Identification of a Functional Glucocorticoid Response Element in theCYP3A1/IGC2Gene

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Abstract
The rat CYP3A subfamily of cytochrome P450 consists of steroid- and drug-metabolizing enzymes inducible by pregnenolone 16alpha-carbonitrile and by supra-physiological doses of dexamethasone. The induction of CYP3A by dexamethasone has been proposed to be mediated by a mechanism distinct from the glucocorticoid receptor mediated response. However, a synergistic induction of CYP3A has been observed with physiological doses of glucocorticoids and other CYP3A inducers. We have identified the presence of a glucocorticoid-responsive element in the CYP3A1/IGC2 gene that mediates the induction with physiological doses of glucocorticoids. A 219-bp dexamethasone responsive fragment of the CYP3A1/IGC2 gene localized at -2100/-1882 bp upstream of the transcription initiation site was identified in transfection experiments with HepG2 cells. Maximum induction was achieved with 50-100 nM dexamethasone. DNase I footprinting analysis revealed two glucocorticoid receptor-protected sequences in the 5' flank of the CYP3A1/IGC2 gene. Point mutations in footprint I (-1982/-1960-bp) completely abolished binding and transcription activation whereas a mutation in footprint II (-2001/-1986-bp) only decreased the binding and had no effect on transcription activation. These results led to the conclusion that the glucocorticoid response element present in footprint I mediated the dexamethasone response in transfection experiments with HepG2 cells. Pregnenolone 16alpha-carbonitrile failed to induce any transcriptional effect mediated by this response element in the HepG2 cells.