Low concentrations of TNF-α promote osteogenic differentiation via activation of the ephrinB2-EphB4 signalling pathway.

Abstract

Low concentrations of tumour necrosis factor-alpha (TNF-α) have been reported to promote osteogenic differentiation. In this study, a series of in vitro experiments was performed to investigate underlying molecular mechanisms involved. MC3T3-E1 murine preosteoblasts were treated with TNF-α at doses of 0, 0.1 or 1ng/mL. The ephrinB2-EphB4 signalling pathway was activated using ephrinB2-fc, or inhibited using lentiviruses encoding siRNAs specifically targeting EphB4. Cell proliferation/survival was evaluated using the Cell Counting Kit-8 (CCK-8) assay, and expression levels of Runx2, BSP, ephrinB2 and EphB4 were determined using RT-PCR and Western blotting. ALP activity in these cells was also determined, and mineral nodule formation was evaluated with alizarin red S staining. Low concentrations of TNF-α had no influence on cell proliferation/survival. However, expression levels of Runx2, BSP, ephrinB2 and EphB4, as well as ALP activity and mineral nodule formation, were significantly enhanced in MC3T3-E1 cells treated with low concentrations of TNF-α. Moreover, activation of the ephrinB2-EphB4 signalling pathway by ephrinB2-fc enhanced TNF-α-induced osteogenic differentiation, while down-regulation of EphB4 level reversed the positive effect of TNF-α. Low concentrations of TNF-α promoted osteogenic differentiation via activation of the ephrinB2-EphB4 signalling pathway.