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)2D3 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)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium 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 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. 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)2D3 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)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 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)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.     

Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12

We have investigated the ability of dexamethasone to regulate interleukin-1beta (IL-1beta)-induced gene expression, histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. Low concentrations of dexamethasone (10(-10) M) repress IL-1beta-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and fail to stimulate secretory leukocyte proteinase inhibitor expression. Dexamethasone (10(-7) M) and IL-1beta (1 ng/ml) both stimulated HAT activity but showed a different pattern of histone H4 acetylation. Dexamethasone targeted lysines K5 and K16, whereas IL-1beta targeted K8 and K12. Low concentrations of dexamethasone (10(-10) M), which do not transactivate, repressed IL-1beta-stimulated K8 and K12 acetylation. Using chromatin immunoprecipitation assays, we show that dexamethasone inhibits IL-1beta-enhanced acetylated K8-associated GM-CSF promoter enrichment in a concentration-dependent manner. Neither IL-1beta nor dexamethasone elicited any GM-CSF promoter association at acetylated K5 residues. Furthermore, we show that GR acts both as a direct inhibitor of CREB binding protein (CBP)-associated HAT activity and also by recruiting HDAC2 to the p65-CBP HAT complex. This action does not involve de novo synthesis of HDAC protein or altered expression of CBP or p300/CBP-associated factor. This mechanism for glucocorticoid repression is novel and establishes that inhibition of histone acetylation is an additional level of control of inflammatory gene expression. This further suggests that pharmacological manipulation of of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.