GLUCOCORTICOID RECEPTOR-INDEPENDENT TRANSCRIPTIONAL INDUCTION OF CYTOCHROME-P450 3A1 BY METYRAPONE AND ITS POTENTIATION BY GLUCOCORTICOID

Metyrapone administration to 21- and 90-day-old male rats causes a tra nscriptional induction of the hepatic glucocorticoid-inducible CYP3A1 gene within an hour as determined by nuclear run-on experiments. Analy ses performed 24 hr after metyrapone administration in both ages of ra t demonstrate that the transcriptional induction of CYP3A1 gene expres sion is followed by significant increases in CYP3A1 mRNA, GYP3A-immuno reactive microsomal protein and total microsomal cytochrome P450 (CYP) . In 21-day-old rats, there is a significant increase in microsomal CY P3A-dependent steroid GP-hydroxylase activity but not in 90-day-old ra ts, possibly because of a slower clearance of this drug, which inhibit s CYP activities. In hepatocytes cultured in serum- and glucocorticoid hormone-free medium, metyrapone alone induces GYP3A1 mRNA expression, which demonstrates that metyrapone transcriptionally induces hepatic CYP3A1 by a direct interaction with the liver. Metyrapone does not com pete with the binding of the synthetic glucocorticoid and potent trans criptional GYPSA1 inducer dexamethasone to the glucocorticoid receptor (GR) in soluble fractions from liver. This suggests that metyrapone i s not a ligand for the GR and induces GYP3A1 by a mechanism independen t of the GR. Addition of glucocorticoid to cultured hepatocytes at lev ers that induce GR-dependent genes potentiate CYP3A1 mRNA induction by metyrapone without inducing CYP3A1 mRNA alone. A GR-dependent mechani sm may therefore mediate the potentiation of CYP3A1 transcriptional in duction by metyrapone. The CYP3A1 transcriptional inducer and glucocor ticoid antagonist pregnenolone 16 alpha-carbonitrile at 100 mu M block s dexamethasone binding to the GF: in 21-day-old rat liver soluble fra ctions but is less effective in 90-day-old rat liver soluble fractions in contrast with 10 mu M glucocorticoid antagonist RU486, which is eq ually effective at blocking dexamethasone binding to the GR. The inabi lity of pregnenolone 16 alpha-carbonitrile to fully compete with dexam ethasone for cytosolic binding in adult animals suggests that there ma y exist variant receptors with different affinities for dexamethasone and pregnenolone 16 alpha-carbonitrile and may explain the mechanism b y which low concentrations of dexamethasone potentiate the transcripti onal induction of CYP3A1 mediated by high concentrations of pregnenolo ne 16 alpha-carbonitrile [J. Biol. Chem. 270:28917-28923 (1995)]. Exam ination of membrane-bound dexamethasone binding activity, with which o ther steroidal and nonsteroidal CYP3A inducers have been shown to comp ete, indicates that binding activity is detectable in 90- but not 21-d ay-old rat liver microsomes. The absence of membrane-bound glucocortic oid binding site activity and the presence of a functional CYP3A1 tran scriptional response in 21-day-old rats suggest that membrane-bound gl ucocorticoid binding site activity is not involved in the transcriptio nal activation of CYP3A1 expression. These data suggest that both gluc ocorticoids and nonsteroidal compounds may trigger the transcriptional induction of CYP3A1 by a GR-independent mechanism that may be potenti ated by a GR-dependent mechanism.