Combined effects of cyclic stretch and TNF-α on the osteogenic differentiation in MC3T3-E1 cells

Abstract

Objective: The study aimed to investigate the combined effects of cyclic stretch and tumor necrosis factor-alpha (TNF-α) on the osteogenic differentiation of MC3T3-E1 cells and the role of the nuclear factor-kappaB (NF-κB) signaling pathway in this process.

Design: MC3T3-E1 cells were treated with TNF-α (0.5 and 10 ng/mL) and cyclically stretched using the Flexcell tension system 4000 with 12 % elongation for 12 h. Furthermore, to explore which cytokines might be regulated by the NF-κB signaling pathway in osteogenic differentiation, the cells were pre-treated with NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), and then cyclically stretched for 12 h in the presence of 10 ng/mL of TNF-α. RT-PCR and western blot were utilized to detect the expression of type Ⅰ collagen (COL1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), NF-κB, and phosphorylated NF-κB (p-NF-κB) at gene and protein levels.

Results: Cyclic stretch alone increased the expression of COL1, OCN, Runx2, ALP, and OPG, decreasing the expression of RANKL and the RANKL/OPG ratio. The upregulation or downregulation induced by cyclic stretch were restrained in the presence of TNF-α. The p-NF-κB/NF-κB ratio increased at any stimulation. Inhibition of NF-κB signaling pathway restrained the expression variations of COL1, OCN, ALP, OPG, and RANKL induced by TNF-α combined with cyclic stretch.

Conclusion: The results indicated that TNF-α inhibited the osteogenic differentiation of MC3T3-E1 cells induced by cyclic stretch and NF-κB signaling pathway might play a role in this process.

Keywords: Cyclic stretch; NF-κB; Osteoblast; Osteogenic differentiation; TNF-α.