High extracellular calcium-induced NFATc3 regulates the expression of receptor activator of NF-κB ligand in osteoblasts.

Nuclear factor of activated T cell (NFAT) is a key transcription factor for receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. However, it is unclear whether NFAT plays a role in the expression of RANKL in osteoblasts. High extracellular calcium ([Ca(2+)](o)) increases intracellular calcium, enhances RANKL expression in osteoblasts/stromal cells, and induces osteoclastogenesis in a coculture of osteoblasts and hematopoietic bone marrow cells. Because intracellular calcium signaling activates the calcineurin/NFAT pathway, we examined the role of NFAT activation on high [Ca(2+)](o)-induced RANKL expression in MC3T3-E1 subclone 4 (MC4) cells. Among the family of NFAT transcription factors, expression of NFATc1 and NFATc3, but not NFATc2, NFATc4 or NFAT5, was observed in MC4 cells. High [Ca(2+)](o) increased the expression levels of NFATc1, NFATc3 and RANKL. Cyclosporin A and FK506, inhibitors of calcineurin phosphatase, blocked high [Ca(2+)](o)-induced expression of NFAT and RANKL. Knockdown of NFATc1 and NFATc3 by siRNA prevented high [Ca(2+)](o)-induced RANKL expression, whereas overexpression of NFATc1 and NFATc3 induced RANKL expression. Furthermore, overexpressed NFATc1 upregulated NFATc3 expression, but NFATc1 knockdown decreased NFATc3 expression. Chromatin immunoprecipitation and reporter assay results showed that NFATc3, but not NFATc1, directly binds to the RANKL promoter and stimulates RANKL expression. In summary, these results demonstrate that high [Ca(2+)](o) increases expression of RANKL via activation of the calcineurin/NFAT pathway in osteoblasts. In addition, high [Ca(2+)](o) induces the activation and expression of NFATc1; NFATc3 expression and activity are subsequently increased; and NFATc3 directly binds to the RANKL promoter to increase its expression.