Figure 4.

The COX inhibitor indomethacin blocks TNFα-induced binding of NFATc1 and cAMP response element-binding protein (CREB) to the RANKL promoter in C2C12 cells. (A) PGE₂ increased the expression of RANKL, NFATc1, CREB, and COX2, whereas indomethacin suppressed TNFα-mediated RANKL, NFATc1, CREB, and COX2 expression. Treatment with PGE₂ in the presence of indomethacin and TNFα partially rescued the expression of RANKL, NFATc1, CREB, and COX2. C2C12 cells were incubated with the indicated reagents for 24 h and subjected to RT-PCR and western blot analyses; (B) PGE₂ increased RANKL promoter–reporter activity in a CREB binding element-dependent manner. C2C12 cells were transfected with RANKL-WT-luc or a CREB-binding site mutant (RANKL-MT(C)-luc) RANKL promoter, incubated for 24 h in the presence of PGE₂, and subjected to a luciferase assay; (C) PGE₂ induced CREB binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with PGE₂ and subjected to a ChIP assay with CREB and control IgG antibodies. The RANKL promoter region containing the CREB-binding element was then amplified; (D) Indomethacin prevented TNFα-induced NFAT and CREB binding to the mouse RANKL promoter. C2C12 cells were incubated for 24 h with the indicated reagents and subjected to a ChIP assay (* p < 0.05, compared to control; # p < 0.05).