Mechanism of 1α,25-dihydroxyvitamin D(3)-dependent repression of interleukin-12B.

Interleukin 12 (IL-12) is a heterodimeric, pro-inflammatory cytokine that plays a central role in activation and differentiation of CD4(+) T cells into interferon-γ secreting T-helper type 1 cells. IL-12B, a gene encoding the larger subunit of active IL-12, has been reported to be down-regulated by the nuclear hormone 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)), but the mechanism of the regulation is unknown. In this study, we have examined the molecular mechanism of transcriptional regulation of the IL-12B gene by 1α,25(OH)(2)D(3) in lipopolysaccharide (LPS)-treated human monocytes (THP-1). Quantitative RT-PCR showed that IL-12B mRNA displays a cyclical expression profile and is down-regulated 2.8-fold during the first 8h and even 12.1-fold 24h after exposure to 1α,25(OH)(2)D(3). Gel shift and quantitative chromatin immunoprecipitation (ChIP) assays demonstrated vitamin D receptor (VDR) binding to genomic regions 480 and 6300bp upstream of the IL-12B transcription start site (TSS). Quantitative ChIP assays also revealed that together with VDR and its partner RXR the above regions recruited the co-repressor NCOR2/SMRT and histone deacetylase 3 leading to a decreased histone 4 acetylation and increased histone 3 trimethylation at the IL-12B promoter and its TSS. We suggest that these repressive epigenetic changes eventually cause down-regulation of IL-12 expression. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Primary effect of 1α,25(OH)2D3 on IL-10 expression in monocytes is short-term down-regulation.

The biologically most active vitamin D compound, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), influences the status of inflammation by modulating the expression of several cytokine genes. In this study, we have examined the mechanism of transcriptional regulation of interleukin 10 (IL-10) by 1α,25(OH)2D3 in lipopolysaccharide (LPS)-treated human monocytes (THP-1). Quantitative PCR showed that IL-10 mRNA expression was significantly down-regulated (2.8-fold) during the first 8h of 1α,25(OH)2D3 treatment, while after 48 h it was up-regulated (3-fold). Gel shift and quantitative chromatin immunoprecipitation (ChIP) assays showed that the vitamin D receptor (VDR) binds in a cyclical fashion to a promoter region 1500-1700 bp upstream of the IL-10 transcription start site (TSS) containing two conserved VDR binding sites. Targeting of VDR binding sites by enhancer specific duplex RNAs revealed that only the more distal element is functional and chromosome conformation capture analysis suggested that this region loops 1α,25(OH)2D3-dependently to the TSS. Quantitative ChIP and micrococcal nuclease assays also revealed 1α,25(OH)2D3-dependent cyclical epigenetic changes and nucleosome remodeling at this promoter region. In conclusion, in LPS-treated THP-1 cells the primary effect of 1α,25(OH)2D3 on IL-10 expression is down-regulation, which is achieved via a cyclical recruitment of VDR to the promoter.

The number of vitamin D receptor binding sites defines the different vitamin D responsiveness of the CYP24 gene in malignant and normal mammary cells.

Primary 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3))-responding genes are controlled by the vitamin D receptor (VDR) binding to specific sites (VDREs) that are located within the regulatory regions of these genes. According to previous studies, the gene encoding 25-dihydroxyvitamin D(3) 24-hydroxylase, CYP24, which is the strongest known 1alpha,25(OH)(2)D(3)-responsive gene, has multiple VDREs that locate within the proximal and the distal promoter. However, it has remained unclear, what is the biological role of these regions and how they participate in the regulation of transcription. In this study, we found a different CYP24 expression profile in normal (MCF-10A) and malignant (MCF-7) human mammary cells. Moreover, CYP24 mRNA showed to be three times more stable in MCF-7 cells than in MCF-10A cells. We studied the mechanism of this difference using expression profiling, quantitative chromatin immunoprecipitation and chromosome conformation capture assays. Interestingly, the number of functional VDREs was higher in MCF-7 cells than in MCF-10A cells. Three functional VDREs in MCF-7 cells are connected to linear mRNA accumulation, whereas only one VDRE seems to lead to stepwise CYP24 mRNA accumulation in MCF-10A cells. The distal VDREs were involved in transcriptional regulation via ligand-dependent, dynamic chromatin looping, which brings cyclically the distal elements together either individually or simultaneously next to the transcription start site. In conclusion, our data suggest that in comparison to normal cells, clearing of 1alpha,25(OH)(2)D(3) is enhanced in malignant cells due to differences in transcriptional regulation of CYP24 and metabolism of CYP24 mRNA.

Synthesis and biological evaluation of phenolic 4,5-dihydroisoxazoles and 3-hydroxy ketones as estrogen receptor alpha and beta agonists.

In this work, 52 diphenyl-4,5-dihydroisoxazoles and -3-hydroxy ketones were prepared and their estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) activities were explored in order to systematize and maximize their biological activity. The biological activity was firstly screened by using ERE reporter assay to find out how aromatic hydroxylation and methylation of the chiral centers of the compounds affect the ability of ER to mediate biological responses. For selected 19 compounds, the relative binding affinities (RBA, relative to 3,17beta-estradiol) and ability to induce transcription of primary E2 target gene pS2 in human MCF-7 breast cancer cells were determined. In the reporter assay, many compounds showed even stronger activity than E2 and some of them showed RBA larger than 1%. The highest RBAs were determined for the enantiomers of 1-hydroxy-6-(4-hydroxy-phenyl)-1-phenyl-hexan-3-one (50a and 50b). Isomer 50a showed high binding affinity both to ERalpha (with RBA approximately 200%) and ERbeta (with RBA approximately 60%), while the RBAs of 50b were ca. 40% of those. Some of the other compounds (with RBA approximately 1-16%) showed also notable ERalpha binding selectivity. When four most promising ligands (50a, 50b, 45a, and 45b) were studied with respect to their ability to induce the transcription of primary E2 target gene pS2, the compounds acted as agonists or partial agonists. Computer modeling was used to predict receptor binding conformations and to rationalize the RBA differences of the compounds.

The genes encoding cytokines IL-2, IL-10 and IL-12B are primary 1alpha,25(OH)2D3 target genes.

A number of studies have described the effects of 1alpha,25(OH)2D3 in immune system. Most of the known effects of 1alpha,25(OH)2D3 are indirect since only two functional VDREs that regulate transcription of cytokine gene has been reported until today. In this study we have examined a possibility of direct transcriptional regulation of IL-2, IL-10 and IL-12B genes in activated Jurkat or THP-1 cells via liganded VDR by using gene expression analysis and chromatin immunoprecipitation assays. According to our data the IL-2, IL-10 and IL-12B genes respond to 1alpha,25(OH)2D3 treatment by 3-6 h. In addition, all of these genes contain several genomic regions that recruit VDR in a ligand dependent fashion. These data suggest that the above cytokines are under direct transcriptional regulation by 1alpha,25(OH)2D3.