EphB4/TNFR2/ERK/MAPK Signaling Pathway Comprises A Signaling Axis To Mediate The Positive Effect Of TNF-? On Osteogenic Differentiation Of MC3T3-E1 Cells

Background:Low concentrations of tumor necrosis factor-alpha(TNF-?)and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells.Furthermore,our previous study showed that EphB4 mediates the osteogenic effect of low concentrations of TNF-?.However,whether TNFR2 and EphB4 interact during TNF-?-induced osteogenic differentiation have not been reported.In this study,we analyzed the interaction between TNFR2 and EphB4 during TNF-a-induced osteogenic differentiation of MC3T3-E1 cells using signaling inhibition strategy to further clarify the mechanism of TNF-a-induced osteogenic differentiation.Materials and Methods:(1)Effect of low concentrations of TNF-? on TNFR2 and osteogenic genes expression in MC3T3-E1 cells.Cells were cultured in basal medium or osteogenic induction medium containing TNF-?(0.5 ng/ml),and the expressions of TNFR2,RUNX2,BSP mRNA and protein were detected by qRT-PCR,Western blot,and immunofluorescence.The ALP activity was evaluated using an Alkaline Phosphatase Assay Kit.(2)Crosstalk between TNFR2 and EphB4 signaling to mediate TNF-?-induced pro-osteogenic effect of MC3T3-E1 cells.Lentivirus was applied to establish TNFR2 knockdown cells,TNFR2 neutralizing antibody was applied to block TNFR2 signaling,ephrinB2-EphB4 positive signal was inhibited by EphB4 inhibitor(NVP-BHG712),and then cells were cultured in osteogenic induction medium containing TNF-?.qRT-PCR and western blot detected EphB4,TNFR2,RUNX2,BSP mRNA and protein expression levels.(3)TNF-a-activated MAPK signaling pathway related with EphB4/TNFR2 signaling pathways.MC3T3-E1 cells were treated with TNF-? for 0,5,15,30 and 60 min,and then MAPK pathway protein expression levels were detected by western blot.The control cells,TNFR2 knockdown cells and NVP-BHG712 pretreated cells were stimulated with TNF-? for 15 min.Western blot was performed to detect MAPK pathway protein expression levels.Cells were cultured in the basal medium and pretreated with JNK,p38 and ERK inhibitors for 60 min,respectively,and then the medium was replaced with osteogenic induction medium supplemented with TNF-? and the corresponding inhibitors.Western blot was performed to detect TNFR2,EphB4,RUNX2 and BSP protein expression levels.Results:The ALP activity,as well as the mRNA and protein levels of RUNX2,BSP,EphB4 and TNFR2,was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-?.After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2,the pro-osteogenic effect of TNF-? was partly reversed,while the up-regulation of EphB4 by TNF-? remained unchanged.With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation,NVP-BHG712,TNF-a-enhanced expressions of TNFR2,BSP and RUNX2 were significantly decreased.Further investigation into the signaling pathways revealed that TNF-? significantly increased levels of p-JNK1+2+3,p-ERK1/2 and p-p38.However,only the p-ERK1/2 level was significantly inhibited in TNFR2-knockdown cells.In addition,the ERK pathway inhibitor,U0126(10 ?M),significantly reversed the positive effect of TNF-? on the protein levels of RUNX2 and BSP.Conclusions:The EphB4,TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-? on osteogenic differentiation of MC3T3-E1 cells.