Osteoblast-specific Gene Expression Research Articles

Osteoclast-derived exosomes inhibit osteogenic differentiation through Wnt/β-catenin signaling pathway in simulated microgravity model

In the microgravity environment of space, astronauts undergo osteoporosis due to an imbalance in bone remodeling. Recent studies have shown that exosomes derived from osteoclasts could mediate cell-to-cell communication in bone remodeling. However, the role of osteoclast-derived exosomes in bone remodeling in microgravity remains a mystery. Our objective is to investigate the cellular processes modulated by exosomes from RAW264.7 cell-derived osteoclasts and the underlying mechanism of action for the exosomes-mediated osteogenesis of osteoblast-like MC3T3-E1 cells in simulated microgravity. We took advantage of the random positioning machine (RPM) to simulate microgravity for the model and investigated the role of exosomes from RAW264.7 cell-derived osteoclasts in osteoclast-osteoblast communication. The results showed that this type of mature osteoclast-derived exosomes (OC-Exos) in the RPM evidently inhibited the cellular proliferation of osteoblasts via inducing cellular apoptosis and altering their cell cycle distribution. Alkaline phosphatase and mineralization activity of MC3T3-E1 cells were significantly lower after treatment with OC-Exos from RPM and mRNA expression of osteoblast-specific genes were down-regulated. Further study confirmed that OC-Exos in RPM obstructed the differentiation of MC3T3-E1 cells by interfering with Wnt/β-catenin signaling pathway. Together, these observations demonstrated that OC-Exos played an essential role in the regulation of bone remodeling in the microgravity environment. These results shed light on a novel pathway of cross-talk between osteoclasts and osteoblasts, which will be helpful to explain the possible mechanisms underlying osteoporosis in microgravity.

Knockdown of FOXA2 enhances the osteogenic differentiation of bone marrow-derived mesenchymal stem cells partly via activation of the ERK signalling pathway

Forkhead box protein A2 (FOXA2) is a core transcription factor that controls cell differentiation and may have an important role in bone metabolism. However, the role of FOXA2 during osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) remains largely unknown. In this study, decreased expression of FOXA2 was observed during osteogenic differentiation of rat BMSCs (rBMSCs). FOXA2 knockdown significantly increased osteoblast-specific gene expression, the number of mineral deposits and alkaline phosphatase activity, whereas FOXA2 overexpression inhibited osteogenesis-specific activities. Moreover, extracellular signal-regulated protein kinase (ERK) signalling was upregulated following knockdown of FOXA2. The enhanced osteogenesis due to FOXA2 knockdown was partially rescued by an ERK inhibitor. Using a rat tibial defect model, a rBMSC sheet containing knocked down FOXA2 significantly improved bone healing. Collectively, these findings indicated that FOXA2 had an essential role in osteogenic differentiation of BMSCs, partly by activation of the ERK signalling pathway.

Comparison of Cell Proliferation and Adhesion of Human Osteoblast Differentiated Cells on Electrospun and Freeze-Dried PLGA/Bioglass Scaffolds

Background: A link between bone loss or bone mineral density and neurodegenerative disease such as Alzheimer’s and Parkinson`s diseases has been recently demonstrated. Human endometrium is an anactive tissue that includes human endometrial stem cells, which are able to differentiate into various cell lines. The goal of this study was to differentiate these cells into osteoblast cells and to culture the differentiated cells onto the scaffolds out of PLGA/Bioglass to be considered as a new method for bone regeneration in neurodegenerative disease. Methods: Endometrial cells were treated with osteogenic media including β-glycerol phosphate, ascorbic acid, and dexamethasone for 21 days in order to be differentiated to the bone. Differentiated osteoblast cells were then cultured onto the scaffolds. Morphology of the cells was examined using SEM and expression of osteoblast markers was studied by real-time PCR and immunocytochemistry. Moreover, biocompatibility of the scaffolds was measured by MTT. Results: The results showed that the scaffold made by the freeze-drying method presented a better biocompatibility and capability to up-regulate the expression of osteoblast-specific genes. Conclusions: Since, hEnSCs are recently found in the stem cell origin for bone tissue repair in vitro, especially when expanded on PLGA/Bioglass scaffolds. Therefore, usage of hEnSCs for bone reconstruction is a new therapeutic approach for interim bone loss in neurodegenerative diseases.

MiR-181d promotes steroid-induced osteonecrosis of the femoral head by targeting SMAD3 to inhibit osteogenic differentiation of hBMSCs.

We aimed to investigate whether miR-181d may be involved in steroid-induced osteonecrosis of the femoral head (ONFH) and its underlying mechanism. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-181d in bone marrow of 5 cases of steroid-induced femoral head necrosis and 5 cases of femoral head necrosis secondary to femoral neck fractures. Marrow-derived mesenchymal stem cells (MSCs) were obtained from bone marrow samples and identified. Subsequently, the effects of miR-181d on osteogenic differentiation were evaluated by alizarin red staining and alkaline phosphatase. Meanwhile, qRT-PCR was performed to detect the levels of osteoblast-specific genes. The expression of miR-181d in the bone marrow of patients with steroid-induced osteonecrosis of the femoral head was significantly higher than that in the control group. When the expression of miR-181d in MSCs was upregulated, the ALP staining became lighter and the number of calcified nodules, as well as the expression of osteoblast-specific genes, decreased significantly. Meanwhile, the opposite results were observed when miR-181d expression was inhibited. Western blot and luciferase reporting assay proved that miR-181d could negatively regulate the expression of SMAD3. MiR-181d can inhibit the differentiation of hBMSCs into osteoblasts by regulating the expression of SMAD3.

MiR-29a promotes osteogenic differentiation of mesenchymal stem cells via targeting HDAC4.

To investigate the role of miR-29a in regulating the differentiation mesenchymal stem cells into osteoblasts. For the first step, the changes of expression of miR-29a during the process of mesenchymal stem cells (MSCs) differentiation into osteoblast were detected. Then, we infected the MSCs with mimics or inhibitors of miR-29a to explore the roles of miR-29a in the differentiation. Further, the prediction and verification of the possible target genes of miR-29a were achieved by bioinformatics analysis and luciferase reporter assay. MiR-29a was up-regulated during the process of MSCs differentiation into osteoblasts. Overexpression or inhibition of miR-29a using mimics or inhibitors had no significant effect on cell proliferation. Furthermore, the differentiation was enhanced when miR-29a was artificially overexpressed in vitro, whereas silencing of miR-29a attenuated this process. It was evidenced by alkaline phosphatase (ALP) staining, matrix mineralization, and increased expression of osteoblast-specific genes. Furthermore, we determined that the gene HDAC4 might be a direct target of miR-29a. In the current study, miR-29a promotes osteogenesis via suppressing HDAC4, indicating that targeting miR-29a may be feasible in the management of osteoporosis.

Simvastatin induces osteogenic differentiation of MSCs via Wnt/β-catenin pathway to promote fracture healing.

This study was designed to investigate whether Simvastatin could facilitate osteogenic differentiation of rat marrow mesenchymal stem cells (MSCs) by modulating the Wnt/β-catenin pathway, thus promoting fracture healing. MSCs were isolated from rat bone marrow specimens and their purity was identified. The third generation of MSCs was cultured in osteoinduction medium containing simvastatin of gradient concentration, and the highest dose of simvastatin that did not cause cell proliferation was determined by the result of the CCK8 assay. The effects of simvastatin on osteogenic differentiation of MSCs were evaluated by ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific gene expression. Finally, Wnt pathway antagonist DKK1 and β-catenin disturbing agent were added to MSCs to detect the ALP activity, Alizarin red staining, alkaline phosphatase staining and osteoblast-specific genes of MSCs respectively, and to evaluate whether simvastatin promoted osteogenic differentiation of MSCs by activating Wnt/β-catenin pathway. After osteoinduction, simvastatin of 0.3 nmol/L was found to be the highest dose that did not induce the proliferation of MSCs. After treated with 0.3 nmol/L simvastatin for 7 days, the ALP activity of cells and the number of cell calcified nodules significantly increased. Meanwhile, the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were clearly up-regulated. However, when the MSCs were treated with DKK1 for 7 days, the ALP activity and the expression of osteoblast-related genes, including ALP, Runx2, OCN, and OPN, were found decreased. Simvastatin markedly up-regulated the expression of the β-catenin protein, while transfection of β-catenin shRNA inhibited the expression of osteoblast-related genes including ALP, Runx2, OCN, and OPN. Simvastatin can promote the differentiation of rat MSCs into osteoblast-like cells, and its mechanism may be related to the Wnt/β-catenin pathway.

Osteoblasts Are Rapidly Ablated by Virus-Induced Systemic Inflammation following Lymphocytic Choriomeningitis Virus or Pneumonia Virus of Mice Infection in Mice.

A link between inflammatory disease and bone loss is now recognized. However, limited data exist on the impact of virus infection on bone loss and regeneration. Bone loss results from an imbalance in remodeling, the physiological process whereby the skeleton undergoes continual cycles of formation and resorption. The specific molecular and cellular mechanisms linking virus-induced inflammation to bone loss remain unclear. In the current study, we provide evidence that infection of mice with either lymphocytic choriomeningitis virus (LCMV) or pneumonia virus of mice (PVM) resulted in rapid and substantial loss of osteoblasts from the bone surface. Osteoblast ablation was associated with elevated levels of circulating inflammatory cytokines, including TNF-α, IFN-γ, IL-6, and CCL2. Both LCMV and PVM infections resulted in reduced osteoblast-specific gene expression in bone, loss of osteoblasts, and reduced serum markers of bone formation, including osteocalcin and procollagen type 1 N propeptide. Infection of Rag-1-deficient mice (which lack adaptive immune cells) or specific depletion of CD8+ T lymphocytes limited osteoblast loss associated with LCMV infection. By contrast, CD8+ T cell depletion had no apparent impact on osteoblast ablation in association with PVM infection. In summary, our data demonstrate dramatic loss of osteoblasts in response to virus infection and associated systemic inflammation. Further, the inflammatory mechanisms mediating viral infection-induced bone loss depend on the specific inflammatory condition.

MiR 376c inhibits osteoblastogenesis by targeting Wnt3 and ARF-GEF-1 -facilitated augmentation of beta-catenin transactivation.

Wnt signaling pathway plays important role in all aspects of skeletal development which include chondrogenesis, osteoblastogenesis, and osteoclastogenesis. Induction of the Wnt-3 signaling pathway promotes bone formation while inactivation of the pathway leads to bone related disorders like osteoporosis. Wnt signaling thus has become a desired target to treat osteogenic disorders. MicroRNAs (miRNAs) represent an important category of elements that interact with Wnt signaling molecules to regulate osteogenesis. Here, we show that miR-376c, a well-characterized tumor suppressor which inhibits cell proliferation and invasion in osteosarcoma by targeting to transforming growth factor-alpha, suppresses osteoblast proliferation, and differentiation. Over-expression of miR-376c inhibited osteoblast differentiation, whereas inhibition of miR-376c function by antimiR-376c promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Target prediction analysis tools and experimental validation by luciferase 3' UTR reporter assay along with qRT-PCR identified Wnt-3 and ARF-GEF-1 as direct targets of miR-376c. It was seen that over-expression of miR-376c leads to repression of canonical Wnt/β-catenin signaling. Our overall results suggest that miR-376c targets Wnt-3 and ARF-GEF-1 suppresses ARF-6 activation which prevents the release of β-catenin and its transactivation thereby inhibiting osteoblast differentiation. Although miR-376c is known to be a tumor repressor; we have identified a second complementary function of miR-376c where it inhibits Wnt-3-mediated osteogenesis and promotes bone loss.

Injectable nanosilica-chitosan microparticles for bone regeneration applications.

This study was aimed at assessing the effects of silica nanopowder incorporation into chitosan-tripolyphosphate microparticles with the ultimate goal of improving their osteogenic properties. The microparticles were prepared by simple coacervation technique and silica nanopowder was added at 0% (C), 2.5% (S1), 5% (S2) and 10% (S3) (w/w) to chitosan. We observed that this simple incorporation of silica nanopowder improved the growth and proliferation of osteoblasts along the surface of the microparticles. In addition, the composite microparticles also showed the increased expression of alkaline phosphatase and osteoblast specific genes. We observed a significant increase ( p < 0.05) in the expression of alkaline phosphatase by the cells growing on all sample groups compared to the control (C) groups at day 14. The morphological characterization of these microparticles through scanning electron microscopy showed that these microparticles were well suited to be used as the injectable scaffolds with perfectly spherical shape and size. The incorporation of silica nanopowder altered the nano-roughness of the microparticles as observed through atomic force microscopy scans with roughness values going down from C to S3. The results in this study, taken together, show the potential of chitosan-tripolyphosphate-silica nanopowder microparticles for improved bone regeneration applications.

Erythropoietin inhibits osteoblast function in myelodysplastic syndromes via the canonical Wnt pathway.

The effects of erythropoietin on osteoblasts and bone formation are controversial. Since patients with myelodysplastic syndromes often display excessively high erythropoietin levels, we aimed to analyze the effect of erythropoietin on osteoblast function in myelodysplastic syndromes and define the role of Wnt signaling in this process. Expression of osteoblast-specific genes and subsequent osteoblast mineralization was increased in mesenchymal stromal cells from healthy young donors by in vitro erythropoietin treatment. However, erythropoietin failed to increase osteoblast mineralization in old healthy donors and in patients with myelodysplasia, whereas the basal differentiation potential of the latter was already significantly reduced compared to that of age-matched controls (P<0.01). This was accompanied by a significantly reduced expression of genes of the canonical Wnt pathway. Treatment of these cells with erythropoietin further inhibited the canonical Wnt pathway. Exposure of murine cells (C2C12) to erythropoietin also produced a dose-dependent inhibition of TCF/LEF promoter activity (maximum at 500 IU/mL, −2.8-fold; P<0.01). The decreased differentiation capacity of erythropoietin-pretreated mesenchymal stromal cells from patients with myelodysplasia could be restored by activating the Wnt pathway using lithium chloride or parathyroid hormone. Its hematopoiesis-supporting capacity was reduced, while reactivation of the canonical Wnt pathway in mesenchymal stromal cells could reverse this effect. Thus, these data demonstrate that erythropoietin modulates components of the osteo-hematopoietic niche in a context-dependent manner being anabolic in young, but catabolic in mature bone cells. Targeting the Wnt pathway in patients with myelodysplastic syndromes may be an appealing strategy to promote the functional capacity of the osteo-hematopoietic niche.

Intermittent Administration of Parathyroid Hormone 1-34 Enhances Osteogenesis of Human Mesenchymal Stem Cells by Regulating Protein Kinase Cδ.

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.

Blockade of Interleukin 6 by Rat Anti-mouse Interleukin 6 Receptor Antibody Promotes Fracture Healing.

Level of interleukin 6 (IL-6) is associated with fracture healing. This study was performed to explore the effect of IL-6 blockade on fracture healing. Clinical serum levels of IL-6 and tumor necrosis factor-α (TNF-α) were evaluated by enzyme-linked immunosorbent assay (ELISA). For animal experiments, the IL-6 levels after fracture and treatment with rat anti-mouse IL-6 receptor antibody (MR16-1) were assessed. Then, mice were assigned into four or seven groups: control group, fracture group, isotype IgG group, and MR16-1 groups. Serum levels of IL-6 and TNF-α, relative flexural rigidity, and mRNA levels of osteoblast-specific genes were respectively assayed by ELISA, three-point bending test, and quantitative reverse transcription PCR (qRT-PCR). Serum levels of IL-6 and TNF-α after fracture in humans and mice were increased. The increase in IL-6 and TNF-α levels in murine serum was attenuated by MR16-1 treatment. The three-point bending test showed the relative flexural rigidity of the femur was decreased after fracture, whereas the decrease was alleviated by MR16-1 treatment. The qRT-PCR results demonstrated mRNA levels of osteoblast-specific genes were upregulated after fracture and then further upregulated by MR16-1 treatment in a dose-dependent manner. Collectively, the serum level of IL-6 was elevated after fracture both in clinical and murine samples. IL-6 blockade by MR16-1 promoted fracture healing, which might be associated with changes in expression of osteoblast-specific genes.

Knockdown of SIRT7 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells partly via activation of the Wnt/\u03b2-catenin signaling pathway

Sirtuin 7 (SIRT7) is a NAD+-dependent deacetylase in the sirtuin family. In a previous study, human bone marrow mesenchymal stem cells (hBMSCs) with reduced SIRT7 activity were developed to evaluate the effect of SIRT7 on osteogenesis. SIRT7 knockdown significantly enhanced osteoblast-specific gene expression, alkaline phosphatase activity, and mineral deposition in vitro. Additionally, SIRT7 knockdown upregulated β-catenin. The enhanced osteogenesis due to SIRT7 knockdown was partially rescued by a Wnt/β-catenin inhibitor. Furthermore, SIRT7 knockdown hBMSCs combined with a chitosan scaffold significantly promoted bone formation in a rat tibial defect model, as determined by imaging and histological examinations. These findings suggest that SIRT7 has an essential role in osteogenic differentiation of hBMSCs, partly by activation of the Wnt/β-catenin signaling pathway.

Acceleration of osteoblast differentiation by a novel osteogenic compound, DMP-PYT, through activation of both the BMP and Wnt pathways

Osteoblast differentiation is regulated through the successive activation of signaling molecules by a complex interplay of extracellular signals such as bone morphogenetic protein (BMP) and Wnt ligands. Numerous studies have identified natural as well as synthetic compounds with osteogenic activity through the regulation of either BMP/SMADs or the Wnt/β-catenin pathway. Here, we attempted to isolate small molecules that concurrently activated both SMADs and β-catenin, which led to the discovery of a novel potent osteogenic compound, DMP-PYT. Upon BMP2 stimulation, DMP-PYT substantially increased osteoblast differentiation featured by enhanced expression of osteoblast-specific genes and accelerated calcification through activation of BMPs expression. DMP-PYT promoted BMP2-induced SMAD1/5/8 phosphorylation and β-catenin expression, the latter in a BMP2-independent manner. DMP-PYT alone enhanced nuclear localization of β-catenin to promote the DNA-binding and transcriptional activity of T-cell factor, thereby resulting in increased osteoblast differentiation in the absence of BMP2. Most importantly, DMP-PYT advanced skeletal development and bone calcification in zebrafish larvae. Conclusively, DMP-PYT strongly stimulated osteoblast differentiation and bone formation in vitro and in vivo by potentiating BMP2-induced activation of SMADs and β-catenin. These results suggest that DMP-PYT may have beneficial effects for preventing and for treating osteoporosis.

Pilose antler peptide potentiates osteoblast differentiation and inhibits osteoclastogenesis via manipulating the NF-κB pathway

Bones are inflexible yet ever-changing metabolic organs, and bone homeostasis is maintained through two delicately regulated processes: bone construction and bone reabsorption. An imbalance in bone metabolism is linked to most orthopedic diseases, including osteoporosis and rheumatoid arthritis. Importantly, tumor necrosis factor-α (TNF-α) blocks osteoblast differentiation and stimulates osteoclast formation, resulting in delayed deposition of new bone and accelerated bone resorption, especially in rheumatoid arthritis patients with inflammatory conditions. Pilose antler peptide (PAP) isolated and purified from deer antlers has been shown to have beneficial effects on chronic inflammation. In the present study, we studied the impact of PAP on osteoblast differentiation and evaluated the regulatory mechanism, with particular emphasis on the effect of PAP on TNF-α-mediated NF-κB signaling. Mouse primary osteoblast cells were activated with bone morphogenetic protein-2 (BMP-2) for osteoblast differentiation. A significant stimulatory effect of PAP in osteoblastogenesis was observed using ALP activity and Alizarin Red S staining assays. Meanwhile, PAP significantly rescued TNF-α-induced impairment of osteoblast formation as well as mineralization. Furthermore, we found a similar trend upon analyzing osteoblast-specific gene expression. PAP significantly rescued TNF-α-mediated decrease in expression of osteoblast-specific genes. A molecular mechanism assay indicated that PAP significantly inhibited TNF-α-mediated stimulation of NF-κB signaling activity, as well as nuclear translocation of its subunit p65. Moreover, over-expression of p65 reversed the stimulatory effects of PAP on osteoblast differentiation. Furthermore, we also identified that PAP dose dependently inhibit osteoclastogenesis, and this effect might be achieved via suppressing NF-κB activity. In summary, this study shows that PAP promotes osteoblast differentiation and blocks TNF-α-mediated suppression of osteoblastogenesis in vitro via the NF-κB/p65 pathway, as well as inhibits osteoclastsogenesis in vitro. Therefore, PAP, a novel drug with both antiresorptive and osteoanabolic activity, shows therapeutic potential as an alternative treatment for osteolytic diseases, including rheumatoid arthritis and osteoporosis.

Accelerated osteogenic differentiation of human bone-derived cells in ankylosing spondylitis.

Ankylosing spondylitis (AS) is characterized by excessive bone formation with syndesmophytes, leading to bony ankylosis. The contribution of osteoblasts to the pathogenesis of ankylosis is poorly understood. The aim of this study was to determine molecular differences between disease controls (Ct) and AS bone-derived cells (BdCs) during osteogenic differentiation with or without inflammation using AS patient serum. We confirmed osteoblastic differentiation of Ct and AS BdCs under osteogenic medium by observing morphological changes and measuring osteoblastic differentiation markers. Osteoblast differentiation was detected by alkaline phosphatase (ALP) staining and activity, and alizarin red and hydroxyapatite staining. Osteoblast-specific markers were analyzed by quantitative reverse-transcriptase-polymerase chain reaction, immunoblotting, and immunostaining. To examine the effects of inflammation, we added AS and healthy control serum to Ct and AS BdCs, and then analyzed osteoblast-specific markers. AS BdCs showed elevated basal intercellular and extracellular ALP activity compared to Ct. When osteoblast differentiation was induced, AS BdCs exhibited higher expression of osteoblast-specific marker genes and faster mineralization than Ct, indicating that these cells differentiated more rapidly into osteoblasts. ALP activity and mineralization accelerated when serum from AS patients was added to Ct and AS BdCs. Our results revealed that AS BdCs showed significantly increased osteoblastic activity and differentiation capacity by regulating osteoblast-specific transcription factors and proteins compared to Ct BdCs. Active inflammation of AS serum accelerated osteoblastic activity. Our study could provide useful basic data for understanding the molecular mechanism of ankylosis in AS.

CHD1 regulates cell fate determination by activation of differentiation-induced genes.

The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination.

Chemical inhibitors of c-Met receptor tyrosine kinase stimulate osteoblast differentiation and bone regeneration

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been recently introduced to negatively regulate bone morphogenetic protein (BMP)-induced osteogenesis. However, the effect of chemical inhibitors of c-Met receptor on osteoblast differentiation process has not been examined, especially the applicability of c-Met chemical inhibitors on in vivo bone regeneration. In this study, we demonstrated that chemical inhibitors of c-Met receptor tyrosine kinase, SYN1143 and SGX523, could potentiate the differentiation of precursor cells to osteoblasts and stimulate regeneration in calvarial bone defects of mice. Treatment with SYN1143 or SGX523 inhibited HGF-induced c-Met phosphorylation in MC3T3-E1 and C3H10T1/2 cells. Cell proliferation of MC3T3-E1 or C3H10T1/2 was not significantly affected by the concentrations of these inhibitors. Co-treatment with chemical inhibitor of c-Met and osteogenic inducing media enhanced osteoblast-specific genes expression and calcium nodule formation accompanied by increased Runx2 expression via c-Met receptor-dependent but Erk-Smad signaling independent pathway. Notably, the administration of these c-Met inhibitors significantly repaired critical-sized calvarial bone defects. Collectively, our results suggest that chemical inhibitors of c-Met receptor tyrosine kinase might be used as novel therapeutics to induce bone regeneration.

Alcohol Induces Cellular Senescence and Impairs Osteogenic Potential in Bone Marrow-Derived Mesenchymal Stem Cells.

Chronic and excessive alcohol consumption is a high-risk factor for osteoporosis. Bone marrow-derived mesenchymal stem cells (BM-MSCs) play an important role in bone formation; however, they are vulnerable to ethanol (EtOH). The purpose of this research was to investigate whether EtOH could induce premature senescence in BM-MSCs and subsequently impair their osteogenic potential. Human BM-MSCs were exposed to EtOH ranging from 10 to 250 mM. Senescence-associated β-galactosidase (SA-β-gal) activity, cell cycle distribution, cell proliferation and reactive oxygen species (ROS) were evaluated. Mineralization and osteoblast-specific gene expression were evaluated during osteogenesis in EtOH-treated BM-MSCs. To investigate the role of silent information regulator Type 1 (SIRT1) in EtOH-induced senescence, resveratrol (ResV) was used to activate SIRT1 in EtOH-treated BM-MSCs. EtOH treatments resulted in senescence-associated phenotypes in BM-MSCs, such as decreased cell proliferation, increased SA-β-gal activity and G0/G1 cell cycle arrest. EtOH also increased the intracellular ROS and the expression of senescence-related genes, such as p16INK4α and p21. The down-regulated levels of SIRT1 accompanied with suppressed osteogenic differentiation were confirmed in EtOH-treated BM-MSCs. Activation of SIRT1 by ResV partially counteracted the effects of EtOH by decreasing senescence markers and rescuing the inhibited osteogenesis. EtOH treatments induced premature senescence in BM-MSCs in a dose-dependent manner that was responsible for EtOH-impaired osteogenic differentiation. Activation of SIRT1 was effective in ameliorating EtOH-induced senescence phenotypes in BMSCs and could potentially lead to a new strategy for clinically preventing or treating alcohol-induced osteoporosis. Ethanol (EtOH) treatments induce premature senescence in marrow-derived mesenchymal stem cells in a dose-dependent manner that is responsible for EtOH-impaired osteogenic differentiation. Activation of SIRT1 is effective in ameliorating EtOH-induced senescence phenotypes, which potentially leads to a new strategy for clinically treating alcohol-induced osteoporosis.

Roles of the Endoplasmic Reticulum Stress Transducer OASIS in Ossification of the Posterior Longitudinal Ligament.

In vitro molecular research on the posterior longitudinal ligament fibroblasts. To investigate different expression of old astrocyte specifically induced substance (OASIS) between spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament (OPLL) and from non-OPLL patients and demonstrate knockdown of OASIS protein expression by RNA interference inhibiting expression of type I collagen (COL I) in OPLL cells. OPLL is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that ligament fibroblasts from OPLL patients have osteogenic characteristics. However, the relevant intracellular signaling pathways remain unclear. Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of osteocalcin, alkaline phosphatase, and COL I were detected in OPLL and non-OPLL cells by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of OASIS was detected by Western blotting. And then, after 72 hours, when RNA interference against OASIS was performed in OPLL cells, expression of the osteoblast-specific genes was compared again between the transfection group and the nontransfection group. Spinal ligament fibroblasts were observed 7 to 10 days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of osteocalcin, alkaline phosphatase, and COL I and protein expressions of OASIS from OPLL cells were significantly greater than those from non-OPLL cells. In addition, knockdown of OASIS protein expression inhibited the mRNA expressions of COL I remarkably in the transfection group compared with the nontransfection group, at 72 hours after RNA interference targeting OASIS was performed in OPLL cells. The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and OASIS expression plays an important role in the development of OPLL through the expression of COL I.