title: Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor.
abstract: T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte-macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3 receptor (VDR). We therefore examined vitamin D3-mediated repression of activated IL-2 expression by cotransfecting Jurkat cells with IL-2 promoter/reporter constructs and a VDR overexpression vector and by DNA binding. We delineated an element conferring both DNA binding by the receptor in vitro and 1,25(OH)2D3-mediated repression in vivo to a short 40-bp region encompassing an important positive regulatory element, NF-AT-1, which is bound by a T-cell-specific transcription factor, NFATp, as well as by AP-1. VDR DNA-binding mutants were unable to either bind to this element in vitro or repress in vivo; the VDR DNA-binding domain alone, however, bound the element but also could not repress IL-2 expression. These results indicate that DNA binding by VDR is necessary but not sufficient to mediate IL-2 repression. By combining partially purified proteins in vitro, we observed the loss of the bound NFATp/AP-1-DNA complex upon inclusion of VDR or VDR-retinoid X receptor. Order of addition and off-rate experiments indicate that the VDR-retinoid X receptor heterodimer blocks NFATp/AP-1 complex formation and then stably associates with the NF-AT-1 element. This direct inhibition by a nuclear hormone receptor of transcriptional activators of the IL-2 gene may provide a mechanistic explanation of how vitamin derivatives can act as potent immunosuppressive agents.
authors.affiliation: Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
authors.forename: I
authors.initials: I
authors.lastname: Alroy
authors.forename: T L
authors.initials: TL
authors.lastname: Towers
authors.forename: L P
authors.initials: LP
authors.lastname: Freedman
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D047488:Retinoid X Receptors; D013601:T-Lymphocytes; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects*
chemical_list: D004268:DNA-Binding Proteins; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004247:DNA; D002117:Calcitriol