FUNCTIONAL CHARACTERIZATION OF FOUR NATURALLY OCCURRING VARIANTS OF HUMAN PREGNANE X RECEPTOR (PXR): ONE VARIANT CAUSES DRAMATIC LOSS OF BOTH DNA BINDING ACTIVITY AND THE TRANSACTIVATION OF THE CYP3A4 PROMOTER/ENHANCER REGION

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Metabolism of administered drugs is determined by expression and activity of many drug-metabolizing enzymes, such as the cytochrome P450 (P450s) family members. Pregnane X receptor (PXR) is a master transcriptional regulator of many drug/xenobiotic-metabolizing enzymes, including P450s and drug transporters. In this study, we describe the functional analysis of four naturally occurring human PXR (hPXR) variants (R98C, R148Q, R381W, and I403V) that we have recently identified. By a reporter gene assay using the CYP3A4 promoter/enhancer reporter in COS-7 or HepG2 cells, it was found that the R98C variant failed to transactivate the CYP3A4 reporter. The R381W and I403V variants also showed varying degrees of reduction in transactivation, depending on the dose of PXR activators, rifampicin, clotrimazole, and paclitaxel. The transcriptional activities of the R148Q variant were not significantly different from that of the wild-type hPXR. The electrophoretic mobility shift assay revealed that only the R98C variant lacked DNA binding. Furthermore, the cellular localization of the hPXR proteins was analyzed. All four variants as well as the wildtype hPXR localized exclusively to the nucleus, regardless of the presence or absence of rifampicin. These data suggest that the R98C, R381W, and I403V hPXR variants, especially R98C, may influence the expression of drug-metabolizing enzymes and transporters, which are transactivated by PXR.

Pregnane X Receptor (PXR) Regulates P-Glycoprotein at the Blood-Brain Barrier: Functional Similarities between Pig and Human PXR

“…On the other hand, literature on the cellular localization of PXR provides conflicting findings. Based on immunostaining data, some groups have reported that hPXR is exclusively localized in the nucleus (Kawana et al, 2003;Koyano et al, 2004), but other groups provided contradictory results (Kawana et al, 2003;Squires et al, 2004). For example, Squires et al (2004) showed in mouse liver that PXR was localized in the cytoplasm and only translocated into the nucleus when PXR was activated.…”

Metabolism-Based Drug-Drug Interactions: What Determines Individual Variability in Cytochrome P450 Induction?

“…Thus, these natural PXR protein variants may play a role in the observed interindividual variability of CYP3A4 expression and may be involved in rare, atypical responses to drugs or altered sensitivities to carcinogens (Hustert et al, 2001). Four additional, naturally occurring hPXR variants (R98C, R148Q, R381W, and I403V) have been identified and their functions characterized recently by Koyano et al (2004). An important finding was that the variant R98C, which alters an amino acid immediately after the fourth cysteine residue of the second zinc finger of the DBD, caused dramatic loss of both DNA binding activity and the transactivation of the CYP3A4 promoter/enhancer region.…”

PXR Pharmacogenetics: Association of Haplotypes with Hepatic CYP3A4 and ABCB1 Messenger RNA Expression and Doxorubicin Clearance in Asian Breast Cancer Patients

“…Recent studies indicate that the overlap in PXR and CAR target genes extends well beyond the members of the CYP3A, CYP2C, GSTs, UGTs, and the canalicular ABCC2 transporter (29). Functional polymorphisms have been identified for both PXR and CAR, but interethnic genetic variation in humans has not yet been well characterized (3,30). Interethnic differences in PXR haplotype constitution as observed in the present report suggest that future studies should also take into consideration polymorphisms in other coregulators such as CAR while investigating for interethnic and interindividual variations in drug disposition related to PXR target genes.…”