Regulation of RANKL promoter activity is associated with histone remodeling in murine bone stromal cells

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)(2)D-3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)(2)D-3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodiurn butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 muM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)(2)D-3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)(2)D-3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)(2)D-3 stimulated RANKL, we added both TSA and 1,25(OH)(2)D-3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)(2)D-3-induced RANKL mRNA expression by 70% at all doses of 1,25(OH)(2)D-3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)(2)D-3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression. (C) 2004 Wiley-Liss, Inc.