Figure 6.

NFATc1 Activity Is Essential for Osteoclast Formation but Only Contributory to NFATc1 Gene Activation

A, A peptide inhibitor of NFATc1 activity prevents osteoclast formation in RAW264.7. Cells were cultured in the presence of sRANKL (sRL) (300 ng/ml) with either vehicle, palmitic-linked NFATc1 inhibitor peptide (50 μm), or scrambled control peptide (50 μm). TRAP-positive multinucleated osteoclasts were quantitated after 5 d in culture. The data represent the average of triplicate determinations ± sem. **, P ≤ 0.05 compared with scrambled control (one-way ANOVA). B, NFATc1 inhibitor peptide partially blocks RANKL-induced NFATc1 mRNA and the up-regulation of the NFATc1 target mRNA TRAP in RAW264.7 cells. Cells were treated for the indicated times without or with sRL (300 ng/ml) and dimethylsulfoxide vehicle, scrambled peptide, or NFATc1-inhibitory peptide. RNA was collected at the times indicated and evaluated for the presence of either β-actin, NFATc1 (upper panel), or TRAP (lower panel). Real-time PCR was performed on reverse transcribed products and expressed as the ratio of NFATc1/β-actin or TRAP/β-actin. The data represent the average of triplicate determinations ± sem. *, P ≤ 0.05 compared with time-matched scrambled control; #, P ≤ 0.05 compared with the time-matched vehicle control (one-way ANOVA). These data are representative of two independent experiments. NT, No treatment; Scram, scrambled; Veh, vehicle.