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.
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.
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