Nuclear receptor involvement in the regulation of rat cytochrome P450 3A23 expression

Abstract

Many genes of the cytochrome P450 3A (CYP3A) subfamily, including several human and rat isoforms, are inducible by glucocorticoids. In the rat CYP3A23 gene, a 110-base pair segment of the proximal 5'-flanking region mediates dexamethasone activation. Three binding sites (DexRE-1, DexRE-2, and Site A), identified by DNase I footprinting analysis, were characterized for their relative contribution to both basal activity and dexamethasone inducibility. Site-directed mutagenesis of DexRE-1 (-144 to -169) and DexRE-2 (-118 to -136) demonstrated that each contained a core imperfect AGGTCA direct repeat, which comprised a consensus nuclear receptor binding site, and was essential for dexamethasone responsiveness but was not required for basal activity. Competition gel shift and supershift analyses revealed that both sites can bind the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor. Site A (-85 to -110) was shown to be important for both basal activity and dexamethasone responsiveness. Point mutants displayed a reduced (2-3-fold) induction response, compared with 15-fold for wild-type, which was accompanied by a 40-60% drop in basal activity. Site A was shown to bind the liver-enriched nuclear receptor hepatocyte nuclear factor 4. Our studies demonstrate that the mechanism mediating glucocorticoid-inducible transcriptional activity of CYP3A23 involves multiple binding sites for members of the nuclear receptor superfamily.