| Background and ObjectivesBone is a metabolically dynamic and active tissue,undergoing constant renewal in response to hormonal,nutritional and mechanical influences.Osteoblasts derive from mesenchymal cells and play important roles in bone formation and remodeling.On one hand,as one basic component of bone tissue,osteoblasts secrete many osteogenic proteins,form extracellular matrix and regulate bone mineralization.On the other hand,osteoblasts control osteoclastic differentiation and activation via secreting many cytokines or directly contacting with osteoclasts.The disturbances of osteoblasts participate in the pathogenesis of many diseases characterized by abnormal bone remodeling.Therefore,identifying roles of pathogenesis in osteoblasts is essential for the prevention and treatment of these diseases.Periodontitis,which is characterized by alveolar bone resportion,is a chronic,bacterial infectious and inflammatory disease,and plaque biofilm is the major etiological factor.The invasion of periodontal pathogens leads to the expressions of many proinflammatory mediators and the activation of inflammatory cascades.TNF-a has been demonstrated to be one of critical proinflammatory cytokines in the pathogenesis of periodontitis.The inhibition of TNF-a on osteoclastic differentiation has been widely demonstrated.However,the roles of TNF-a in bone healing and regeneration are controversial.It has been demonstrated that TNF-a inhibits osteogenic differentiation of mesenchymal stem cells and osteoblasts in vitro,and an experiment also indicated that TNF-a inhibits bone formation in vivo.However,some researchers found that TNF-a promotes osteogenic differentiation of many cells,including bone mesenchymal stem cells(BMSCs),osteoblasts and dental pulp stem cells(DPSCs).TNF-a can activate several signaling pathways which interact with each other to achieve a balance in controlling osteogenic differentiation.In the pathogenesis of periodontitis,the duration and impact of inflammation and the levels of inflammatory mediators including TNF-a are diverse.In the progression of bone fracture healing,TNF-a plays an important role in bone regeneration and promotes bone healing.Therefore,we should identify the effects of TNF-a at different levels on osteogenic differentiation and the underlying mechanisms,which may provide a new insight for periodontitis treatment.NF-?B signaling has been demonstrated to be an important downstream pathway of TNF-?.NF-?B signaling activated by TNF-? can regulate transcription of many target genes.Inhibition of NF-?B signaling using antagonists could reverse the adverse effects of TNF-a on osteogenic differentiation of MC3T3-E1 cells.In ST2 cells,the activation of NF-?B signaling could inhibit osteogenic differentiation via inhibiting the expression of Runx2.Therefore,the activation of NF-?B signaling by TNF-a plays a central role in the inhibition of osteogenic differentiation induced by TNF-a.Recently,ephrinB2-EphB4 signaling has been demonstrated to be an important pathway in maintaining bone homeostasis.Activation of EphB4 by ephrinB2 existed on the surface of osteoclasts is referred to as forward signaling,which stimulates osteogenic differentiation.However,reverse signaling is the activation of ephrinB2 by EphB4 existed on the surface of osteoblasts,which inhibits the differentiation of osteoclast precursor cells.Therefore,ephrinB2-EphB4 signaling promotes bone formation,while inhibits excessive bone resorption.Multiple factors participate in the progression of inflammation,and long term of inflammatory response would influence the signaling pathways which control bone metastasis.Therefore,inflammatory microenvironment may regulate osteogenic differentiation via ephrinB2-EphB4 forward pathway.The present study investigated effects of TNF-a at various concentrations on osteogenic differentiation of MC3T3-E1 cells.The roles of ephrinB2-EphB4 forward signaling in osteogenic differentiation of MC3T3-E1 cells in inflammatory microenvironment.In order to identify the underlying mechanisms,the effects of NF-?B inhibition on osteogenic differentiation and the expression of EphB4 in MC3T3-E1 cells were also evaluated.Thus,we could know the effects of inflammation on osteogenic differentiation clearer and open up new avenues of periodontitis treatment.Materials and Methods1.Effects of TNF-a on osteogenic differentiation of MC3T3-E1 murine preosteoblastsMC3T3-E1 murine preosteoblasts were treated with TNF-a at doses of 0,0.1,1 or 10 ng/ml,respectively.The changes in the mRNA and protein expression levels of osteogenic parameters(Runx2 and BSP)were determined using RT-PCR and Western blot assays after treated with TNF-a for 24 or 48 h;The changes in ALP activity were evaluated by ALP activity kits after treated with TNF-a for 7 or 14 d;In vitro mineral nodule formation of MC3T3-E1 cells was monitored using alizarin red S staining after treated with TNF-a for 28 d,and the relative amount of calcium was evaluated using cetylpyridinium.2.Effects of ephrinB2-EphB4 forward signaling on TNF-a mediated osteogenic differentiationThe changes in the mRNA and protein levels of EphB4 were evaluated using RT-PCR and Western blot assays after treated with TNF-a at doses of 0,0.1,1 or 10 ng/ml for 24 or 48 h.The pLenti6.3-ephb4siRNA or pLenti6.3-ctrl lentiviral vector was constructed and transfected into human 293 T cells with ViraPower packing mix using Lipofectamine 2000.Forty-eight hours after transfection,the supernatant was collected.The ephrinB2-EphB4 forward signaling in MC3T3-E1 cells was inhibited by knocking-down the expression levels of EphB4 using pLenti6.3-ephb4siRNA encoding siRNA specifically targeting EphB4.The changes in mRNA and protein expression levels were determined using RT-PCR and Western blot assays.MC3T3-Elcells infected with EphB4 were treated with TNF-a at concentration of 1 ng/ml.The changes in the protein expression levels of osteogenic parameters(Runx2 and BSP)were determined using Western blot assays after treated with TNF-a for 48 h;The changes in ALP activity were evaluated by ALP activity kits after treated with TNF-a for 7 or 14 d;In vitro mineral nodule formation of MC3T3-E1 cells was monitored using alizarin red S staining after treated with TNF-a for 28 d,and the relative amount of calcium was evaluated using cetylpyridinium.EphrinB2-fc was added into the medium to stimulate the ephrinB2-EphB4 forward signaling.The protein expression levels of osteogenic parameters(Runx2 and BSP)were evaluated by Western blot assays after treated with ephrinB2-fc and/or TNF-a at concentrations of 10 ng/ml for 48 h.The changes in ALP activity were evaluated by ALP activity kits after treated with ephrinB2-fc and/or TNF-a at concentrations of 10 ng/ml for 7 d;In vitro mineral nodule formation of MC3T3-E1 cells was monitored using alizarin red S staining after treated with ephrinB2-fc and/or TNF-a at concentrations of 10 ng/ml for 28 d,and the relative amount of calcium was evaluated using cetylpyridinium.3.Effects of activated NF-?B signaling on the regulation of ephrinB2-EphB4 forward signalingThe protein expression levels of p-NF-?B p65 were evaluated using Western blot assays in MC3T3-E1 cells treated with TNF-a at concentration of 10 ng/ml in the presence/absence of PDTC for 0,30 or 60 min.The changes in protein expression levels of p-NF-?B,Runx2,BSP,as well as EphB4,were determined using Western blot after treated with TNF-a at concentration of 10 ng/ml in the presence/absence of PDTC for 24 or 48 h.Results1.Effects of TNF-a on osteogenic differentiation of MC3T3-E1 cellsAfter treated with TNF-a at concentrations of 0.1 or 1 ng/ml for 24 or 48 h,the mRNA and protein expression levels of osteogenic parameters(Runx2 and BSP)were significantly up-regulated when compared with the control group(0 ng/ml TNF-?),while the mRNA and protein expression levels of Runx2 and BSP were significantly down-regulated after treated with TNF-a at concentration of 10 ng/ml for 24 or 48 h when compared with the control group(0 ng/ml TNF-?).After treated with TNF-? at concentrations of 0.1 or 1 ng/ml for 7 or 14 d,ALP activity in MC3T3-E1 cells was significantly increased when compared with the control group(0 ng/ml TNF-?),while ALP activity was significantly inhibited after treated with TNF-? at concentration of 10 ng/ml for 7 or 14 d when compared with the control group(0 ng/ml TNF-a).Mineral nodule formation was significantly enhanced in MC3T3-E1 cells treated with TNF-a at concentrations of 0.1 or 1 ng/ml for 28 d.Treatment with TNF-a at concentrations of 10 ng/ml for 28 d significantly inhibited mineral nodule formation in MC3T3-E1 cells.The amount of calcium was increased after treated with TNF-a at concentrations of 0.1 or 1 ng/ml,while was decreased after treated with TNF-a at concentrations of 10 ng/ml.2.Effect of ephrinB2-EphB4 forward signaling on TNF-a mediated osteogenic differentiation(1)After treated with TNF-a at concentrations of 0.1 or 1 ng/ml for 24 or 48 h,the mRNA and protein expression levels of EphB4 were significantly up-regulated when compared with the control group(0 ng/ml TNF-a),while the mRNA and protein expression levels of EphB4 were significantly down-regulated after treated with TNF-a at concentration of 10 ng/ml for 24 or 48 h when compared with the control group(0 ng/ml TNF-a).(2)MC3T3-E1 cells infected with EphB4 knocked-down lentiviruses were named as pLenti6.3-ephb4siRNA MC3T3-E1 cells.MC3T3-E1 cells infected with the negative control lentiviruses served as controls(named as pLenti6.3-ctrl MC3T3-E1 cells).The mRNA and protein expression of EphB4 were significantly down-regulated in pLenti6.3-ephb4siRNA MC3T3-E1 cells when compared with pLenti6.3-ctrl MC3T3-El cells.(3)After treated with TNF-a at concentration of 1 ng/ml,the protein expression levels of Runx2 and BSP,the ALP activity and mineral nodule formation in pLenti6.3-ephb4siRNA MC3T3-E1 cells were significantly decreased when compared with pLenti6.3-ctrl MC3T3-E1 cells.(4)The protein levels of Runx2 and BSP,the ALP activity and mineral nodule formation were significantly down-regulated in MC3T3-E1 cells treated with 10 ng/ml of TNF-a.The levels of osteogenic parameters were higher in MC3T3-E1 cells treated with both of TNF-a and ephrinB2-fc when compared with the cells treated with IgG-fc and TNF-a.3.Effects of activated NF-?B signaling on EphB4 expression(1)The protein levels of p-NF-?B p65 were significantly up-regulated in MC3T3-E1 cells treated with TNF-a compared to those detected at 0 min,and the up-regulation of p-NF-?B p65 levels was significantly weaken in the presence of NF-?B inhibitor PDTC.(2)The protein levels of Runx2 and BSP were significantly down-regulated in MC3T3-El cells treated with TNF-a at concentration of 10 ng/ml for 24 or 48 h when compared with the control group.The pretreatment of PDTC prevents the TNF-a-mediated down-regulation of Runx2 and BSP in MC3T3-E1 cells.(3)The protein expression levels of EphB4 were significantly inhibited in MC3T3-E1 cells treated with TNF-a at concentration of 10 ng/ml for 24 or 48 h when compared with the control group,while the inhibitory effects of TNF-a on EphB4 expression were rescured by PDTC.Conclusions(1)Lower concentrations of TNF-? promote osteogenic differentiation and mineralization in MC3T3-El cells,while higher concentrations of TNF-a inhibit osteogenic differentiation and mineralization in MC3T3-E1 cells.(2)The inhibition of ephrinB2-EphB4 forward signaling reverses low concentrations of TNF-a-induced osteogenic differentiation in MC3T3-E1 cells.The stimulation of ephrinB2-EphB4 forward signaling rescures high concentrations of TNF-a-induced osteogenic differentiation in MC3T3-E1 cells.TNF-a regulates osteogenic differentiation via ephrinB2-EphB4 forward signaling.(3)The inhibition of NF-?B rescues the negative effects of high concentrations of TNF-a on osteogenic differentiation,as well as EphB4 expression.High concentration of TNF-a inhibits ephrinB2-EphB4 forward signaling by activated NF-?B signaling. |
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