TRANSCRIPTIONAL REPRESSION OF THE INTERLEUKIN-2 GENE BY VITAMIN-D-3 -DIRECT INHIBITION OF NFATP AP-1 COMPLEX-FORMATION BY A NUCLEAR HORMONE-RECEPTOR/

T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3], the active metabolite of vitamin D-3, and is associa ted with a decrease in interleukin 2 (IL-2), gamma interferon, and gra nulocyte-macrophage colony-stimulating factor mRNA levels. We report h ere that 1,25(OH)(2)D-3-mediated repression in Jurkat cells is cyclohe ximide resistant, suggesting that it is a direct transcriptional repre ssive effect on IL-2 expression by the vitamin D-3 receptor (VDR). We therefore examined vitamin D-3-mediated repression of activated IL-2 e xpression by cotransfecting Jurkat cells with IL 2 promoter/reporter c onstructs and a VDR overexpression vector and by DNA binding. We delin eated an element conferring both DNA binding by the receptor in vitro and 1,25(OH)(2)D-3 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 e lement 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 s ufficient to mediate IL-2 repression, By combining partially purified proteins in vitro, we observed the loss of the bound NFATp/AP-1-DNA co mplex upon inclusion of VDR or VDR-retinoid X receptor. Order of addit ion and off-rate experiments indicate that the VDR-retinoid X receptor heterodimer blocks NFATp/AP-1 complex formation and then stably assoc iates with the NF-AT-1 element. This direct inhibition by a nuclear ho rmone receptor of transcriptional activators of the IL-2 gene may prov ide a mechanistic explanation of how vitamin derivatives fan act as po tent immunosuppressive agents.