ACCELERATED COMMUNICATION Regulation of the Human CYP2B6 Gene by the Nuclear Pregnane X Receptor

Cytochromes P450 (P450s) are involved in the oxidative metabolism of a plethora of structurally unrelated compounds, including therapeutic drugs. Two orphan members of the nuclear receptor superfamily, the pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) have been implicated in this phenomenon. In the present study, we examined the transcriptional regulation of the human CYP2B6 gene. In primary cultures of human hepatocytes, CYP2B6 was highly inducible by a number of compounds known to be human PXR ligands, including rifampicin and hyperforin. PXR was shown to be capable of activating the phenobarbital-responsive enhancer module (PBREM) region of the CYP2B6 gene, a 51-base-pair enhancer element that mediates induction of CYP2B6 expression by CAR. The two nuclear receptor-binding motifs within the PBREM effectively bound PXR as a heterodimer with the 9-cis retinoic acid receptor (NR2B1). Taken together, these observations demonstrate that the CYP2B6 gene is directly regulated by PXR and further establish this receptor as a key regulator of drug-metabolizing P450s. Cytochromes P450 (P450s) are a superfamily of hemethiolate–containing proteins involved in the oxidative metabolism of a diverse range of compounds, including steroid hormones, bile acids, fatty acids, and prostaglandins. In addition, many P450 enzymes participate in the conversion of carcinogens, environmental pollutants, and drugs to more polar metabolites, thereby facilitating their excretion and preventing the accumulation of these potentially harmful compounds (Nelson et al., 1996). For many years, it has been understood that xenobiotic compounds can induce the expression of certain P450 genes, notably members of the CYP1A, CYP2B, CYP3A, and CYP4A subfamilies (Waxman, 1999). This adaptive response increases the organism’s ability to metabolize and ultimately eliminate toxic and carcinogenic compounds. Whereas induction of CYP1A genes by aromatic hydrocarbons and CYP4A subfamily members by peroxisome proliferators are known to be mediated by the aryl hydrocarbon receptor and peroxisome proliferator activated receptor (NR1C1), respectively, the molecular mechanisms by which structurally dissimilar compounds induce CYP2B and CYP3A genes remained obscure (Denison and Whitlock, 1995). Recently, a number of laboratories identified two orphan members of the nuclear receptor family, the pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3), as xenobiotic-responsive transcription factors (Bertilsson et al., 1998; Blumberg et al., 1998; Honkakoski et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998; Moore et al., 2000b; Tzameli et al., 2000) Human PXR (also known as SXR or PAR) binds and is activated by many known CYP3A4 inducers, including the macrocyclic antibiotic rifampicin, the antimycotic clotrimazole, the barbiturate phenobarbital (PB), and the putative antidepressant component of St. John’s wort, hyperforin (Bertilsson et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998; Jones et al., 2000; Moore et al., 2000a). PXR interacts with its cognate response elements in the 5 -flanking regions of CYP3A genes as a heterodimer with the 9-cis retinoic acid receptor (RXR ; NR2B1). Typically, these elements contain two copies of the AG(G/T)TCA hexad organized as a direct repeat with a three-nucleotide spacer (DR3) or an everted repeat (ER) separated by 6 bp (ER6) (Bertilsson ABBREVIATIONS: P450; cytochrome P450, PXR; pregnane X receptor; CAR; constitutive androstane receptor; RXR ; 9-cis retinoic acid receptor ; DRn; direct repeat with n-bp spacer; bp, base pair(s); ER6; everted repeat with 6-base-pair spacer; PB; phenobarbital; PBRU, phenobarbitalresponsive unit; PBREM; phenobarbital-responsive enhancer module; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; SPAP, secreted placental alkaline phosphatase; EMSA, electrophoretic mobility-shift assay. 0026-895X/01/6003-427–431$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 3 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 1044/928011 Mol Pharmacol 60:427–431, 2001 Printed in U.S.A. 427 at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from et al., 1998; Blumberg et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998; Goodwin et al., 1999). Further compelling evidence for the role of PXR in the induction of CYP3A genes is provided by experiments performed in mice harboring a homozygous disruption of the pxr gene. Thus, mice lacking functional PXR fail to up-regulate Cyp3a11 expression in response to the classic rodent CYP3A inducers pregnenolone 16 -carbonitrile and dexamethasone (Xie et al., 2000a; Staudinger et al., 2001). Notably, induction of Cyp3a11 expression by PB was intact in the PXR-null mice (Xie et al., 2000b; Staudinger et al., 2001). In addition to PXR, CAR also plays a central role in the regulation of xenobiotic-inducible P450 genes, although the biology of this receptor is very different from that of PXR. CAR exhibits a high level of constitutive transcriptional activity and can activate expression of reporter gene constructs in the absence of exogenously added ligand (Baes et al., 1994; Choi et al., 1997). The androstane metabolites androstanol and androstenol act as inverse agonists for the mouse and, to a lesser extent, human CAR, whereas the potent Cyp2b10 inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is a highaffinity mouse CAR agonist (Forman et al., 1998; Moore et al., 2000b; Tzameli et al., 2000). As with PXR, there seems to be a species divergence in CAR pharmacology (Jones et al., 2000; Moore et al., 2000b). Elegant studies by Negishi and coworkers have demonstrated that induction of CYP2B subfamily members by PB and PB-like inducers is mediated by CAR (Honkakoski et al., 1998; Kawamoto et al., 1999). In untreated liver, CAR resides in the cytoplasm of the hepatocyte. However, exposure of the cell to PB or PB-like inducers promotes the rapid translocation of CAR to the nucleus, where it trans-activates expression of its target genes. Importantly, this process is uncoupled by the phosphatase inhibitor okadaic acid, suggesting that the PB-induced nuclear translocation of CAR is a phosphorylation-sensitive event (Kawamoto et al., 1999). Although PB does not seem to interact directly with CAR (Moore et al., 2000b), targeted disruption of the mouse car gene results in total ablation of Cyp2b10 inducibility by both PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (Wei et al., 2000). After translocating to the nucleus, CAR interacts with a conserved 51-bp enhancer element located 2 kilobase pairs upstream of the CYP2B1, CYP2B2, CYP2B6, and Cyp2b10 transcription initiation sites (Trottier et al., 1995; Honkakoski et al., 1998; Sueyoshi et al., 1999; Smirlis et al., 2001). These regions, termed phenobarbital-responsive units (PBRU) or phenobarbital-responsive enhancer modules (PBREM), contain two DR4 elements (NR1 and NR2) that act as high-affinity binding sites for CAR and its obligate heterodimerization partner RXR . Notably, PXR is also reported to trans-activate DR4 elements; moreover, PXR and CAR bind and activate common response elements in the human CYP3A4 and rodent CYP3A23, CYP2B1, and Cyp2b10 genes, suggesting that interplay between these two receptors is likely to be a central theme in the regulation of xenobioticinducible P450s (Blumberg et al., 1998; Sueyoshi et al., 1999; Xie et al., 2000b; Geick et al., 2001; Smirlis et al., 2001; B. Goodwin, E. Hodgson, and C. Liddle, submitted). In this study, we examined the role of PXR in the regulation of the human CYP2B6 gene. CYP2B6 is involved in the metabolism of a number of clinically important drugs (Ekins and Wrighton 1999); moreover, its expression is reported to be induced by compounds that are PXR ligands, including rifampicin, PB, troglitazone, and dexamethasone (Strom et al., 1996; Chang et al., 1997; Sahi et al., 2000; Gerbal-Chaloin et al., 2001). We show that PXR directly regulates