Orphan nuclear receptor binding site in the human inducible nitric oxide synthase promoter mediates responsiveness to steroid and xenobiotic ligands

Abstract

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are members of the nuclear receptor superfamily that regulate target gene transcription in a ligand-dependent manner. CAR and PXR have a rather broad, overlapping set of ligands that range from natural steroids to xenobiotics and also recognize similar DNA binding sites, referred to as response elements (REs), primarily in promoter regions of cytochrome P450 (CYP) genes. In this study, a CAR and PXR RE, composed of a direct repeat of two GGTTCA motifs in a distance of 4 nucleotides (DR4), was identified in the promoter of the human inducible nitric oxide (NO) synthase (iNOS) gene, which is the first nuclear receptor binding site reported for this promoter. In a heterologous promoter context, the DR4-type sequence also acts as a functional RE for the nuclear receptors for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25OH2D3) and 3,5,3'-triiodothyronine (T3), VDR and T3R. However, in a direct competition of CAR, PXR, VDR, and T3R, the PXR-retinoid X receptor (RXR) complex appears to be the dominant regulator on the iNOS DR4-type RE. In the natural iNOS promoter context, the DR4-type RE specifically mediates downregulation of promoter activity by the testosterone metabolite androstanol through CAR-RXR heterodimers and upregulation by the xenobiotic drug clotrimazole through PXR-RXR heterodimers. These results were confirmed on the level of mRNA expression. Since an iNOS-induced production of NO is known to influence inflammation and apoptosis, a CAR- and PXR-regulated iNOS activity may explain a modulatory effect of steroids and xenobiotics on these cellular processes.