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.

No abstract

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 1␣,25-dihydroxyvitamin D 3 (1␣,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 1␣,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 1␣,25(OH) 2 D 3 is enhanced in malignant cells due to differences in transcriptional regulation of CYP24 and metabolism of CYP24 mRNA. This change enhances the interaction of VDR with its heterodimeric partner, the retinoid X receptor (RXR). The liganddependent conformational change also modulates interactions of NRs with a number of different nuclear proteins, such as co-activators (CoAs) and co-repressors (CoRs) (4). CoR proteins, such as NCoR1 (5) and SMRT/NCoR2 (6), link non-liganded, DNA-bound NRs to enzymes with histone deacetylase (HDAC) activity, which causes chromatin condensation. After a ligand-dependent conformational change, interactions with CoA proteins, such as SRC-1, are favored (7). These CoAs link the ligand-activated NRs to enzymes displaying histone acetyltransferase (HAT) activity, such as CBP, which leads to chromatin decondensation (8). In a second step, mediator proteins, such as Med1, build a bridge to the basal transcriptional machinery containing RNA polymerase II (pol II) and TATAbox binding protein (TBP).The liganded VDR-RXR heterodimer binds with high affinity and selectivity to vitamin D response elements (VDREs) in the regulative regions of VDR target genes (9). Traditionally, response elements (REs) are thought to be located relatively close to the transcription start site (TSS) of target genes. However, several studies have recently suggested that both positively and negatively regulated genes may have multiple REs * This work was supported by The Academ...

No abstract