Functional analysis of four naturally occurring variants of human constitutive androstane receptor
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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2018, Comprehensive Toxicology: Third EditionA comprehensive analysis and functional characterization of naturally occurring non-synonymous variants of nuclear receptor PXR
2016, Biochimica et Biophysica Acta - Gene Regulatory MechanismsCitation Excerpt :This implied that these functional variations are not attributable to variations in protein expression levels. Natural allelic variants of several key nuclear receptors are reported to affect gene regulation and their downstream target genes [81,82]. In similar context, naturally occurring non-synonymous SNPs in human PXR variants is also likely to alter receptor functioning.
Small-molecule modulators of PXR and CAR
2016, Biochimica et Biophysica Acta - Gene Regulatory MechanismsCitation Excerpt :The liver expresses high levels of CAR, and a lower level of CAR expression has been detected in brain, intestinal, heart, adrenal, testis, prostate, and kidney tissues [56,85,86]. CAR exists in many alternatively spliced variants [57,87,88] with diverse tissue expression profiles [85,89–92], heterodimerization properties [89,93], and ligand specificities [79,94–98]. The organ-specific expression of CAR splice variants requires further exploration in order to understand the variations in drug metabolism and DDIs.
Tunicate pregnane X receptor (PXR) orthologs: Transcript characterization and natural variation
2015, Marine GenomicsCitation Excerpt :Overall, this interpretation would be the most parsimonious explanation of the SNP types and frequencies detected in the tunicate VDR/PXR coding sequences. However, despite evidence of strong purifying selection in the tunicate VDR/PXR coding sequences, it remains possible that single amino acid differences between alleles do have significant functional consequences, as has been reported for human PXR and CAR polymorphisms (Hustert et al., 2001; Ikeda et al. 2005; Tirona et al., 2004). Clearly further research would be required to assess the biological relevance of the allelic variants reported in this study.
Human cytochrome P450 epoxygenases: Variability in expression and role in inflammation-related disorders
2014, Pharmacology and TherapeuticsCitation Excerpt :Accumulating evidence indicates that mutations in NR binding sites in CYP epoxygenase gene promoters could be associated with dysregulation of these genes. Several single nucleotide polymorphisms (SNPs) have been identified, so far, in genes encoding NRs (del Senno, Aguiari & Piva, 1992; Zhang et al., 2001; Auerbach et al., 2003; Savkur et al., 2003; Jinno et al., 2004; Lamba et al., 2004; Ikeda et al., 2005; Wortham et al., 2007; Lamba, 2008; Zhou et al., 2010; Endo-Umeda et al., 2012; Chai et al., 2013). The genetic variations in NR coding regions have been shown to result in altered levels of NR proteins (Wortham et al., 2007) and, by this means, can contribute to a variety of local and systemic disorders (Schubert et al., 1999; Gennari et al., 2005; Lonard et al., 2007; Neugebauer et al., 2009; Urano et al., 2009; Castaño et al., 2010; Stender et al., 2011; Müllenbach et al., 2012; Dongiovanni & Valenti, 2013; Zadeh-Vakili et al., 2013), and also to inter-individual differences in drug response through changes in the expression of genes involved in drug metabolism, including CYPs (Lamba et al., 2003; Koyano et al., 2004; Lamba et al., 2005; Lee et al., 2008).
Genetic variations of NR1I3 and NR2B1 in Asian populations
2013, Drug Metabolism and Pharmacokinetics
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Present address: Epidemiology and Prevention Division, National Cancer Center Research Institute, Japan.