Calcineurin/nuclear factor of activated T-cells (NFAT) activation is involved in tumor necrosis factor α (TNFα)-induced receptor activator of nuclear factor-κB ligand ( RANKL ) expression in C2C12 cells. ( A ) TNFα increased RANKL expression in a time-dependent manner. C2C12 cells were incubated in the presence of 10 ng/mL TNFα for the indicated time periods and subjected to quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses. Quantitative data are presented as means ± standard deviations (SD); ( B ) TNFα induces NFAT transcriptional activity. C2C12 cells were transfected with a reporter plasmid containing an NFAT response element, exposed to TNFα for 24 h, and subjected to a luciferase assay. Data are presented as firefly luciferase activity levels relative to Renilla activity; ( C ) Inhibition of the calcineurin/NFAT pathway blocked TNFα-mediated RANKL expression. C2C12 cells pretreated with FK506 (10 μg/mL) or cyclosporin A (10 μg/mL) were treated with TNFα for 24 h and subjected to RT-PCR and western blot analyses; ( D ) TNFα increases NFAT binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with TNFα, after which a chromatin immunoprecipitation (ChIP) assay was performed using an antibody against NFATc1, with IgG serving as a negative control. The RANKL promoter region containing the NFAT binding element was amplified via PCR. Quantitative ChIP data were normalized to the input and are presented as values relative to vehicle-treated control samples (CON) ( E ) TNFα increased RANKL promoter–reporter activity in an NFAT binding element-dependent manner. C2C12 cells were transfected with a wild-type ( RANKL -WT-luc) or NFAT-binding site mutant ( RANKL -MT(N)-luc) RANKL promoter reporter, incubated for 24 h in the presence of TNFα or NFAT overexpression vector, and subjected to a luciferase assay (* p