Tumor necrosis factor‐alpha inhibits osteogenic differentiation of pre‐osteoblasts by downregulation of EphB4 signaling via activated nuclear factor‐kappaB signaling pathway

Background and Objective The majority of experiments show that tumor necrosis factor‐alpha (TNF‐α) inhibits osteogenic differentiation of mesenchymal stem cells and pre‐osteoblasts by activated nuclear factor‐kappaB (NF‐κB) signaling. However, the underlying mechanisms by which NF‐κB signaling inhibits osteogenic differentiation are not fully understood. The aim of the present study was to investigate whether EphB4 signaling inhibition mediates the effects of TNF‐α‐activated NF‐κB signaling on osteogenic differentiation of pre‐osteoblasts. Material and Methods Murine MC3T3‐E1 pre‐osteoblasts were treated with 10 ng/mL of TNF‐α. NF‐κB inhibitor, pyrrolidine dithiocarbamate, was used to achieve NF‐κB signaling inhibition. EphB4 signaling was activated using ephrinB2‐fc. The mRNA expressions of runt related transcription factor 2 (Runx2), bone sialoprotein (BSP) and EphB4 were determined using reverse transcription‐polymerase chain reaction. The protein levels of Runx2, BSP, Col Ia1, osteopontin, EphB4, p‐NF‐κB p65 and NF‐κB p65 were evaluated using western blot assays. Alkaline phosphatase (ALP) activity in MC3T3‐E1 cells was evaluated by ALP activity kit, and mineral nodule formation was evaluated by Alizarin Red S staining. Results TNF‐α inhibited EphB4 expression, while it suppressed Runx2, BSP expression from gene and protein levels as well as ALP activity and mineral nodule formation in MC3T3‐E1 cells. Activation of EphB4 signaling by ephrinB2‐fc promoted osteogenic differentiation of MC3T3‐E1 cells, whereas TNF‐α impaired the osteogenic differentiation enhanced by ephrinB2‐fc. Pyrrolidine dithiocarbamate blocked the activation of NF‐κB signaling induced by TNF‐α, while it prevented the downregulation of Runx2, BSP and EphB4, induced by TNF‐α. Conclusion TNF‐α inhibits osteogenic differentiation of pre‐osteoblasts by downregulation of EphB4 signaling via activated NF‐κB signaling pathway.