transcription via Dlx3/Osx pathway.

Although accumulating evidence indicates that both β-catenin and osterix (Osx) are essential for bone and tooth development, few studies have investigated the interaction of these two key proteins in the context of cementogenesis. In this study, we used transgenic mice with constitutively active β-catenin and inactive Osx in the dental mesenchyme to address this question. We found that cementoblasts with constitutively active β-catenin require Osx to produce excessive cellular cementum, and that ablation of Osx prevents this abnormal accumulation. Importantly, cementoblasts transduced with retrovirus expressing constitutively active β-catenin exhibited upregulation of Osx expression through direct binding to the promoter region of Osx. Osx regulates Lef1 expression and consequently could regulate T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding activity in Wnt/β-catenin signaling. However, the loss of Tcf/Lef binding activity by Osx ablation was not rescued by transduction of retrovirus expressing constitutively active β-catenin or ectopic Lef1 overexpression. These results suggest that the Tcf/Lef binding activity of Wnt/β-catenin signaling is Osx-dependent during cementogenesis. Moreover, Osx differentially regulates the expression of various Tcf family members, suggesting that Osx regulates cementogenesis by utilizing various Tcf/Lef-dependent mechanisms. This is the first report to show that downstream Osx signaling through Tcf/Lefs is critical for cementogenesis.

The molecular pathogenesis of osteonecrosis of the femoral head (ONFH) has been remained obscure so that its prevalence has been increasing in recent decades. Different transcription factors play critical roles in maintaining the balance between osteogenesis and adipogenesis. However, it has been unclear that the genes variants of the transcription factors exert the effects on the imbalance between steogenesis and adipogenesis during the development of ONFH. Here, we selected the 11SNPs from steogenesis, adipogenesis-specific transcription factors RUNX2, Osterix, and PPARγ genes, chondrogenesis or adipogenesis key factors COL2A1, IGFBP3 genes and analysed the genotypes, alleles, haplotypes and their association with the risk and clinical phenotypes of ONFH through Mass ARRAY® platformin in 200 ONFH patients and 177controls. The patients with ONFH (132 males, 68 females; age: 53.46±11.48yr) were consecutively enrolled at the Department of Orthopedics, the Second Clinical College of Jilin University, from March 2014 to June 2015 and were diagnosed and classified into 10 cases of stage II (5.6%), 54 cases of stage III (30.2%) and 115 cases (64.2%) of stage IV and alcohol-induced (71 cases (39.7%)), idiopathic (64 cases (34.0%)), and steroid-induced osteonecrosis (47 cases (26.3%)) subgroup, respectively. Our results showed that all models of logistical regression analysis, the co-dominants, dominants, and recessives of PPARγrs2920502, significantly associated with the increased risk of ONFH (p=0.004, p=0.013, p=0.016), respectively. Both the minor homozygous CC genotype and the allele C of rs2920502 were evidently correlated with the enhanced risk of ONFH (p=0.005, p=0.0005),respectively. The recessives models of IGFBP3rs2132572 (G/A) as well as RUNX2 rs3763190(G/A) were statistically associated with the higher ONFH risk, p=0.030, p=0.029, respectively; the minor homozygous(AA) of IGFBP3rs2132572 (G/A) was also related to the increased risk of bilateral hips lesions, p=0.039. Moreover, the ages on set of major homozygous(GG) and heterozygous(GT) of COL2A1rs2070739(G/A) were significantly younger than that of the minor homozygous(AA) of the SNP(p=0.008) while the A-T-G-A haplotype of COL2A1 gene revealed significant association with the decreased the risk of bilateral hip lesions, p=0.01, OR:0.258. More important, the serum HDL-c level and the ratio of LDL-c/HDL-c in the ONFH group were significantly decreased and increased compared with those of the control group (p=0.02, p=0.0001), respectively. Particularly, the CC genotype of PPARγ rs2920502 was statistically correlated with the enhanced serum TG level, p=0.011.These results suggest that the variants of PPARγ, RUNX2, COL2A1, and IGFBP3 genes closely associated with the development of ONFH., (Copyright © 2017. Published by Elsevier Inc.)

Age-related bone loss in mice results from a decrease in bone formation and an increase in cortical bone resorption. The former is accounted by a decrease in the number of postmitotic osteoblasts which synthesize the bone matrix and is thought to be the consequence of age-dependent changes in mesenchymal osteoblast progenitors. However, there are no specific markers for these progenitors, and conclusions rely on results from in vitro cultures of mixed cell populations. Moreover, the culprits of such changes remain unknown. Here, we have used Osx1-Cre;TdRFP mice in which osteoprogenitors express the TdRFP fluorescent protein. We report that the number of TdRFP-Osx1 cells, freshly isolated from the bone marrow, declines by more than 50% between 6 and 24 months of age in both female and male mice. Moreover, TdRFP-Osx1 cells from old mice exhibited markers of DNA damage and senescence, such as γH2AX foci, G1 cell cycle arrest, phosphorylation of p53, increased p21 CIP 1 levels, as well as increased levels of GATA4 and activation of NF-κB - two major stimulators of the senescence-associated secretory phenotype (SASP). Bone marrow stromal cells from old mice also exhibited elevated expression of SASP genes, including several pro-osteoclastogenic cytokines, and increased capacity to support osteoclast formation. These changes were greatly attenuated by the senolytic drug ABT263. Together, these findings suggest that the decline in bone mass with age is the result of intrinsic defects in osteoprogenitor cells, leading to decreased osteoblast numbers and increased support of osteoclast formation., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Although the biological role of melatonin in osteogenic differentiation has been suggested, the mechanism of osteoblast differentiation remains unclear. Thus, the present study investigated the underlying molecular mechanisms based on osteoblast-specific transcription factors. We found that melatonin enhanced BMP-4-induced osteogenic differentiation and increased the expression of osteogenic markers, especially Osterix, which is an essential transcription factor for the differentiation of preosteoblasts into mature osteoblasts in the late stage of osteoblast differentiation. Melatonin treatment increased the expression of Osterix during osteoblast differentiation and stabilized its expression by the inhibition of ubiquitin-proteasome-mediated degradation of Osterix, leading to up-regulated Osterix transcriptional activity on the osteogenic promoter and promoting alkaline phosphatase activity and bone mineralization. Furthermore, treatment with protein kinase A (PKA) inhibitor H89 and protein kinase C (PKC) inhibitor Go6976 blocked the melatonin-induced transcriptional activity and phosphorylation of Osterix, indicating that melatonin regulates Osterix expression via the PKA and PKC signaling pathways. Overall, these findings suggest that melatonin directly regulates the late stage of osteoblast differentiation by enhancing Osterix expression; this provides further evidence of melatonin as a potent agent for treating osteoporosis.

Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L.) Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP) elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP) activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB). Blockage of cAMP/CREB downstream signals with protein kinase A (PKA) inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.

An imbalance between bone resorption by osteoclasts and bone formation by osteoblasts can cause bone loss and bone-related disease. In a previous search for natural products that increase osteogenic activity, we found that 5,6-dehydrokawain (1) from Alpinia zerumbet promotes osteoblastogenesis. In this study, we synthesized and evaluated series of 5,6-dehydrokawain analogs. Our structure-activity relationships revealed that alkylation of para or meta position of aromatic ring of 1 promote osteogenic activity. Among the potential analogs we synthesized, (E)-6-(4-Ethylstyryl)-4-methoxy-2H-pyran-2-one (14) and (E)-6-(4-Butylstyryl)-4-methoxy-2H-pyran-2-one (21) both significantly up-regulated Runx2 and Osterix mRNA expression at 10µM. These osteogenic activities could be mediated by bone morphogenetic protein (BMP) and activation of p38 MAPK signaling pathways. Compounds 14 and 21 also inhibited RANKL-induced osteoclast differentiation of RAW264 cells. These results indicated that novel 5,6-dehydrokawain analogs not only increase osteogenic activity but also inhibit osteoclast differentiation, and could be potential lead compounds for the development of anti-osteoporosis agents., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Excess glucocorticoid administration impairs osteogenic activities, which raises the risk of osteoporotic disorders. Epigenetic methylation of DNA and histone regulates the lineage commitment of progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 6a (UTX) with regard to the glucocorticoid impediment of osteogenic differentiation. Osteogenic progenitor cells responded to supraphysiological glucocorticoid by elevating CpG dinucleotide methylation proximal to transcription start sites within Runx2 and osterix promoters and Wnt inhibitor Dickkopf-1 (Dkk1) expression concomitant with low UTX expression. 5'-Aza-deoxycystidine demethylation of Runx2 and osterix promoters abolished the glucocorticoid inhibition of mineralized matrix accumulation. Gain of UTX function attenuated the glucocorticoid-induced loss of osteogenic differentiation, whereas UTX silencing escalated adipogenic gene expression and adipocyte formation. UTX sustained osteogenic gene transcription through maintaining its occupancy to Runx2 and osterix promoters. It also mitigated the trimethylation of histone 3 at lysine 27 (H3K27me3), which reduced H3K27me3 enrichment to Dkk1 promoter and thereby lowered Dkk1 transcription. Modulation of β-catenin and Dkk1 actions restored UTX signaling in glucocorticoid-stressed cells. In vivo, UTX inhibition by exogenous methylprednisolone and GSK-J4 administration, an effect that disturbed H3K27me3, β-catenin, Dkk1, Runx2, and osterix levels, exacerbated trabecular microarchitecture loss and marrow adiposity. Taken together, glucocorticoid reduction of UTX function hindered osteogenic differentiation. Epigenetic hypomethylation of osteogenic transcription factor promoters and H3K27 contributed to the UXT alleviation of Dkk1 transcription and osteogenesis in glucocorticoid-stressed osteogenic progenitor cells. Control of UTX action has an epigenetic perspective of curtailing glucocorticoid impairment of osteogenic differentiation and bone mass., Key Messages: UTX attenuates glucocorticoid deregulation of osteogenesis and adipogenesis. UTX reduces Runx2 promoter methylation and H3K27me3 enrichment in the Dkk1 promoter. β-catenin and Dkk1 modulate the glucocorticoid inhibition of UTX signaling. UTX inhibition exacerbates bone mass, trabecular microstructure and fatty marrow. UTX signaling is indispensable in fending off glucocorticoid-impaired osteogenesis.

Background: External root resorption, commonly starting from cementum, is a severe side effect of orthodontic treatment. In this pathological process and repairing course followed, cementoblasts play a significant role. Previous studies implicated that parathyroid hormone (PTH) could act on committed osteoblast precursors to promote differentiation, and inhibit apoptosis. But little was known about the role of PTH in cementoblasts. The purpose of this study was to investigate the effects of intermittent PTH on cementoblasts and its influence after mechanical strain treatment., Results: Higher levels of cementogenesis- and differentiation-related biomarkers (bone sialoprotein (BSP), osteocalcin (OCN), Collagen type I (COL1) and Osterix (Osx)) were shown in 1-3 cycles of intermittent PTH treated groups than the control group. Additionally, intermittent PTH increased alkaline phosphatase (ALP) activity and mineralized nodules formation, as measured by ALP staining, quantitative ALP assay, Alizarin red S staining and quantitative calcium assay. The morphology of OCCM-30 cells changed after mechanical strain exertion. Expression of BSP, ALP, OCN, osteopontin (OPN) and Osx was restrained after 18 h mechanical strain. Furthermore, intermittent PTH significantly increased the expression of cementogenesis- and differentiation-related biomarkers in mechanical strain treated OCCM-30 cells., Conclusions: Taken together, these data suggested that intermittent PTH promoted cementum formation through activating cementogenesis- and differentiation-related biomarkers, and attenuated the catabolic effects of mechanical strain in immortalized cementoblasts OCCM-30.

During osteoporosis bone formation by osteoblasts is reduced and/or bone resorption by osteoclasts is enhanced. Currently, only a few factors have been identified in the regulation of bone integrity by osteoblast-derived osteocytes. In this study, we show that specific disruption of menin, encoded by multiple endocrine neoplasia type 1 (Men1), in osteoblasts and osteocytes caused osteoporosis despite the preservation of osteoblast differentiation and the bone formation rate. Instead, an increase in osteoclast numbers and bone resorption was detected that persisted even when the deletion of Men1 was restricted to osteocytes. We demonstrate that isolated Men1-deficient osteocytes expressed numerous soluble mediators, such as C-X-C motif chemokine 10 (CXCL10), and that CXCL10-mediated osteoclastogenesis was reduced by CXCL10-neutralizing antibodies. Collectively, our data reveal a novel role for Men1 in osteocyte-osteoclast crosstalk by controlling osteoclastogenesis through the action of soluble factors. A role for Men1 in maintaining bone integrity and thereby preventing osteoporosis is proposed.

Background/aims: Osterix (Osx), a key regulator of osteoblast differentiation and bone formation, has been recently reported to be associated with the progression of breast cancer. However, the precise roles of Osx in breast cancer remain unclear., Methods: Drug sensitivity of the cancer cells was assessed using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Target genes were obtained by high-throughput Illumina sequencing and were confirmed in vitro and in vivo. Apoptosis was analysed by Hoechst staining and western blotting. A tissue microarray including 129 samples from breast cancer patients was used for immunohistochemistry (IHC) assays., Results: Overexpression of Osx decreased the chemosensitivity of breast cancer cells, while knockdown of Osx increased the chemosensitivity of breast cancer cells. In particular, we found that the decreased chemosensitivity effect was significantly associated with elevated expression of the polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14). Silencing of GALNT14 in Osx-overexpressed cells restored the decreased chemosensitivity. Conversely, overexpression of GALNT14 in Osx-knockdown cells abrogated the increased chemosensitivity in breast cancer cells. In addition, we revealed that Osx decreased GALNT14-dependent chemosensitivity by enhancing anti-apoptosis. GALNT14 expression exhibited a significant association with breast cancer stages as well as the disease-free survival (DFS) rate., Conclusion: Osx plays an important role in the chemosensitivity and inhibition of Osx expression may represent a therapeutic strategy to enhance the chemosensitivity of breast cancer., (© 2017 The Author(s). Published by S. Karger AG, Basel.)