The Orphan Human Pregnane X Receptor Mediates the Transcriptional Activation ofCYP3A4by Rifampicin through a Distal Enhancer Module

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 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.

Retinoids induce cytochrome P450 3A4 through RXR/VDR-mediated pathway

“…Using VDR knockout mice might provide a direct evidence for the role of VDR in regulating Cyp3a11 gene expression. However, the liver expresses high levels of PXR and CAR, and these two nuclear receptors play the dominant roles in regulating Cyp3a11 gene expression [4,12]. Thus, using VDR knockout mice might not demonstrate an obvious reduction of retinoid-mediated induction of Cyp3a11.…”

“…It has been well characterized that CYP3A4 gene is regulated by PXR [5][6][7][8], CAR [4], and VDR [9,10]. Induction of Cyp3a11 (homologous of human CYP3A4) mRNA by retinoids in hepatocytes which are deficient in both PXR and CAR would strongly suggest the role of VDR in regulation of Cyp3a11.…”

“…Several members of the nuclear receptor superfamily including CAR [4], PXR [5][6][7][8], VDR [9,10], and the glucocorticoid receptor (GR) [11] have been shown to be responsible for endobiotic-and xenobiotic-mediated CYP3A induction. Human PXR (hPXR) or steroids and xenobiotic receptor (SXR) [12,13], CAR [4], and VDR [9,10] control CYP3A4 expression by targeting the same cis-acting elements located in the regulatory region of target genes. One of the shared motifs is pER6, an everted repeat separated by 6 nucleotides, in the proximal region of the CYP3A4 promoter [7].…”

“…One of the shared motifs is pER6, an everted repeat separated by 6 nucleotides, in the proximal region of the CYP3A4 promoter [7]. The second is dXREM, a distal xenobiotic-responsive enhancer module [4]. The cross-talk among these intracellular signaling pathways in regulating CYP3A4 gene expression is a key defensive mechanism in response to a diversity of harmful xenobiotic challenges.…”

Nuclear receptor mediated induction of cytochrome P450 3A4 by anticancer drugs: a key role for the pregnane X receptor

“…Only a few agonists are known for CAR and VDR, while PXR is activated by a wide variety of structurally unrelated compounds that include rifampicin, phenobarbital and hyperforin, but also anticancer drugs like paclitaxel [10] and tamoxifen [11]. Upon agonist binding, the nuclear receptors heterodimerize to the retinoid X receptor (NR2B1) and bind to distinct motifs within the promoter area of CYP3A4 [12]. Nuclear receptor activation is therefore one of the major mechanisms behind drug-drug interactions due to induction of CYP3A4.…”