Isoproterenol induces RANKL expression by activating NFAT and ATF4 in mouse osteoblastic cells.

Abstract

Isoproterenol (ISO), a nonspecific beta-adrenergic receptor agonist is known to induce bone loss and increase of osteoclast action. Although ISO has been shown to increase receptor activator of NF-kappaB ligand (RANKL), the details of the regulatory mechanisms remain unclear. In the present study, we investigated the role of calcineurin/ NFAT and cAMP/PKA pathways in ISO-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. ISO increased expression levels of RANKL mRNA and protein and RANKL promoter reporter activity. ISO also promoted the phosphorylation of ATF4 and CREB and the transcriptional activity of NFAT. ISOmediated RANKL expression was downregulated by the inhibitors of calcineurin and PKA. Mutations in CRE-like or NFAT-binding element suppressed ISO-induced RANKL promoter activity. ChIP analysis demonstrated that ISO increased the NFAT and ATF4-binding activity on the mouse RANKL promoter but not CREBbinding activity. Association of NFATc1 and ATF4 was not observed in co-immunoprecipitation study. Knockdown of NFATc1 or ATF4 blocked ISO-mediated RANKL expression. ATF4 knockdown also prevented ISO induction of NFATc1 expression. These results suggest that ISO-induced RANKL expression depends on the activation of NFAT and ATF4.