Abstract
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 theCYP2B6 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 α (NR2B1). Taken together, these observations demonstrate that theCYP2B6 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 heme-thiolate–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 theCYP1A, CYP2B, CYP3A, andCYP4A subfamilies (Waxman, 1999). This adaptive response increases the organism's ability to metabolize and ultimately eliminate toxic and carcinogenic compounds. Whereas induction ofCYP1A genes by aromatic hydrocarbons and CYP4Asubfamily members by peroxisome proliferators are known to be mediated by the aryl hydrocarbon receptor and peroxisome proliferator activated receptor α (NR1C1), respectively, the molecular mechanisms by which structurally dissimilar compounds induce CYP2B andCYP3A 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-cisretinoic acid receptor α (RXRα; 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 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 Cyp3a11expression in response to the classic rodent CYP3A inducers pregnenolone 16α-carbonitrile and dexamethasone (Xie et al., 2000a; Staudinger et al., 2001). Notably, induction ofCyp3a11 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 Cyp2b10inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is a high-affinity 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 ittrans-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 theCYP2B1, CYP2B2, CYP2B6, andCyp2b10 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 RXRα. Notably, PXR is also reported to trans-activate DR4 elements; moreover, PXR and CAR bind and activate common response elements in the humanCYP3A4 and rodent CYP3A23, CYP2B1, andCyp2b10 genes, suggesting that interplay between these two receptors is likely to be a central theme in the regulation of xenobiotic-inducible 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 regulatesCYP2B6 expression.
Experimental Procedures
Materials.
Rifampicin, dexamethasone, sodium phenobarbital, and charcoal-stripped, delipidated FBS were obtained from the Sigma Chemical Co. (St. Louis MO). Cell culture reagents, unless otherwise stated, were provided by Invitrogen (Carlsbad, CA). Hyperforin was purchased from Apin Chemicals Ltd. (Abingdon, Oxon, UK). SR12813 was synthesized in house.
Primary Culture of Human Hepatocytes and Northern Blot Analysis.
Primary human hepatocytes were obtained from Dr. Stephen Strom (University of Pittsburgh, Pittsburgh, PA) and maintained exactly as described elsewhere (Moore et al., 2000a). At 48 h after isolation, cells were treated for a further 48 h with various inducers that were added to the culture medium as 1000× stocks in DMSO. Sodium phenobarbital was dissolved directly into the medium. Control cultures received vehicle (0.1% DMSO) alone. Total RNA was isolated using a commercially available reagent (TriZOL; Invitrogen). CYP2B6 and CYP3A4 mRNA levels were examined by Northern blot analysis using standard techniques. Blots were sequentially hybridized with CYP2B6 [bases 3–659 of the published cDNA; GenBank accession numberAF182277), CYP3A4 (bases 790 to 1322 of the published cDNA; GenBank accession number M18907), and β-actin (CLONTECH Laboratories Inc., Palo Alto, CA) cDNA probes.
Preparation of CYP2B6 PBREM Reporter Gene Constructs.
Luciferase reporter gene constructs were prepared by annealing oligonucleotides corresponding to the wild-type and mutantCYP2B6 PBREM (Fig. 2A) before insertion into theBglII site of pGL3-tk-Luc, which contains bases −105 to +51 of the herpes simplex virus thymidine kinase promoter linked to a luciferase reporter gene.
Activation of the CYP2B6 PBREM by PXR and CAR. A, structure of the CYP2B6 PBREM. The two DR-4 elements (NR1 and NR2) are delineated by shaded boxes. The repeated half-sites within these elements are indicated by arrows. The mutated bases in the pGL3-CYP2B6-PBREMmut1, pGL3-CYP2B6-PBREMmut2, and pGL3-CYP2B6-PBREMmut1 + 2 are shown in lower case. The position relative to the CYP2B6 transcription initiation site is also shown. trans-Activation of the wild-type and mutantCYP2B6 PBREM constructs by PXR (B) and CAR (C) was examined by transient transfection in HuH7 cells as described underExperimental Procedures. PXR-mediated activation of theCYP2B6 PBREM cells was characterized by treatment of transfected cells with rifampicin (10 μM) or vehicle (0.1% DMSO) for 24 h prior to harvest. Data represent the mean ± S.D. of four individual transfections.
Transient Transfection Assays.
Analysis of PXR- and CAR-dependent trans-activation of the CYP2B6PBREM reporter gene constructs was performed in a human liver-derived cell line, HuH7. Cells (20,000 per well) were inoculated into a 96-well plate in Dulbecco's modified Eagle's/Ham's F12 media nutrient mixture supplemented with 10% charcoal/dextran-treated FBS (HyClone Laboratories Inc., Logan, UT) and transfected 24 h later with LipofectAMINE Plus reagent (Invitrogen). Transfection mixes contained 8 ng of luciferase reporter gene construct, 2 ng of human CAR or PXR expression vectors, pSG5-hCAR (Moore et al., 2000b) and pSG5-ΔATG-hPXR (Lehmann et al., 1998), 8 ng of pβ-actin-SPAP, and 52 ng pBluescript (Stratagene, La Jolla, CA). Transfection was allowed to proceed for 3 h. Cells were maintained for a further 24 h in the presence of drug (added as a 1000× stock in DMSO) in Dulbecco's modified Eagle/Ham's F12 media nutrient mixture supplemented with 10% heat-inactivated, charcoal-stripped, delipidated FBS. An aliquot of medium was withdrawn for SPAP assay and the cells lysed before luciferase determination. Luciferase activities were normalized to SPAP expression.
Electrophoretic Mobility-Shift Assay.
Electrophoretic mobility-shift assay (EMSA) was performed exactly as described elsewhere (Goodwin et al., 1999). In vitro translated human RXRα, CAR, and PXR were prepared using a TnT rabbit reticulocyte system (Promega, Madison, WI). Binding reactions were preincubated on ice for 10 min before the addition of32P-end-labeled probe corresponding to the NR1 and NR2 motifs. After a further 20 min on ice, samples were resolved on a pre-electrophoresed 5% acrylamide gel in 0.25× Tris/borate/EDTA buffer (22.5 mM Tris-borate, 0.5 mM EDTA).
Results
Induction of CYP2B6 Expression by PXR Ligands.
To examine whether PXR regulates CYP2B6 expression, human hepatocytes were treated with a panel of known PXR ligands (Fig.1A). In line with earlier observations, rifampicin (∼15-fold) and PB (∼50-fold) effectively induced CYP2B6 mRNA levels (Strom et al., 1996; Chang et al., 1997; Gervot et al., 1999; Gerbal-Chaloin et al., 2001). In addition, treatment of human hepatocytes with dexamethasone or the high-affinity PXR ligands SR12813 and hyperforin (Jones et al., 2000; Moore et al., 2000a) resulted in the induction of CYP2B6 expression (∼6-fold, 5-fold, and 4-fold, respectively). In parallel with CYP2B6 mRNA levels, expression of CYP3A4 was also examined. As expected, CYP3A4 mRNA levels were strongly induced by rifampicin (13-fold), SR12813 (∼5-fold), hyperforin (∼3.5-fold), dexamethasone (7-fold), and PB (∼13-fold) (Fig. 1B). Similarly, rifampicin, SR12813, hyperforin, and dexamethasone inducedCYP2B6 and CYP3A4 expression. However, the PB-mediated induction of CYP2B6 (∼50-fold) expression was significantly higher than that of CYP3A4 (∼13-fold), in line with the important role of CAR in the regulation ofCYP2B6 (Sueyoshi et al., 1999).
Induction of CYP2B6 andCYP3A4 expression in primary cultures of human hepatocytes. Northern blot analysis of CYP2B6 (A) and CYP3A4 (B) mRNA levels was performed with total RNA (10 μg) prepared from human hepatocytes treated for 48 h with vehicle alone (0.1% DMSO; lane 1), rifampicin (10 μM; lane 2), SR12813 (1 μM; lane 3), hyperforin (10 μM; lane 4), dexamethasone (10 μM; lane 5), or PB (1 mM; lane 6). Relative mRNA abundance (corrected for β-actin expression) is depicted graphically at the bottom of each panel.
PXR Activates the CYP2B6 PBREM.
Transcriptional activation of the CYP2B6 gene by CAR is mediated by the 51-bp PBREM. This region is located 1.7 kilobase pairs upstream of theCYP2B6 transcription initiation site and contains two imperfect DR4 elements, designated NR1 and NR2 (Fig.2A) (Sueyoshi et al., 1999). Importantly, PXR-RXRα heterodimers are reported to be capable of binding andtrans-activating DR4 elements (Blumberg et al., 1998). Taken together, these observations suggested that induction ofCYP2B6 expression by compounds that activate PXR may be mediated by the PBREM region. Thus, we examined the ability of PXR to activate reporter gene constructs harboring the CYP2B6 PBREM linked to a minimal herpes simplex virus thymidine kinase promoter and luciferase reporter. Chimeric CYP2B6-PBREM reporter gene constructs containing wild-type and mutated DR4 motifs (Fig. 2A) were transiently transfected into a liver-derived cell line (HuH7) and the ability of rifampicin, a human PXR ligand, to activate expression of these constructs was determined. In the absence of exogenously expressed PXR, there was no detectable induction of reporter gene expression by rifampicin (Fig. 2B). Cotransfection of the wild-typeCYP2B6-PBREM construct (pGL3-CYP2B6-PBREM) with a human PXR expression vector resulted in an ∼2-fold increase in reporter gene activity. Treatment of transfected cells with rifampicin (10 μM) elicited a further 3-fold induction of luciferase expression (Fig. 2B). The relative contribution of the NR1 and NR2 DR4 motifs to the PXR response was examined by mutating the hexad half-sites in one or both of these elements. Although mutation of the NR1 site (pGL3-CYP2B6-PBREMmutNR1) completely abrogated PXR-dependent activation, the pGL3-CYP2B6-PBREMmutNR2 construct, which contains a mutated NR2 element, retained some responsiveness to PXR. As expected, mutation of both the NR1 and NR2 sites (pGL3-CYP2B6-PBREMmut1 + 2) destroyed the PXR response (Fig. 2B). These data suggest that the NR1 motif is quantitatively the more important element to the PXR response.
In parallel transfection experiments, CAR effectively activated (∼8-fold) the wild-type CYP2B6 PBREM reporter (Fig. 2C). Disruption of the NR1 site caused a total loss in CAR activation; mutation of the NR-2 resulted in a substantial reduction in the CAR-dependent reporter activity. However, some residual CAR-responsiveness remained (Fig. 2C). The relative importance of the NR1 element in the CAR response is in agreement with earlier reports by Negishi and colleagues (Sueyoshi et al., 1999).
The NR1 and NR2 Motifs Bind PXR-RXRα Heterodimers.
The ability of the NR1 and NR2 sites to bind PXR was examined by EMSA. Both the NR1 and NR2 elements strongly complexed PXR-RXRα heterodimers (Fig. 3A). In close agreement with the cell-based reporter gene assays described above, competition binding studies demonstrated that the NR1 site bound PXR-RXRα with higher affinity than the NR2 motif (Fig. 3B), confirming that this site is the predominant PXR response element within the PBREM. As reported previously (Sueyoshi et al., 1999), CAR-RXRα heterodimers bound both NR1 and NR2 (Fig. 3A). In similarity to the PXR-RXRα binding profile, the NR1 site interacted with the CAR-RXRα heteromer with significantly higher affinity than the NR2 element (Fig. 3B). The mutated derivatives of the NR1 and NR2 sites used in the transient transfection studies failed to compete for either PXR-RXRα or CAR-RXRα binding.
PXR binding to the NR1 and NR2 response elements. The ability of CAR-RXRα and PXR-RXRα-heterodimers to bind the DR4 elements (NR1 and NR2) in the CYP2B6 PBREM was examined by EMSA as outlined under Experimental Procedures. A, EMSA with radiolabeled double-stranded oligonucleotides corresponding to the NR1 and NR2-binding motifs (see Fig. 2A). Reactions received in vitro translated RXRα, CAR, and PXR as indicated. B, competition EMSA was performed using the NR1 motif as probe. Unlabeled competitor oligonucleotides corresponding to the wild-type and mutant NR1 and NR2 sites (see Fig. 2A) were added to the preincubation reaction at the indicated molar excess.
Discussion
A number of earlier studies suggested that the humanCYP2B6 gene might be regulated in a PXR-dependent manner (Ekins and Wrighton 1999, and references therein). In this report, we show that CYP2B6 is regulated directly by PXR. In primary cultures of human hepatocytes, the human PXR ligands rifampicin, hyperforin, SR12813, dexamethasone, and PB effectively induced expression of both CYP2B6 and CYP3A4, a well-documented PXR target gene. Trans-activation ofCYP2B6 by PXR was shown to be mediated by the PBREM region of the gene, a 51-bp enhancer module that controls induction ofCYP2B6 by CAR (Sueyoshi et al., 1999).
The PBREM/PBRU is highly conserved among PB-inducible CYP2Bsubfamily members, namely CYP2B1, CYP2B2,CYP2B6, and Cyp2b10 (Sueyoshi et al., 1999); moreover, similar to CYP2B6, both the mouseCyp2b10 PBREM and rat CYP2B1 PBRU are activated by PXR (Xie et al., 2000b; Smirlis et al., 2001). TheCYP2B6 PBREM contains two DR4 elements that are capable of binding both PXR-RXRα and CAR-RXRα heteromers. CAR was originally reported to bind a DR5 element in the RARβ2 promoter; subsequently, however, CAR-mediated trans-activation through DR3, DR4, and ER6 elements has been documented (Baes et al., 1994; Choi et al., 1997;Honkakoski et al., 1998; Sueyoshi et al., 1999; Xie et al., 2000; Smirlis et al., 2001; B. Goodwin, E. Hodgson, and C. Liddle, submitted). It is now apparent that these configurations of nuclear receptor half-sites are also capable of binding PXR-RXRα heterodimers (Blumberg et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998;Goodwin et al., 1999; Sueyoshi et al., 1999; Xie et al., 2000b;Geick et al., 2001; Smirlis et al., 2001). Taken together, these observations demonstrate that CAR and PXR are capable of regulating common genes through the same cis-acting elements, suggesting that cross talk between these two signaling pathways is an important factor in mounting an appropriate response to a xenobiotic challenge.
Although rifampicin and PB induced CYP3A4 to a similar extent (∼13-fold), CYP2B6 was substantially more responsive to PB (∼50-fold) than rifampicin (∼15-fold). Thus, although both CAR and PXR directly regulate CYP2B6expression, CAR seems to assume a dominant role in the PB-mediated induction of this gene. It is possible that the arrangement of the nuclear receptor binding motifs within the CYP2B6 PBREM provides an optimal platform for CAR-mediated transactivation. In addition to promoting nuclear translocation of CAR, PB is an effective activator of human PXR (Lehmann et al., 1998; Goodwin et al., 1999;Jones et al., 2000). We have previously shown that CAR and PXR share common ligands (Moore et al., 2000b); therefore, it is likely that certain xenobiotics, including PB, are capable of inducing P450 expression through multiple signaling pathways. The existence of multiple xenobiotic receptors with distinct but overlapping ligand specificities increases the organism's ability to detect and respond to a potentially harmful substance.
In summary, we have shown that the human CYP2B6 gene is directly regulated by PXR. These results provide evidence for a functional redundancy between the nuclear receptors PXR and CAR in the protective response to xenobiotic challenge in humans. Importantly, our findings extend the range of potential drug interactions caused by compounds that activate PXR to include CYP2B6 substrates. This knowledge can be used to understand more fully the metabolism of drugs currently on the market and to design safer drugs for the future.
Footnotes
- Abbreviations:
- P450
- cytochrome P450, PXR
- pregnane X receptor
- CAR
- constitutive androstane receptor
- RXRα
- 9-cis retinoic acid receptor α
- DRn
- direct repeat with n-bp spacer
- bp, base pair(s)
- ER6
- everted repeat with 6-base-pair spacer
- PB
- phenobarbital
- PBRU
- phenobarbital-responsive unit
- PBREM
- phenobarbital-responsive enhancer module
- FBS
- fetal bovine serum
- DMSO
- dimethyl sulfoxide
- SPAP
- secreted placental alkaline phosphatase
- EMSA
- electrophoretic mobility-shift assay
- Received April 18, 2001.
- Accepted June 4, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
