Mouse Osteoblastic Cell Line MC3T3-E1 Research Articles

Activation of Nuclear Factor Erythroid 2-Related Factor 2 Transcriptionally Upregulates Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 Expression and Inhibits Ectopic Calcification in Mice.

Calcification plays a key role in biological processes, and breakdown of the regulatory mechanism results in a pathological state such as ectopic calcification. We hypothesized that ENPP1, the enzyme that produces the calcification inhibitor pyrophosphate, is transcriptionally regulated by Nrf2, and that Nrf2 activation augments ENPP1 expression to inhibit ectopic calcification. Cell culture experiments were performed using mouse osteoblastic cell line MC3T3-E1. Nrf2 was activated by 5-aminolevulinic acid and sodium ferrous citrate. Nrf2 overexpression was induced by the transient transfection of an Nrf2 expression plasmid. ENPP1 expression was monitored by real-time RT-PCR. Because the promoter region of ENPP1 contains several Nrf2-binding sites, chromatin immunoprecipitation using an anti-Nrf2 antibody followed by real-time PCR (ChIP-qPCR) was performed. The relationship between Nrf2 activation and osteoblastic differentiation was examined by alkaline phosphatase (ALP) and Alizarin red staining. We used mice with a hypomorphic mutation in ENPP1 (ttw mice) to analyze whether Nrf2 activation inhibits ectopic calcification. Nrf2 and Nrf2 overexpression augmented ENPP1 expression and inhibited osteoblastic differentiation, as indicated by ALP expression and calcium deposits. ChIP-qPCR showed that some putative Nrf2-binding sites in the ENPP1 promoter region were bound by Nrf2. Nrf2 activation inhibited ectopic calcification in mice. ENPP1 gene expression was transcriptionally regulated by Nrf2, and Nrf2 activation augmented ENPP1 expression, leading to the attenuation of osteoblastic differentiation and ectopic calcification in vitro and in vivo. Nrf2 activation has a therapeutic potential for preventing ectopic calcification.

Globo-series Gb4 activates ERK and promotes the proliferation of osteoblasts

ObjectivesGlobo-series Gb4 (globoside) is involved in the immune system and disease pathogenesis. We recently reported that systemic Gb4 deficiency in mice led to decreased bone formation due to a reduction in osteoblast number. However, it remains unclear whether Gb4 expressed in osteoblasts promotes their proliferation. Therefore, we investigated the role of Gb4 in osteoblast proliferation in vitro. MethodsWe examined osteoblast proliferation in Gb3 synthase knockout mice lacking Gb4. We investigated the effects of Gb4 synthase knockdown in the mouse osteoblast cell line MC3T3-E1 on its proliferation. Furthermore, we administered Gb4 to MC3T3-E1 cells in which Gb4 was suppressed by a glucosylceramide synthase (GCS) inhibitor and evaluated its effects on their proliferation. To elucidate the mechanisms by which Gb4 promotes osteoblast proliferation, the phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) levels were measured in MC3T3-E1 cells. ResultsOsteoblast proliferation was lower in Gb3 synthase knockout mice lacking Gb4 than in wild-type mice. Proliferation was inhibited by Gb4 synthase knockdown in MC3T3-E1 cells. Furthermore, the administration of Gb4 to MC3T3-E1 cells, in which a GCS inhibitor suppressed Gb4, promoted their proliferation. Moreover, it increased the phosphorylated ERK1/2 levels in MC3T3-E1 cells. ConclusionsOur results suggest that Gb4 expressed in osteoblasts promotes their proliferation through ERK1/2 activation.

A Novel Porous Butyryl Chitin-Animal Derived Hydroxyapatite Composite Scaffold for Cranial Bone Defect Repair.

Bone defects, a common orthopedic problem in clinical practice, are a serious threat to human health. As alternative materials to autologous bone grafts, synthetic cell-free functionalized scaffolds have been the focus of recent research in designing scaffolds for bone tissue engineering. Butyryl chitin (BC) is a derivative of chitin (CT) with improved solubility. It has good biocompatibility, but few studies have investigated its use in bone repair. In this study, BC was successfully synthesized with a degree of substitution of 2.1. BC films were prepared using the cast film method and showed strong tensile strength (47.8 ± 4.54 N) and hydrophobicity (86.4 ± 2.46°), which was favorable for mineral deposition. An in vitro cytological assay confirmed the excellent cell attachment and cytocompatibility of the BC film; meanwhile, in vivo degradation indicated the good biocompatibility of BC. Hydroxyapatite (HA), extracted from bovine cancellous bone, had good cytocompatibility and osteogenic induction activity for the mouse osteoblast cell line MC3T3-E1. With the aim of combining the advantages of BC and HA, a BC-HA composite scaffold, with a good pore structure and mechanical strength, was prepared by physical mixing. Administered into skull defects of rats, the scaffolds showed perfect bone-binding performance and effective structural support, and significantly promoted the regeneration of new bone. These results prove that the BC-HA porous scaffold is a successful bone tissue engineering scaffold and has strong potential to be further developed as a substitute for bone transplantation.

The Association between Accumulation of Toxic Advanced Glycation End-Products and Cytotoxic Effect in MC3T3-E1 Cells.

In diabetic patients, the metabolism of excess glucose increases the toxicity of the aldehyde group of sugar. Aldehydes, including glyceraldehyde (GA), react with intracellular proteins to form advanced glycation end-products (AGEs), which deteriorate bone quality and cause osteoporosis. One of the causes of osteoporotic fractures is impaired osteoblast osteogenesis; however, the cytotoxic effects of aldehydes and the subsequent formation of AGEs in osteoblasts have not yet been examined in detail. Therefore, the present study investigated the cytotoxicity of intracellular GA and GA-derived AGEs, named toxic AGEs (TAGE), in the mouse osteoblastic cell line MC3T3-E1. Treatment with GA induced MC3T3-E1 cell death, which was accompanied by TAGE modifications in several intracellular proteins. Furthermore, the downregulated expression of Runx2, a transcription factor essential for osteoblast differentiation, and collagen correlated with the accumulation of TAGE. The GA treatment also reduced the normal protein levels of collagen in cells, suggesting that collagen may be modified by TAGE and form an abnormal structure. Collectively, the present results show for the first time that GA and TAGE exert cytotoxic effects in osteoblasts, inhibit osteoblastic differentiation, and decrease the amount of normal collagen. The suppression of GA production and associated accumulation of TAGE has potential as a novel therapeutic target for osteoporosis under hyperglycemic conditions.

MiR-193a-3p Promotes Fracture Healing via Targeting PTEN Gene.

The aim of this study was to investigate the role and potential mechanism of miR-193a-3p in fracture healing. The 70 fragility fracture patients and 45 healthy controls were enrolled in this study. Quantitative real-time PCR (qRT-PCR) was used for the measurement of the expression levels of miR-193a-3p and PTEN. MTT assay and flow cytometry were used to detect cell viability and apoptosis in the mouse osteoblastic cell line MC3T3-E1. Luciferase reporter assay was performed to confirm the correlation of miR-193a-3p with PTEN. The serum expression level of miR-193a-3p showed no significant change in fracture patients 7days after fixation treatment, but over time, there was a significant decrease in the expression at 14days and 21days after treatment (P < 0.01). Overexpression of miR-193a-3p significantly enhanced cell viability and inhibited cell apoptosis in MC3T3-E1 cells (P < 0.001). Serum PTEN level in fracture patients was increased gradually during the fracture healing process (P < 0.01). PTEN was demonstrated to be a target gene of miR-9-5p and reversed the effect of miR-193a-3p on cell viability and apoptosis (P < 0.001). miR-193a-3p promoted fracture healing via regulating PTEN and may serve as a novel potential target for enhancing bone repair of fragility fracture.

Cytotoxicity, Oxidative Stress, and Autophagy Effects of Tantalum Nanoparticles on MC3T3-E1 Mouse Osteoblasts.

As a bone implant material, porous tantalum (Ta) has better corrosion resistance and more suitable elastic modulus than titanium. Surface nanomodification can accelerate the integration of Ta implants with bone tissue, which has broad application prospects in the field of dental implantology. Due to mechanical stress and load wear, nanoscale Ta fragments are inevitably exfoliated from the implant surface and brought into direct contact with osteoblasts surrounding the implant. These wear fragments may affect the biological characteristics of osteoblasts and thus the stability of implants. To date, the interaction of nanoscale Ta fragments with osteoblasts has not been clearly investigated. In the current study, we used the mouse osteoblast cell line MC3T3-E1 to explore the effects of Ta nanoparticles (Ta-NPs) on the cytotoxicity, oxidative stress and autophagy of osteoblasts. We found that a low concentration (12.5 μg/mL) of Ta-NPs can promote the proliferation of osteoblasts, while the Ta-NPs began to induce a decrease in cell viability at concentrations ≥25 μg/mL. Increased cell mortality, reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (MMP) occurred in a dose-dependent manner after Ta-NP treatment. Moreover, with Ta-NP stimulation, the ratio of LC3-II/LC3-I increased, and the level of p62 protein was reduced. However, the degradation of p62 was not continuously increased when the concentration of Ta-NPs was ≥25 μg/mL. These results indicate that Ta-NPs induced osteoblast damage via oxidative stress. Autophagy activation may be a key factor in the cellular response to Ta-NP toxicity and could have an important impact on determining the survival or death of osteoblasts.

LncRNA MEG3 promotes proliferation and differentiation of osteoblasts through Wnt/β-catenin signaling pathway.

We aimed at detecting the expression of long non-coding ribonucleic acid (lncRNA) maternally expressed 3 (MEG3) in the serum of fracture patients, and at investigating its impacts on the proliferation and differentiation of osteoblasts and the specific molecular mechanism of action. The serum samples of 48 fracture patients diagnosed in our hospital (Fracture group) and 30 healthy people receiving physical examination (Health group) were collected. The expression level of serum lncRNA MEG3 in Fracture group and Health group was measured via reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, a small interfering RNA (siRNA) was applied to construct mouse osteoblast cell line MC3T3-E1 with a stable knockout of MEG3. The growth status of the cell was observed, and the impacts of MEG3 knockout on the osteoblast proliferation were determined using cell counting kit-8 (CCK-8), a proliferation activity detection kit. Meanwhile, 5-ethynyl-2'-deoxyuridine (EdU) staining was applied to detect the proportion of EdU positive cells in the osteoblasts in Control group and MEG3 knockout group (MEG3 siRNA group). In addition, RT-PCR was performed to measure the messenger RNA (mRNA) levels of differentiation-related genes. Finally, RT-PCR and Western blotting assay were adopted to analyze the expression of the Wnt/β-catenin signaling pathway. The expression of serum lncRNA MEG3 in fracture patients was increased markedly (p<0.05). Results of in-vitro cell experiment indicated that intervention with MEG3 siRNA could obviously promote the proliferation and differentiation of osteoblast cell line MC3T3-E1. The results of RT-PCR and Western blotting assay revealed that the role of MEG3 in promoting differentiation and proliferation might be mediated by the activation of the Wnt/β-catenin signaling pathway in osteoblasts. LncRNA MEG3 can promote the proliferation and differentiation of osteoblasts by activating the Wnt/β-catenin signaling pathway, so it is expected to become a new target for accelerating the fracture healing.

Knockdown of Long Non-coding RNA TUG1 Suppresses Osteoblast Apoptosis in Particle-induced Osteolysis by Up-regulating BMP-7

B ackground: Periprosthetic osteolysis may lead to the failure of hip arthroplasty. The role of long non-coding RNA TUG1 in inducing periprosthetic osteolysis after hip arthroplasty remains unknown. M ethods: Clinical tissues were obtained from the patients undergoing hip arthroplasty. Mouse osteoblast cell line MC3T3-E1 was simulated by CoCrMo metal particles (CoPs). Real-time PCR was performed to determine the expression of TUG1 and BMP-7. Western blot was performed to determine the BMP-7 protein expression. Cell apoptosis was determined using flow cytometry. RNA pull-down was performed to determine the interaction between TUG1 and BMP-7. In vivo experiments were performed to verify the role of TUG1. R esults : Overexpressed TUG1 and down-regulated BMP-7 was observed in both clinical periprosthetic tissues and CoPs-stimulated osteoblasts. Knockdown of TUG1 significantly inhibited the apoptosis of CoPs-stimulated osteoblasts by targeting BMP-7. In vivo experiments verified that knockdown of TUG1 increased bone mineral density of the mouse osteolysis model. C onclusion: Knockdown of TUG1 inhibited the apoptosis of CoPs-stimulated osteoblasts by negatively regulating BMP-7, which might provide a new insight in treating periprosthetic osteolysis after hip arthroplasty. K eywords: periprosthetic osteolysis; TUG1; BMP-7; osteoblast apoptosis

Understanding the functional role of genistein in the bone differentiation in mouse osteoblastic cell line MC3T3-E1 by RNA-seq analysis

Genistein, a phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood. To obtain insight into the effect of genistein on bone differentiation, RNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in genistein-treated vs. untreated MC3T3-E1 mouse osteoblastic cells. Osteoblastic cell differentiation was monitored by measuring osteoblast differentiation factors (ALP production, bone mineralization, and expression of osteoblast differentiation markers). From RNA-seq analysis, a total of 132 DEGs (including 52 up-regulated and 80 down-regulated genes) were identified in genistein-treated cells (FDR q-value < 0.05 and fold change > 1.5). KEGG pathway and Gene Ontology (GO) enrichment analyses were performed to estimate the biological functions of DEGs and demonstrated that these DEGs were highly enriched in functions related to chemotactic cytokines. The functional relevance of DEGs to genistein-induced osteoblastic cell differentiation was further evaluated by siRNA-mediated knockdown in MC3T3-E1 cells. These siRNA knockdown experiments (of the DEGs validated by real-time qPCR) demonstrated that two up-regulated genes (Ereg and Efcab2) enhance osteoblastic cell differentiation, while three down-regulated genes (Hrc, Gli, and Ifitm5) suppress the differentiation. These results imply their major functional roles in bone differentiation regulated by genistein.

Direct-writing of 3D periodic TiO2 bio-ceramic scaffolds with a sol-gel ink for in vitro cell growth

In this work, three-dimensional (3D) periodic TiO2 bio-ceramic scaffolds were created by continuous filament writing with TiO2 sol-gel ink followed by sintering. The TiO2 sol-gel ink has the advantage of creating 3D bio-ceramic scaffolds with finer feature size than commonly used ceramic powder based ink systems. The structure, pore sizes, and interconnectivity could be controlled through 3D structure design and printing parameter adjustment. To evaluate the potential of these 3D TiO2 bio-ceramic scaffolds for bone tissue engineering, the mouse osteoblastic cell line MC3T3-E1 was cultured on them. The observed good viability demonstrated that 3D TiO2 bio-ceramic scaffolds created by direct-writing technique provide a biocompatible environment that favors cell growth and attachment.

Degradation behavior and compatibility of micro, nanoHA/chitosan scaffolds with interconnected spherical macropores

Hydroxyapatite/Chitosan (HA/CS) composite have significant application in biomedical especially for bone replacement. Inorganic particle shape and size of composite affect the scaffold mechanical property, biological property, and degradation. The aim of this study was to fabricate HA/CS scaffold with good pore connectivity and analyze their biological, degradation properties. Microhydroxyapatite/chitosan (mHA/CS) and nanohydroxyapatite/chitosan (nHA/CS) composite scaffolds with interconnected spherical pore architectures were fabricated. Composite scaffolds structure parameters were analyzed using micro CT. Cell proliferation and morphology were tested and compared between two scaffolds using mouse osteoblastic cell line MC3T3-E1. To research the composite degradation in lysozyme PBS solution, degradation rate and reducing sugar content were tested, and scaffolds morphology were observed by SEM. The results showed that microHA and nanoHA were fabricated by being calcined and synthesis methods, and their infrared spectra are very similar. EDAX composition analysis demonstrated that both of microHA and nanoHA were calcium deficiency HA. Micro-CT results demonstrated the scaffolds had interconnected spherical pores, and the structure parameters were similar. Cell viabilities were significant increased with cultured time, but there were no significant difference between microHA/CS and nanoHA/CS scaffolds. Scaffold structure was gradually destroyed and inorganic composition HA particles are more prominent with degradation time.Significance: (1) Inorganic particle shape and size of composite affect the scaffold mechanical property, biological property, and degradation. NanoHA/CS and microHA/CS scaffolds with good pore connectivity were fabricated and their biological, degradation properties were studied in this manuscript. (2) The scaffold with interconnected porosity construct provides the necessary support for cells to proliferate and maintain their differentiated function, and its architecture related to the structure and morphology of new bone. Polymer scaffolds were fabricated by the technique of compression molding and particulate leaching method, and paraffin microspheres were used as the porogen. (3) MicroHA/CS and nanoHA/CS composite scaffolds are potential materials for use in bone tissue engineering.

Starch-derived absorbable polysaccharide hemostat enhances bone healing via BMP-2 protein

Surgical hemostasis is critical in reducing the likelihood of excessive bleeding and blood transfusion. In treating some cases, commonly used hemostatic agent showed limited efficacy and prolonged degradation and clearance, causing an inhibition of bone healing. Starch absorbable polysaccharide (SAPH) is a novel hemostatic agent made from a plant starch, which can be completely absorbed and achieve better hemostatic effects than many commonly used hemostatic agents. However, whether SAPH can induce a promotion of bone healing remains unknown. In this study, we used a model of rabbit parietal bone defect and a mouse osteoblast cell line MC3T3-E1 to evaluate the effects of SAPH on bone healing. We found that SAPH significantly decreased bone healing scores, reduced defective area of parietal bone, and increased the areas of bone trabeculae and cavitas medullaris. In addition, SAPH enhanced MC3T3-E1 osteoblasts proliferation, up-regulated the expressions of alkaline phosphatase (ALP) and osteocalcin and increased the level of bone morphogenetic protein 2 (BMP-2) in MC3T3-E1 osteoblasts. These SAPH-induced benefits in MC3T3-E1 osteoblasts were significantly abolished by the application of BMP-2-siRNA. These findings suggested that SAPH enhances bone healing, promotes the proliferation, differentiation and maturation of osteoblast by up-regulating BMP-2 expression in osteoblastic cells.

Biocompatibility of artificial bone based on vancomycin loaded mesoporous silica nanoparticles and calcium sulfate composites.

The aim of this study was to evaluate the in vitro and in vivo biocompatibility of artificial bone based on vancomycin loaded mesoporous silica nanoparticles and calcium sulfate composites. In vitro cytotoxicity tests by cholecystokinin octapeptide (CCK-8) assay showed that the 5 %Van-MSN-CaSO4 and Van-CaSO4 bone cements were cytocompatible for mouse osteoblastic cell line MC3T3-E1. The microscopic observation confirmed that MC3T3-E1cells incubated with Van-CaSO4 group and 5 %Van-MSN-CaSO4 group exhibited clear spindle-shaped changes, volume increase and maturation, showing that these cements supported adhesion of osteoblastic cells on their surfaces. In addition, the measurement of alkaline phosphatase activity revealed the osteoconductive property of these biomaterials. In order to assess in vivo biocompatibility, synthesized cements were implanted into the distal femur of twelve adult male and female New Zealand rabbits. After implantation in artificial defects of the distal femur, 5 %Van-MSN-CaSO4 and Van-CaSO4 bone cements did not damage the function of main organs of rabbits. In addition, the Van-MSN-CaSO4 composite allowed complete repair of bone defects with new bone formation 3 months after implantation. These results show potential application of Van-MSN-CaSO4 composites as bone graft materials for the treatment of open fracture in human due to its mechanical, osteoconductive and potential sustained drug release characteristics and the absence of adverse effects on the body.

The influence of MicroRNA-150 in Osteoblast Matrix Mineralization.

This study investigated the influence of miR-150 expression on osteoblast matrix mineralization and its mechanisms. The mouse osteoblast cell line MC3T3-E1 was used as an in vitro model of bone formation. On the fifth day of mineralization, transfection experiments using agomiR-150, agomiR-NC, antagomiR-150 antagomiR-NC, and mock groups were set up to test the effects of miR-150 in MC3T3-E1 model. The mRNA and protein levels of OC, ALP, type I collagen, and OPN were measured by qRT-PCR and ELISA. Matrix mineralization was detected by alizarin red S (ARS) staining and flow cytometry was employed to quantify apoptosis in each group. RT-PCR and Western blot were applied to detect the expression of target gene MMP14. Our results demonstrated that the endogenous expression levels of miR-150, OC, ALP, type I collagen, and OPN in MC3T3-E1 cells increased steadily. Exogenous expressions of agomiR-150 and antagomiR-150 can significantly up-/down-regulate, respectively, the expression level of miR-150 in MC3T3-E1 cells. Compared with the mock group, higher expression levels of OC, ALP, type I collagen, and OPN mRNA were observed in the agomiR-150 group, while lower mRNA expression levels of OC, ALP, type I collagen, and OPN were found in the antagomiR-150 group. Based on these results, potential miR-150 targeted genes are discussed. Our results showed that miR-150 supports the osteoblastic phenotype related to osteoblast function and bone mineralization. Thus, miR-150 may have potential therapeutic applications in promoting bone formation in certain disease settings, such as in osteoporosis and in elderly patients.

Cadherin-11 regulates the metastasis of Ewing sarcoma cells to bone.

Ewing sarcoma (ES) is a small round-cell tumor of the bones and soft tissues. ES frequently causes distant metastases, particularly in the lung and bone, which worsens patient prognosis. Cadherin-11 (Cad-11) is an adhesion molecule that is highly expressed in osteoblasts. Its expression is associated with bone metastases in prostate and breast cancer patients, and is known to occur in ES. Here we investigated the effects of Cad-11 on bone metastases of ES. Human ES cell lines RD-ES, SK-ES-1, SK-N-MC, and TC-71 cells were transduced with lentivirus containing Cad-11 shRNA or control shRNA (ES/Cad-11 and ES/Ctr). RD-ES and TC-71 were infected with a lentivirus luciferase vector. Adhesion assays were performed using these cells and recombinant Cad-11-Fc chimera or mouse osteoblast cell line MC3T3-E1. Cell motility was investigated via wound-healing assay. Intracardiac injection of RD-ES/Cad-11 and RD-ES/Ctr was used to create a mouse model of experimental bone metastasis. The association between Cad-11 expression and bone metastases and clinical prognosis in ES patients was analyzed by immunohistochemistry. We found knockdown of Cad-11 in ES cells resulted in reduced attachment ability and cell motility. In a mouse model of metastasis, RD-ES/Cad-11 cells caused fewer metastases than RD-ES/Ctr cells. The expression of Cad-11 in ES patients was significantly related to bone metastases (P < 0.05, logistic regression) and poorer overall survival (P < 0.05, log-rank test). These findings may explain that Cad-11 in ES cells may be essential for cell adhesion and motility, and is a promising molecular target for patients with ES.

The Effects of Dickkopf-4 on the Proliferation, Differentiation, and Apoptosis of Osteoblasts

The Dickkopf family of proteins is comprised of four members (Dkk1, Dkk2, Dkk3, Dkk4) that are known to modulate Wnt/β-catenin signaling, which is activated during bone formation. Although the effects of Dkk1 on Wnt/β-catenin signaling have been well studied, little is known about the effects of Dkk4. Therefore, to evaluate the role of Dkk4 in osteoblastogenesis, we used the mouse osteoblastic cell line MC3T3-E1, in which Dkk4 expression was suppressed by small interfering RNA knockdown. Our results showed that the suppression of Dkk4 expression promoted osteoblast proliferation and differentiation and suppressed apoptosis. In colony-forming unit alkaline phosphatase assay, Dkk4 knockdown cells possessed markedly higher alkaline phosphatase activity compared with Dkk1 knockdown cells. Reduced Dkk4 expression also led to the up-regulation of β-catenin levels, β-catenin/T cell factor activity, and Wnt-target genes. In contrast, overexpression of Dkk4 in MC3T3-E1 cells led to inhibition of osteoblast differentiation. Our findings reveal that Dkk4 functions as an inhibitor of osteoblastogenesis through Wnt/β-catenin signaling, providing new insights into the relationship between Wnt/β-catenin signaling and Dkk4 in bone formation.

OP0187 Anti-IL-6 Receptor Antibody Promotes Anti-Arthritic Effects of Prednisolone but Not Side-Effects in Mice

BackgroundGlucocorticoids (GCs) such as prednisolone (PSL) and dexamethasone (DEX) are the main therapies used for chronic inflammatory diseases. GCs are potent and effective, but induce many side-effects such as osteoporosis,...

Expression of Bim, Bax and Bak in the process of gingipain-induced osteoblast apoptosis

To establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak). Gingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis. Arginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1. 1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.

Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats

Anti-diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose-dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1. Inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin-induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP-positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats.

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

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