IJMS, Vol. 18, Pages 495: TNF? Increases RANKL Expression via PGE2-Induced Activation of NFATc1 (International Journal of Molecular Sciences)

Tumor necrosis factor ? (TNF?) is known to upregulate the expression of receptor activator of NF-?B ligand (RANKL). We investigated the role of the calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway in TNF?-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. TNF?-induced RANKL expression was blocked by the calcineurin/NFAT pathway inhibitors. TNF? increased NFAT transcriptional activity and subsequent RANKL promoter binding. Mutations in the NFAT-binding element (MT(N)) suppressed TNF?-induced RANKL promoter activity. TNF? 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. TNF? 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 TNF? 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 TNF?-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 TNF?-induced RANKL expression depends on PGE2 production and subsequent transcriptional activation/enhanced binding of NFATc1 and CREB to the RANKL promoter.