Constitutive androstane receptor transcriptionally activates human CYP1A1 and CYP1A2 genes through a common regulatory element in the 5′-flanking region

Abstract

Phenobarbital has long been known to increase cellular levels of CYP1A1 and CYP1A2 possibly through a pathway(s) independent of aryl hydrocarbon receptor. We have investigated the role of constitutive androstane receptor (CAR), a xenobiotic-responsive nuclear receptor, in the transactivation of human CYP1A1 and CYP1A2. These genes are located in a head-to-head orientation, sharing a 5′-flanking region. Reporter assays were thus performed with dual-reporter constructs, containing the whole or partially deleted human CYP1A promoter between two different reporter genes. In this system, human CAR (hCAR) enhanced the transcription of both genes through common promoter regions from −461 to −554 and from −18089 to −21975 of CYP1A1. With reporter assays using additional deleted and mutated constructs, electrophoresis mobility shift assays and chromatin immunoprecipitation assays, an ER8 motif (everted repeat separated by eight nucleotides), located at around −520 of CYP1A1, was identified as an hCAR-responsive element and a binding motif of hCAR/human retinoid X receptor α heterodimer. hCAR enhanced the transcription of both genes also in the presence of an aryl hydrocarbon receptor ligand. Finally, hCAR activation increased CYP1A1 and CYP1A2 mRNA levels in cultured human hepatocytes. Our results indicate that CAR transactivates human CYP1A1 and CYP1A2 in human hepatocytes through the common cis-element ER8. Interestingly, the ER8 motif is highly conserved in the CYP1A1 proximal promoter sequences of various species, suggesting a fundamental role of CAR in the xenobiotic-induced expression of CYP1A1 and CYP1A2 independent of aryl hydrocarbon receptor.

Introduction

CYP1A1 and CYP1A2 are involved in the metabolic activation of procarcinogenic and hepatotoxic compounds as well as in the detoxification of drugs, nutrients, and environmental pollutants. These enzymes are highly inducible after exposure to polycyclic aromatic hydrocarbons such as 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. This induction results from the enhancement of transcription of both genes through the activation of aryl hydrocarbon receptor (AhR) [1]. In response to ligand exposure, AhR translocates to the nucleus and heterodimerizes with AhR nuclear translocator. The heterodimer binds to the cis-element termed xenobiotic-responsive element (XRE) in the 5′-flanking region of target genes to activate their transcription. Human CYP1A1 and CYP1A2 are located in a head-to-head orientation in chromosome 15 and share a ∼23 kb 5′-flanking region, in which twelve XREs are found. We have recently demonstrated that both genes share a common regulatory region and that the region from −461 to −1826 of CYP1A1 containing five XREs works bidirectionally to enhance the transcription of human CYP1A1 and CYP1A2 [2], [3]. Mouse and rat CYP1A1 and CYP1A2 are also located in a head-to-head orientation and share a corresponding 5′-flanking region. These data suggest the commonality of the AhR-XRE system for CYP1A1 and CYP1A2 induction in various species.

Constitutive androstane receptor (CAR), a member of nuclear receptor superfamily, plays a pivotal role in the drug-induced expression of CYP2A, CYP2B, CYP2C, and CYP3A subfamily enzymes, in concert with pregnane X receptor [4]. CAR is normally retained in cytoplasm and translocates to the nucleus in response to the exposure of drugs such as phenobarbital (PB). In nucleus, CAR heterodimerizes with retinoid X receptor α (RXRα), binds to cis-elements in the promoter region of target genes, and enhances their transcription.

Involvement of CAR in the drug-induced expression of CYP1A1 and CYP1A2 is also suggested. Treatment with PB resulted in the increased hepatic Cyp1a2 mRNA levels in Ahr-null mice [5] and DBA/2 mice that are aryl hydrocarbon-nonresponsive [6]. The molecular mechanism, however, remains unclear. In the present study, we have investigated the role of CAR in the expression of CYP1A1 and CYP1A2 using the dual-reporter assay system recently developed in our laboratory [3]. We here demonstrate that human CAR (hCAR) transactivates human CYP1A1 and CYP1A2, which is independent of AhR.