IDENTIFICATION OF A FUNCTIONAL GLUCOCORTICOID RESPONSE ELEMENT IN THECYP3A1 IGC2 GENE/

The rat CYP3A subfamily of cytochrome P450 consists of steroid-and dru g-metabolizing enzymes inducible by pregnenolone 16 alpha-carbonitrile and by supra-physiological doses of dexamethasone. The induction of C YP3A 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 glucocor ticoids. A 219-bp dexamethasone responsive fragment of the CYP3A1/IGC2 gene localized at -2100/-1882 bp upstream of the transcription initia tion 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 s equences in the 5' flank of the CYP3A1/IGC2 gene. Point mutations in f ootprint I (-1982/-1960-bp) completely abolished binding and transcrip tion activation whereas a mutation in footprint II (-2001/-1986-bp) on ly 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 16 alpha-carb onitrile failed to induce any transcriptional effect mediated by this response element in the HepG2 cells.