Calcineurin/NFAT signaling pathway mediates titanium particle-induced inflammation and osteoclast formation by inhibiting RANKL and M-CSF in vitro

Abstract

Wear particles serve a central role in periprosthetic osteolysis, which leads to the aseptic loosening of prostheses. In the present study a lentiviral vector was constructed to silence macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor k-B ligand (RANKL) genes, which synergistically inhibit osteoclast formation and differentiation. To confirm the role of the calcineurin/nuclear factor of activated T cells (NFAT) pathway in osteolysis, we transduced murine macrophage/monocyte RAW264.7 cells with M-CSF-short hairpin (sh)RNA-RANKL-shRNA. Tumor necrosis factor-α (TNF-α) protein levels were evaluated using enzyme-linked immunosorbent assay. Transduced RAW264.7 cells were cultured in Transwell chambers in the presence of 0.1 mg/ml titanium particles to investigate the capacity of TNF-α inhibition to reduce wear debris-induced inflammation. RANKL, M-CSF, TNF-α, interleukin (IL)-1β, IL-6 and NFATc1 mRNA levels were also assessed by reverse transcription-quantitative polymerase chain reaction. Osteoclastogenesis was measured by tartrate-resistant acid phosphatase (TRAP) mRNA quantification. Lentiviral-mediated double gene inhibition is known to be able to completely inhibit inflammatory osteolysis, simultaneously decreasing the number of NFATc1- and TRAP-positive cells. The present study confirmed that the combined silencing of M-CSF and RANKL genes can inhibit the osteolysis induced by the wear particles around the prosthesis. The calcineurin/NFAT pathway serves a role in the prevention of prosthesis loosening.