Figure 3.

PGE₂ stimulates RANKL expression by activating the calcineurin/NFAT pathway in C2C12 cells. (A) PGE₂ increased RANKL expression in a time-dependent manner. C2C12 cells were incubated in the presence of PGE₂ (50 nM) for the indicated time periods, followed by quantitative RT-PCR and western blot analyses; (B) PGE₂ increased NFAT transcriptional activity. C2C12 cells were transfected with a reporter plasmid containing an NFAT response element, treated with PGE₂ for 24 h, and subjected to a luciferase assay; (C) PGE₂ induced NFAT binding to the mouse RANKL promoter. C2C12 cells were treated with PGE₂ for 24 h and a ChIP assay was performed; (D) PGE₂ increased RANKL promoter-reporter activity in an NFAT binding element-dependent manner. C2C12 cells were transfected with RANKL-WT-luc or RANKL-MT(N)-luc, incubated for 24 h in the presence of PGE₂, and subjected to a luciferase assay (* p < 0.05, compared to control; # p < 0.05).