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 wild-type 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.
Footnotes
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↵1 Abbreviations used are: P450, cytochrome P450; PXR, pregnane X receptor; PAR, pregnane-activated receptor; DBD, DNA binding domain; LBD, ligand binding domain; ER6, everted repeat-6; RXRα, 9-cis retinoic acid receptor-α; SNP, single nucleotide polymorphism; XREM, xenobiotic-responsive enhancer module; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PBS-GS, PBS containing 10% goat serum; EMSA, electrophoretic mobility shift assay; DAPI, 4,6-diamidino-2-phenylindole; VDR, vitamin D receptor; WT, wild-type.
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This study was supported by the Program for the Promotion of Fundamental Studies in Health Sciences (MPJ-3, -5, and -6) of the Organization for Pharmaceutical Safety and Research of Japan.
- Received July 14, 2003.
- Accepted September 19, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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