The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module.
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 the CYP3A4 5'-flanking region. Rifampicin-inducible transcription of the reporter gene was observed only with the longest construct, which encompassed bases -13000 to +53 of CYP3A4 (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 approximately 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 and cis-acting elements in the proximal promoter of CYP3A4. 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 of CYP3A4 by activators of hPXR.
Goodwin
B
B
Department of Clinical Pharmacology and Storr Liver Unit, University of Sydney at Westmead Hospital, Westmead, Australia.
Hodgson
E
E
Liddle
C
C
D016475:3T3 Cells; D000818:Animals; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001483:Base Sequence; D019556:COS Cells; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004247:DNA; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D019281:Dimerization; D004742:Enhancer Elements, Genetic; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D051379:Mice; D015735:Mifepristone / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D000077297:Pregnane X Receptor; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011987:Receptors, Steroid / Q000502:physiology*; D000072482:Retinoic Acid Receptor alpha; D012293:Rifampin / Q000494:pharmacology*; D012689:Sequence Homology, Nucleic Acid; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D015262:Xenobiotics
D000904:Antibiotics, Antitubercular; D000077297:Pregnane X Receptor; C000607071:RARA protein, human; C000607072:Rara protein, mouse; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D000072482:Retinoic Acid Receptor alpha; D015262:Xenobiotics; D015735:Mifepristone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003850:Deoxyribonuclease I; D012293:Rifampin