Transcriptional Regulation of CYP2C9 Gene: ROLE OF GLUCOCORTICOID RECEPTOR AND CONSTITUTIVE ANDROSTANE RECEPTOR

Sabine Gerbal-Chaloin,Martine Daujat,Jean-Marc Pascussi,Lydiane Pichard-Garcia,Marie-Jose Vilarem,Patrick Maurel

doi:10.1074/jbc.m107228200

Abstract

Although cytochrome P450 2C9 (CYP2C9) is a major CYP expressed in the adult human liver, its mechanism of regulation is poorly known. In previous work, we have shown that CYP2C9 is inducible in primary human hepatocytes by xenobiotics including dexamethasone, rifampicin, and phenobarbital. The aim of this work was to investigate the molecular mechanism(s) controlling the inducible expression of CYP2C9. Deletional analysis of CYP2C9 regulatory region (+21 to -2088) in the presence of various hormone nuclear receptors suggested the presence of two functional response elements, a glucocorticoid receptor-responsive element (-1648/-1684) and a constitutive androstane receptor-responsive element (CAR, -1783/-1856). Each of these were characterized by co-transfection experiments, directed mutagenesis, gel shift assays, and response to specific antagonists RU486 and androstanol. By these experiments we located a glucocorticoid-responsive element imperfect palindrome at -1662/-1676, and a DR4 motif at -1803/-1818 recognized and transactivated by human glucocorticoid receptor and by hCAR and pregnane X receptor, respectively. Identification of these functional elements provides rational mechanistic basis for CYP2C9 induction by dexamethasone (submicromolar concentrations), and by phenobarbital and rifampicin, respectively. CYP2C9 appears therefore to be a primary glucocorticoid-responsive gene, which in addition, may be induced by xenobiotics through CAR/pregnane X receptor activation.

Highlights

Cytochrome P450 2C9 (CYP2C9) is a major Cytochrome P-450 (CYP) expressed in the adult human liver, its mechanism of regulation is poorly known
Identification of a Functional Glucocorticoid-responsive Element in the Regulatory Region of Gene CYP2C9 —In recent work, we observed that induction of CYP2C9 mRNA by dexamethasone paralleled that of Tyrosine aminotransferase (TAT), a gene product known to be controlled by glucocorticoid receptor (GR) [18] in terms of time and concentration dependence in primary human hepatocytes [3]
We report on the presence of two functional responsive elements in the regulatory region of gene CYP2C9, a glucocorticoid-responsive element (GRE) and a constitutive androstane receptor (CAR)-RE (DR4 motif at Ϫ1803/Ϫ1818) (Fig. 11)

Summary

Introduction

Cytochrome P450 2C9 (CYP2C9) is a major CYP expressed in the adult human liver, its mechanism of regulation is poorly known. We have shown that CYP2C9 is inducible in primary human hepatocytes by xenobiotics including dexamethasone, rifampicin, and phenobarbital. We recently demonstrated that CYP2C9 is inducible at the mRNA and protein levels in human hepatocytes in primary cultures in response to xenobiotics shown previously to be CYP3A4 and CYP2B6 inducers such as dexamethasone, rifampicin, and phenobarbital [3]. Recent reports on PXR and CAR, two new members of the steroid receptor superfamily, have considerably clarified our understanding of the inducible regulation of CYP genes from families 2 and 3, in response to xenobiotics in rodents and in man (4 –10). PXR is activated by numerous compounds known to induce CYP3A expression, such as rifampicin, phenobarbital, clotrimazole, and dexamethasone [12]. Phenobarbital, a compound that has been shown to activate CAR through indirect mechanism [13], and other compounds known as CYP inducers (like rifampicin and dexamethasone) are not ligands of CAR [12]

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