Functional analysis of four naturally occurring variants of human constitutive androstane receptor

doi:10.1016/j.ymgme.2005.05.011

Molecular Genetics and Metabolism

Volume 86, Issues 1–2, September–October 2005, Pages 314-319

https://doi.org/10.1016/j.ymgme.2005.05.011 Get rights and content

Abstract

The human constitutive androstane receptor (CAR, NR1I3) is a member of the orphan nuclear receptor superfamily that plays an important role in the control of drug metabolism and disposition. In this study, we sequenced all the coding exons of the NR1I3 gene for 334 Japanese subjects. We identified three novel single nucleotide polymorphisms (SNPs) that induce non-synonymous alterations of amino acids (His246Arg, Leu308Pro, and Asn323Ser) residing in the ligand-binding domain of CAR, in addition to the Val133Gly variant, which was another CAR variant identified in our previous study. We performed functional analysis of these four naturally occurring CAR variants in COS-7 cells using a CYP3A4 promoter/enhancer reporter gene that includes the CAR responsive elements. The His246Arg variant caused marked reductions in both transactivation of the reporter gene and in the response to 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), which is a human CAR-specific agonist. The transactivation ability of the Leu308Pro variant was also significantly decreased, but its responsiveness to CITCO was not abrogated. The transactivation ability and CITCO response of the Val133Gly and Asn323Ser variants did not change as compared to the wild-type CAR. These data suggest that the His246Arg and Leu308Pro variants, especially His246Arg, may influence the expression of drug-metabolizing enzymes and transporters that are transactivated by CAR.

Introduction

The constitutive androstane receptor (CAR) encoded by NR1I3 is expressed predominantly in the liver [1] and it belongs to the nuclear receptor subfamily 1I. This subfamily also includes the pregnane X receptor (PXR) and the vitamin D receptor. CAR regulates transcription of the genes encoding drug/steroid-metabolizing enzymes and transporters, as well as other physiologically important enzymes [2], [3], [4]. CAR also regulates thyroid hormone and bilirubin metabolism [5], [6]. In humans, CAR transactivates several major hepatic drug-metabolizing enzymes, such as the cytochrome P450s (CYPs) and transferase, including CYP2B6 [7], CYP3A4 [7], [8], CYP2C9 [9], [10], CYP2C19 [11], and UGT1A1 [12]. CAR forms a heterodimer with the retinoid X receptor (RXRα) and this binds to DNA motifs such as DR3, DR4, DR5, or ER6, of the target genes. It is noteworthy that, unlike most nuclear receptors, CAR is constitutively active in the absence of any added ligand [1], [13]. However, ligand binding modulates the transcriptional activity of CAR. For example, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) is an agonistic ligand of human CAR and it also triggers nuclear translocation [14].

NR1I3 is located on chromosome 1 and consists of nine exons. The DNA binding domain is encoded by exons 2, 3, and 4. A small hinge domain is encoded by a portion of exon 4, while the ligand-binding domain corresponds to exons 4–8 and a 5′ portion of exon 9 [15], [16]. Recently, we sequenced all the coding exons of the NR1I3 gene for 253 Japanese subjects and identified novel SNPs in the NR1I3 gene [17]. These SNPs included one non-synonymous amino acid change that was localized to the ligand-binding domain of CAR. It is thought that SNPs may induce changes in the function or expression of the NR1I3 gene, and this may explain variations in drug metabolism among humans. In this study, we analyzed the NR1I3 sequence in an additional set of 334 Japanese subjects and found three novel, non-synonymous SNPs. We performed functional analysis of these four CAR variants using a reporter gene assay carried out with COS-7 cells.

Section snippets

Human genomic DNA samples

The 334 subjects used in this study were Japanese cancer patients who were administered irinotecan or paclitaxel. Genomic DNA was extracted from blood leukocytes and was used as a template for the polymerase chain reaction (PCR). This study was approved by all the Ethnic Committees of the National Cancer Center, the National Cardiovascular Center, and the National Institute of Health Sciences. Written informed consent was obtained from all participants.

Conditions for PCR and DNA sequencing

The conditions and primers for PCR and DNA

Four non-synonymous SNPs in CAR

Previously, we identified the variant 398T > G in the CAR gene after screening 253 Japanese patients. This mutation leads to the amino acid alteration Val133Gly [17]. In this study, we surveyed an additional set of 334 Japanese subjects and identified novel non-synonymous SNPs 737A > G (His246Arg), 923T > C (Leu308Pro), and 968A > G (Asn323Ser), found in two, one, and one subjects, respectively. The electropherograms of the three novel SNPs are shown in Fig. 1. All four SNPs were found as heterozygotes

Discussion

CAR, as well as PXR, regulates the expression of genes involved in the metabolism and transport of xenobiotics and endobiotics [20], [21], [22]. It is possible that functional alterations resulting from genetic polymorphisms may influence drug metabolism and therapeutic outcomes. To date, several non-synonymous SNPs that affect the transactivation ability of human PXR have been reported [19], [23], [24]. These PXR variants might cause variations between individuals in their responses to drugs.

Acknowledgments

We thank Ms. Chie Knudsen for her secretarial assistance. This study was supported in part by the Program for the Promotion of Fundamental Studies in Health Sciences and in part by the Program for the Promotion of Studies in Health Sciences of the Ministry of Health, Labor and Welfare of Japan.

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¹: Present address: Epidemiology and Prevention Division, National Cancer Center Research Institute, Japan.

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Functional analysis of four naturally occurring variants of human constitutive androstane receptor

Abstract

Introduction

Section snippets

Human genomic DNA samples

Conditions for PCR and DNA sequencing

Four non-synonymous SNPs in CAR

Discussion

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Hepatology

J. Biol. Chem.

Drug Metab. Pharmacokinet.

Mol. Cell

Mol. Cell

Mol. Cell

J. Biol. Chem.

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements

Mol. Cell. Biol.

Orphan nuclear receptors: from gene to function

Endocr. Rev.

Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital

Mol. Pharmacol.

Induction of drug metabolism: the role of nuclear receptors

Pharmacol. Rev.