The Orphan Human Pregnane X Receptor Mediates the Transcriptional Activation of CYP3A4 by Rifampicin through a Distal Enhancer Module
- Department of Clinical Pharmacology and Storr Liver Unit, University of Sydney at Westmead Hospital, Westmead, Australia
Abstract
Cytochrome P-450 3A4 (CYP3A4), the predominant cytochrome P-450 expressed in adult human liver, is subject to transcriptional induction by a variety of structurally unrelated xenobiotics, including the antibiotic rifampicin. The molecular mechanisms underlying this phenomenon are poorly understood. We transfected a human liver-derived cell line (HepG2) with various CYP3A4-luciferase reporter gene constructs containing a nested set of 5′-deletions of theCYP3A4 5′-flanking region. Rifampicin-inducible transcription of the reporter gene was observed only with the longest construct, which encompassed bases −13000 to +53 ofCYP3A4 (3-fold induction). The responsive region was functional regardless of its position or orientation relative to the proximal promoter of CYP3A4 and was capable of conferring rifampicin-inducible expression on a heterologous promoter. Further deletion mutants localized the induction to bases −7836 to −7607. In vitro DNase I footprint analysis of this region revealed four protected sites (FP1, FP2, FP3, and FP4). Two of these sites, FP3 (bases −7738 to −7715) and FP4 (bases −7698 to −7682), overlapped binding motifs for the orphan human pregnane X receptor (hPXR). Cotransfection of responsive constructs with a hPXR expression vector substantially increased the rifampicin-inducibility to ∼50-fold. In addition, the rifampicin-responsive constructs were strongly activated by a range of CYP3A inducers. Finally, we demonstrate cooperativity between elements within the distal enhancer region andcis-acting elements in the proximal promoter ofCYP3A4. Our results provide evidence for the existence of a potent enhancer module, 8 kb distal to the transcription start point, which mediates the transcriptional induction ofCYP3A4 by activators of hPXR.
Footnotes
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Send reprint requests to: Dr. Christopher Liddle, Department of Clinical Pharmacology and Storr Liver Unit, University of Sydney at Westmead Hospital, Westmead, NSW 2145, Australia. E-mail: chrisl{at}westgate.wh.usyd.edu.au
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This work was supported by a grant from the National Health and Medical Research Council (Australia) and the Robert W. Storr bequest to the Medical Foundation, University of Sydney. B.G. was a recipient of the National Health and Medical Research Council (Australia) Dora Lush Biomedical Scholarship.
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1 Three independent studies have implicated an orphan nuclear receptor in CYP3A regulation. The human PXR (hPXR; Lehmann et al., 1998) and human pregnane activated receptor (hPAR; Bertilsson et al., 1998) are identical in the derived amino acid sequence. The human steroid and xenobiotic receptor (hSXR; Blumberg et al., 1998) contains a single base-pair insertion at position 1225 and a single base deletion at 1279, relative to hPXR, which results in a shift in the reading frame for amino acid residues 215–233. However, hPXR, hPAR, and hSXR almost certainly represent products of the same gene. In this study, we used the “hPXR” nomenclature.
- Abbreviations:
- P-450
- cytochrome P-450
- PXR
- pregnane X receptor
- RXRα
- 9-cis retinoic acid receptor-α
- PXRE
- pregnane X receptor response element
- prPXRE
- proximal pregnane X receptor response element
- HNF
- hepatocyte nuclear factor
- EMSA
- electrophoretic mobility shift assay
- RORα1
- retinoic acid receptor-related receptor α-1
- COUP-TF
- chicken ovalbumin upstream promoter-transcription factor
- PCR
- polymerase chain reaction
- PCN
- pregnenolone 16α-carbonitrile
- tk
- herpes simplex virus thymidine kinase
- hGR
- human glucocorticoid receptor
- GRE
- glucocorticoid-responsive element
- XREM
- xenobiotic-responsive enhancer module
- DMSO
- dimethyl sulfoxide
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- Received July 6, 1999.
- Accepted August 26, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
