TNFα Increases RANKL Expression via PGE₂-Induced Activation of NFATc1

Tumor necrosis factor α (TNFα) is known to upregulate the expression of receptor activator of NF-κB ligand (RANKL). We investigated the role of the calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway in TNFα-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. TNFα-induced RANKL expression was blocked by the calcineurin/NFAT pathway inhibitors. TNFα increased NFAT transcriptional activity and subsequent RANKL promoter binding. Mutations in the NFAT-binding element (MT(N)) suppressed TNFα-induced RANKL promoter activity. TNFα increased prostaglandin E2 (PGE₂) production, which in turn enhanced NFAT transcriptional activity and binding to the RANKL promoter. MT(N) suppressed PGE₂-induced RANKL promoter activity. TNFα and PGE₂ increased the expression of RANKL, NFAT cytoplasmic-1 (NFATc1), cAMP response element-binding protein (CREB), and cyclooxygenase 2 (COX2); which increment was suppressed by indomethacin, a COX inhibitor. Mutations in the CRE-like element blocked PGE₂-induced RANKL promoter activity. PGE₂ induced the binding of CREB to the RANKL promoter, whereas TNFα increased the binding of both CREB and NFATc1 to this promoter through a process blocked by indomethacin. The PGE₂ receptor antagonists AH6809 and AH23848 blocked TNFα-induced expression of RANKL, NFATc1, and CREB; transcriptional activity of NFAT; and binding of NFATc1 or CREB to the RANKL promoter. These results suggest that TNFα-induced RANKL expression depends on PGE₂ production and subsequent transcriptional activation/enhanced binding of NFATc1 and CREB to the RANKL promoter.