BACKGROUND Low concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated. 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-α. 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-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 μM), significantly reversed the positive effect of TNF-α 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-α on osteogenic differentiation.

中文翻译：

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EphB4 / TNFR2 / ERK / MAPK信号通路包含一个信号轴，以介导TNF-α对成骨细胞分化的积极作用。

背景技术据报道，低浓度的肿瘤坏死因子-α（TNF-α）及其受体TNFR2均可促进成骨细胞前体细胞的成骨分化。此外，低浓度的TNF-α会上调EphB4的表达。但是，尚未完全阐明TNF-α诱导成骨分化的分子机制以及TNFR2和EphB4的作用。结果当用0.5 ng / mlTNF-α刺激时，MC3T3-E1鼠成骨前体细胞中ALP活性以及RUNX2，BSP，EphB4和TNFR2的mRNA和蛋白水平显着提高。在通过慢病毒介导的shRNA干扰基因敲低或通过针对TNFR2的中和抗体抑制TNFR2之后，TNF-α的促成骨作用被部分逆转，而TNF-α对EphB4的上调保持不变。通过有效的EphB4自磷酸化抑制剂抑制的EphB4正向信号传导，NVP-BHG712，TNF-α增强的TNFR2，BSP和Runx2的表达显着降低。对信号通路的进一步研究表明，TNF-α显着增加了p-JNK，p-ERK和p-p38的水平。然而，在TNFR2-敲低细胞中仅p-ERK水平被显着抑制。此外，ERK途径抑制剂U0126（10μM）可以显着逆转TNF-α对RUNX2和BSP蛋白质水平的积极作用。结论EphB4，TNFR2和ERK / MAPK信号通路包含一个信号轴，以介导TNF-α对成骨细胞分化的积极作用。通过有效的EphB4自磷酸化抑制剂抑制的EphB4正向信号传导，NVP-BHG712，TNF-α增强的TNFR2，BSP和Runx2的表达显着降低。对信号通路的进一步研究表明，TNF-α显着增加了p-JNK，p-ERK和p-p38的水平。然而，在TNFR2-敲低细胞中仅p-ERK水平被显着抑制。此外，ERK途径抑制剂U0126（10μM）可以显着逆转TNF-α对RUNX2和BSP蛋白水平的积极作用。结论EphB4，TNFR2和ERK / MAPK信号通路包含一个信号轴，以介导TNF-α对成骨细胞分化的积极作用。通过有效的EphB4自磷酸化抑制剂抑制的EphB4正向信号传导，NVP-BHG712，TNF-α增强的TNFR2，BSP和Runx2的表达显着降低。对信号通路的进一步研究表明，TNF-α显着增加了p-JNK，p-ERK和p-p38的水平。然而，在TNFR2-敲低细胞中仅p-ERK水平被显着抑制。此外，ERK途径抑制剂U0126（10μM）可以显着逆转TNF-α对RUNX2和BSP蛋白质水平的积极作用。结论EphB4，TNFR2和ERK / MAPK信号通路包含一个信号轴，以介导TNF-α对成骨细胞分化的积极作用。对信号通路的进一步研究表明，TNF-α显着增加了p-JNK，p-ERK和p-p38的水平。然而，在TNFR2-敲低细胞中仅p-ERK水平被显着抑制。此外，ERK途径抑制剂U0126（10μM）可以显着逆转TNF-α对RUNX2和BSP蛋白质水平的积极作用。结论EphB4，TNFR2和ERK / MAPK信号通路包含一个信号轴，以介导TNF-α对成骨细胞分化的积极作用。对信号通路的进一步研究表明，TNF-α显着增加了p-JNK，p-ERK和p-p38的水平。然而，在TNFR2-敲低细胞中仅p-ERK水平被显着抑制。此外，ERK途径抑制剂U0126（10μM）可以显着逆转TNF-α对RUNX2和BSP蛋白质水平的积极作用。结论EphB4，TNFR2和ERK / MAPK信号通路包含一个信号轴，以介导TNF-α对成骨细胞分化的积极作用。