BACKGROUND: Periodontitis is characterized by alveolar bone resorption, aggravated by osteoblast dysfunction, and associated with intracellular oxidative stress linked to the nuclear factor erythroid 2-related factor 2 (NRF2) level. We evaluated the molecular mechanism of periodontitis onset and development and the role of NRF2 in osteogenic differentiation. METHODS: Primary murine mandibular osteoblasts were extracted and exposed to Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) or other stimuli. Reactive oxygen species (ROS) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining were used to detect intracellular oxidative stress. Alkaline phosphatase staining and alizarin red S staining were used to detect the osteogenic differentiation of osteoblasts. Immunofluorescence and western blotting were used to determine the changes in the mitogen-activated protein kinase (MAPK) pathway and related molecule activities. Immunofluorescence colocalization and co-immunoprecipitation were performed to examine the nuclear translocation of NRF2 and its interaction with dual-specific phosphatase 1 (DUSP1) in cells. RESULTS: Ligated tissue samples showed higher alveolar bone resorption rate and lower NRF2 level than healthy periodontal tissue samples. Pg-LPS increased intracellular oxidative stress levels and inhibited osteogenic differentiation, whereas changes in NRF2 expression were correlated with changes in the oxidative stress and osteogenesis rate. NRF2 promoted the dephosphorylation of the MAPK pathway by nuclear translocation and the upregulation of DUSP1 expression, thus enhancing the osteogenic differentiation capacity of mandibular osteoblasts. The interaction between NRF2 and DUSP1 was observed. CONCLUSIONS: NRF2 and its nuclear translocation can regulate the osteogenic differentiation of mandibular osteoblasts under Pg-LPS conditions by interacting with DUSP1 in a process linked to the MAPK pathway. These findings form the basis of periodontitis treatment.
Animals ; NF-E2-Related Factor 2/physiology* ; Lipopolysaccharides/pharmacology* ; Osteoblasts/drug effects* ; Mice ; Porphyromonas gingivalis/chemistry* ; Cell Differentiation ; Osteogenesis ; Dual Specificity Phosphatase 1/metabolism* ; Mandible/cytology* ; Reactive Oxygen Species/metabolism* ; Oxidative Stress ; Periodontitis/metabolism* ; Cells, Cultured ; Male ; Cell Nucleus/metabolism*

Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.
Animals ; Cell Differentiation ; physiology ; Core Binding Factor Alpha 1 Subunit ; Homeodomain Proteins ; metabolism ; Mesenchymal Stem Cells ; metabolism ; Mice ; Mice, Nude ; Osteoblasts ; metabolism ; Osteocalcin ; drug effects ; Osteogenesis ; physiology ; Transcription Factors ; metabolism ; Up-Regulation

To investigate the microRNA (miR)-150 expression level in human osteosarcoma cell lines (Saos-2, MG-63) and its function in cell proliferation, and to explore the potential molecular mechanisms. 
 Methods: MiR-150 expression levels in human osteosarcoma cell lines (Saos-2, MG-63) and normal osteoblast cell line (NHOst) were detected by relative quantitative real-time PCR (qRT-PCR). MiR-150 was overexpressed in Saos-2 and MG-63 cells by lentivirus infection. Cell proliferation rates were monitored by MTS assay. RUNX2 and β-actin protein levels were examined by Western blot. Inhibitory effect of miR-150 on binding RUNX2 3'-UTR was detected by Dual-Luciferase assay.
 Results: MiR-150 expression level is lower in human osteosarcoma cell lines (Saos-2, MG-63) compared to the normal osteoblast cell line (NHOst) (0.23±0.02 and 0.32±0.03 vs 1.00±0.02), which showed statistical significance (P<0.01). After lentivirus infection, miR-150 level increased in Saos-2 (P<0.01) and MG-63 cells (P<0.01). Overexpression of miR-150 decreased cell proliferation and RUNX2 protein level in Saos-2 and MG-63 cells. The binding of miR-150 to RUNX2 3'-UTR decreased luciferase activity by 69% in Saos-2 cells (P<0.05) and 59% in MG-63 cells (P<0.05). Administration of exogenous RUNX2 recovered the cell proliferation in miR-150 overexpressed Saos-2 and MG-63 cell lines (P<0.01).
 Conclusion: MiR-150 inhibites proliferation in human osteosarcoma cell lines through binding to RUNX2 3'-UTR, resulting in the reducion of RUNX2 protein level.
3' Untranslated Regions ; Actins ; metabolism ; Bone Neoplasms ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; genetics ; Core Binding Factor Alpha 1 Subunit ; drug effects ; genetics ; pharmacology ; physiology ; Gene Expression Regulation, Neoplastic ; drug effects ; genetics ; Humans ; MicroRNAs ; pharmacology ; Osteoblasts ; physiology ; Osteosarcoma ; genetics ; physiopathology

OBJECTIVETo establish osteoblast model, primary cilla model was removed by chloral hyrate, observe effects of osteoblast primary cilla moved on enhancing ALP staining and calcified nodules staining in electromagnetic field.

METHODSThree 3-day-old male SD rats weighed between 6 and 9 g were killed, cranial osteoblast was drawed and adherencing cultured respectively. Cells were subcultured and randomly divided into 4 groups until reach to fusion states. The four groups included chloral hydrate non-involved group (control group), 2 mM, 4 mM and 8 mM chloral hydrate group, and cultured in 37 °C, 5% CO2 incubator for 72 h. Morphology of primary cilla was observed by laser confocal scanning microscope, and incidence of osteoblast primary cilia was analyzed by Image-Pro Plus 6.0 software. Cells in the correct concentration group which can removed cillia most effectively were selected and divided into 3 groups, including control group (C), Electromagnetic fields group (EMFs), and EMFs with 4 mM chloral hydrate group. DMEM nutrient solution contained 10%FBS were added into three groups and cultured for 9 days and formation of ALP were observed by histochemical staining of alkaline phosphatase. After 12 days' cultivation, formation of mineralization nodes was observed by alizarin red staining.

RESULTSCompared with control group and 2mM chloral hydrate group,4 mM chloral hydrate group could effectively remove osteoblast primary cilla (P<0.01). Removal of osteoblast primary cilla could weaken the formation of ALP and mineralization nodes in osteoblast in EMFS. Compared with EMFs group, the area of ALP and mineralization nodes in EMFs with 4 mM chloral hydrate group were decreased obviously (P<0.01).

CONCLUSION4mM chloral hydrate could effectively remove osteoblast primary cilia. Primary cilla participate in EMFs promoting formation of ALP and mineralization nodes in osteoblast and provide new ideas for exploring mechanism of EMFs promoting osteoblast maturation and mineralization.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Culture Techniques ; instrumentation ; methods ; Cells, Cultured ; Chloral Hydrate ; pharmacology ; Cilia ; drug effects ; enzymology ; physiology ; Male ; Osteoblasts ; cytology ; enzymology ; Rats ; Rats, Sprague-Dawley

In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein (ox-LDL, 50 mg/L), and divided into four groups according to the ox-LDL treatment time: control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1 (DDK-1, 100 μg/L) was added in ox-LDL 72-h group. The expression of a-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of β-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/β-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of a-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups (P<0.05 for all); β-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group (P<0.05), and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion (P<0.05). After addition of DKK-1, the expression of α-SMA, bone-related proteins and β-catenin protein was significantly reduced as compared with ox-LDL 72-h group (P<0.05). The Wnt/ β-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.
Actins ; metabolism ; Animals ; Aortic Valve ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Lipoproteins, LDL ; pharmacology ; Myofibroblasts ; drug effects ; Osteoblasts ; physiology ; Phenotype ; Swine ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells (VICs) were isolated, cultured and stimulated with oxidized low density lipoprotein (ox-LDL) for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β (GSK-3β) and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction (PCR). The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h (P<0.05). It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.
Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Aortic Valve ; metabolism ; pathology ; Aortic Valve Stenosis ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Calcinosis ; Cell Differentiation ; drug effects ; genetics ; Cells, Cultured ; Gene Expression ; drug effects ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Lipoproteins, LDL ; pharmacology ; Osteoblasts ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Wnt Signaling Pathway ; genetics ; physiology ; beta Catenin ; genetics ; metabolism

This study was performed to evaluate the contribution of adiponectin to the production of interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and MMP-13 in human endothelial cells and osteoblasts in arthritic joints. Cultured human umbilical vascular endothelial cells (HUVECs) and osteoblasts were stimulated with adiponectin (1 or 10 mug ml-1) or IL-1beta (0.1 ng ml-1) in the presence or absence of hypoxia for 24 h. The protein expression patterns were examined by analyzing culture supernatants using the enzyme-linked immunosorbent assay (ELISA). Adiponectin significantly stimulated the production of VEGF, MMP-1 and MMP-13 in osteoblasts but not in endothelial cells, whereas it significantly stimulated the production of IL-6 and IL-8 in both endothelial cells and osteoblasts. The increase in VEGF production induced by adiponectin was significantly greater than that induced by IL-1beta. The production of IL-6 and IL-8 in adiponectin-stimulated endothelial cells was approximately 10-fold higher than that in IL-1beta-stimulated endothelial cells; in osteoblasts, adiponectin-induced IL-6 and IL-8 secretion was approximately twofold higher than that induced by IL-1beta. In addition, IL-8 production in endothelial cells was approximately sevenfold higher than in osteoblasts. However, IL-6 levels were similar between the two cell types, suggesting that adiponectin may be involved in the production of IL-8 in endothelial cells, which may have an important role in neutrophil recruitment to arthritic joints. Furthermore, the increases in protein expression induced by adiponectin were differentially regulated by hypoxia. In conclusion, adiponectin has a more important role than does IL-1beta in the production of mediators that drive synovitis and joint destruction in endothelial cells and osteoblasts at physiological concentrations.
Adiponectin/pharmacology/*physiology ; Arthritis, Rheumatoid/metabolism ; Cell Hypoxia ; Cell Line ; Human Umbilical Vein Endothelial Cells/drug effects/*metabolism ; Humans ; Interleukin-6/genetics/*metabolism ; Interleukin-8/genetics/*metabolism ; Matrix Metalloproteinase 1/genetics/*metabolism ; Osteoblasts/drug effects/*metabolism ; Vascular Endothelial Growth Factor A/genetics/*metabolism

OBJECTIVETo study influence of serum containing Liuwei Dihuang decoction (see text) on proliferation and differentiation of osteoblast form neonatal SD rats cultured in vitro at different times and different stretch stress.

METHODSAfter osteoblast cultured for 24 hours in the serum containing Liuwei Dihuang decoction (see text) and serum in control group, the 0.5 Hz frequency, 6% and 12% stretch-stress were added. The MTT1 and the activity of ALP were measured at the 12th and 24th hours, and the data were analyzed.

RESULTS1. In the environment of stretch stress to the frequency of 0.5 Hz, and stretched for 24 hours, the osteoblast was stimulated under elongation rate of 6% and 12%; the proliferation and differentiation of osteoblast was more active under elongation rate of 12% than that of 6%. 2. There were no stimulating effects on osteoblast proliferation and differentiation of serum containing Liuwei Dihuiang decoction (see text) acted on osteoblast cells of SD rats cultured in vitro for a shot time.

CONCLUSIONStretch stress environment can enhance osteoblast proliferation and differentiation cultured in vitro.

Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Male ; Osteoblasts ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley ; Serum ; Stress, Mechanical

OBJECTIVEInjection of insulin-like growth factor-1 (IGF-1) can prevent bone loss in sciatic nerve transaction rats. We try to investigate the action mechanism of IGF-1 on bone formation.

METHODSA total of 40 adult male Spragne-Dawley rats were divided into two groups (experimental group and control group) with 20 animals in each. Sciatic neurectomy was performed to model disuse osteoporosis in all rats. IGF-1 was administered in experimental group with the dose of 100 microgramme/kilogram per day for 3 days. Meanwhile, the rats in control group were treated with saline. Bone mineral density was measured by dual-energy X-ray absorptiometry 4 and 6 weeks after neurectomy respectively. Expression of Osterix and Runx2 was determined by reverse transcription-polymerase chain reaction (RT-PCR) assay.

RESULTSThere was a significant increase in the bone mineral density of experimental group compared with control group. There was a significant decrease in the level of receptor activator of nuclear factor-kappaB-ligand but an increase in the level of osteoprotegerin 4 and 6 weeks after neurectomy in the experimental group compared with control one. The expression of Osterix and Runx2 was up-regulated in the bone marrow of experimental group compared with control group.

CONCLUSIONIGF-1 can increase bone formation by stimulation of osteoblast number and activity, and reduce bone resorption by restriction of differentiation of osteoclast, suggesting that IGF-1 may improve the therapeutic efficacy for disuse osteoporosis.

Animals ; Bone Density ; drug effects ; Bone Resorption ; prevention & control ; Cell Differentiation ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Immunohistochemistry ; Injections ; Insulin-Like Growth Factor I ; administration & dosage ; Male ; Osteoblasts ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; surgery ; Transcription Factors ; metabolism ; Up-Regulation ; physiology

BACKGROUNDEstrogen deficiency results in loss of bone mass. Phytoestrogens are plant-derived non-steroidal compounds with estrogen-like activity that bind to estrogen receptors. The main aim of this study was to investigate the effect of the phytoestrogen puerarin on adult mouse osteoblasts.

METHODSOsteoblast cells were harvested from 8-month old female imprinting control region (ICR) mice. The effects of puerarin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The production of nitric oxide (NO) and the expression of bone morphogenetic protein-2 (BMP-2), SMAD4, mitogen-activated protein kinases (MAPK), core binding factor α1/ runt-related transcription factor 2 (Cbfa1/Runx2), osteoprotegerin (OPG), and receptor activator of NF-κB ligand (RANKL) genes were analyzed. The activation of signal pathways was further confirmed by specific pathway inhibitors.

RESULTSThe osteoblast viability reached its maximum at 10(-8) mol/L puerarin. At this concentration, puerarin increases the proliferation and matrix mineralization of osteoblasts and promotes NO synthesis. With 10(-8) mol/L puerarin treatment, BMP-2, SMAD4, Cbfa1/Runx2, and OPG gene expression were up-regulated, while the RANKL gene expression is down-regulated. Concurrent treatment involving the (bone morphogenetic protein) BMP antagonist Noggin or the NOS inhibitor L-NAME diminishes puerarin induced cell proliferation, Alkaline phosphatase (ALP) activity, NO production, as well as the BMP-2, SMAD4, Cbfa1/Runx2, OPG, and RANKL gene expression.

CONCLUSIONSIn this in vitro study, we demonstrate that puerarin is a bone anabolic agent that exerts its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expression. This effect may contribute to its induction of osteoblast proliferation and differentiation, resulting in bone formation.

Animals ; Bone Morphogenetic Protein 2 ; genetics ; physiology ; Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Female ; Isoflavones ; pharmacology ; MAP Kinase Signaling System ; physiology ; Mice ; Mice, Inbred ICR ; Nitric Oxide ; physiology ; Osteoblasts ; drug effects ; metabolism ; Osteogenesis ; drug effects ; Phytoestrogens ; pharmacology ; RANK Ligand ; genetics