Low concentrations of TNF-αpromote osteogenic differentiation via activation of the ephrinB2-EphB4 signalling pathway

Objectives: Low concentrations of tumour necrosis factor-alpha (TNF-alpha) have been reported to promote osteogenic differentiation. In this study, a series of in vitro experiments was performed to investigate underlying molecular mechanisms involved. Materials and methods: MC3T3-E1 murine preosteoblasts were treated with TNF-alpha at doses of 0, 0.1 or 1 ng/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. Results: Low concentrations of TNF-alpha 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-alpha Moreover, activation of the ephrinB2-EphB4 signalling pathway by ephrinB2-Fc enhanced TNF-alpha-induced osteogenic differentiation, while down-regulation of EphB4 level reversed the positive effect of TNF-alpha. Conclusions: Low concentrations of TNF-alpha promoted osteogenic differentiation via activation of the ephrinB2-EphB4 signalling pathway.