Tumor necrosis factor-α inhibits osteoblast differentiation by interfering with the bone morphogentic protein-2 signaling pathway

Objective To investigate the influence of tumor necrosis factor-α(TNF-α) activation on osteoblast differentiation through interfering with the bone morphogenetic protein-2(BMP-2) signaling pathway. Methods Mouse C2C12 myoblast cells stimulated with BMP-2 were used as the in vitro osteoblast differentiation model. The stimulated cells were cultured with growth factors TNF-α and BMP-2 of different concentrations. We had one blank control group and 3 experimental groups. In the BMP-2 group, 100 ng/mL of BMP-2 was added. In the TNF-α group, 5 ng/mL of TNF-α was added. In the BMP-2 + TNF-α group, 100 ng/mL of BMP-2 and 5 ng/mL of TNF-α were added. After culture for 7 days, ALP staining was used to determine the level of osteoblast differentiation. The P-Nitrophenyl phosphate (PNPP) method was used to measure the activity of alkaline phosphatase (ALP). Results The ALP activity was 0. 260 ± 0. 245 in the BMP-2 group, 7. 311 ±0. 772 in the TNF-α group, and 1. 344 ±0. 133 in the BMP-2 +TNF-α group. The differences between any 2 groups were significant ( P ＜ 0. 05). At a constant concentration of 100 ng/mL of BMP-2, the ALP activities were 10. 207 ±0. 459, 6. 183 ±0. 374, 1. 873 ±0. 388, 0. 096 ±0. 023 respectively when TNF-α of 0, 2, 5, 10 ng/mL were added. The differences between any 2 groups were significant ( P ＜ 0. 05) . ALP activities in C2C12 cells were significantly decreased by TNF-α treatment, but significantly increased by BMP-2. Conclusions TNF-α may inhibit osteoblast differentiation induced by BMP-2 in a dose-dependent manner. However, the inhibition of osteoblast differentiation by TNF-α can be counter balanced if the concentration of BMP-2 treatment is high enough. Key words: Osteoblasts;  Bone morphogentic protein;  Tumor necrosis factor-α;  Cell differentiation