Paradoxical transcriptional activation of rat liver cytochrome P-450 3A1 by dexamethasone and the antiglucocorticoid pregnenolone 16 alpha-carbonitrile: analysis by transient transfection into primary monolayer cultures of adult rat hepatocytes.

Abstract

The family 3A cytochromes P-450, among the most abundant members of this supergene family of microsomal hemoproteins expressed in animal and human liver, are inducible by glucocorticoids but also by such antiglucocorticoids as pregnenolone 16 alpha-carbonitrile (PCN). To investigate the mechanism for this nonclassical glucocorticoid effect, we analyzed the ability of 1.5 kilobases of DNA or of its successive subsegments isolated from the 5' flanking region of the rat CYP3A1 structural gene to modulate transcription of a reporter gene consisting of a viral promoter coupled to the chloramphenicol acetyltransferase (CAT) structural gene (expression vector pBLCAT2) and transiently expressed in a homologous cell system consisting of primary monolayer cultures of adult rat hepatocytes in which CYP3A1 mRNA and protein are inducible. The CAT activity measured after chimeric gene constructions were transferred into the cultured rat hepatocytes by lipofection increased as much as 7.2-fold if the cells were treated with dexamethasone (DEX). One CYP3A1 fragment (positions -220 to -56; 164 base pairs), which does not contain a traditional glucocorticoid responsive element, conferred dose-dependent DEX responsiveness independent of its orientation but not its position in pBLCAT2. This construction was activated by addition of PCN to the cultures and was synergistically induced by PCN plus DEX. In contrast, induction of CAT activity in cultures containing MMTVCAT, a plasmid containing the CAT gene controlled by the mouse mammary tumor virus long terminal repeat, was unaffected by PCN treatment, required lower concentrations of DEX for a maximal response, and was inhibited by treatment with DEX plus PCN. We conclude that a primary mechanism for induction of CYP3A1 is stimulated transcription through a pathway activated by steroid hormones.