A Novel Distal Enhancer Module Regulated by Pregnane X Receptor/Constitutive Androstane Receptor Is Essential for the Maximal Induction of CYP2B6 Gene Expression*

Hongbing WangDagger , Stephanie FaucetteDagger , Tatsuya Sueyoshi§, Rick Moore§, Stephen Ferguson§, Masahiko Negishi§, and Edward L. LeCluyseDagger

From the Dagger  Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599 and the § Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, December 9, 2002, and in revised form, February 3, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CYP2B6 plays an important role in the metabolism of a variety of structurally unrelated xenobiotics, including the anticancer drugs cyclophosphamide and ifosfamide. Previous studies have shown that the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are involved in the transcriptional regulation of CYP2B genes through the phenobarbital-responsive enhancer module (PBREM). However, for human CYP2B6 the relatively weak response of the PBREM to PXR and CAR activation in transfection assays fails to describe the potent induction observed in primary human hepatocyte cultures. In this report, a novel nuclear receptor response module located -8.5 kilobases upstream from the CYP2B6 encoding region is described. Several potential PXR/CAR binding motifs were identified within the distal regulatory cluster. In electrophoretic mobility shift assays, one DR4 motif showed the strongest binding to both PXR and CAR. Transient transfection assays in HepG2 cells demonstrated that the novel distal response cluster could be activated by PXR and CAR. In primary human hepatocytes, both PBREM and the distal responsive element were activated individually by endogenous nuclear receptors upon exposure to prototypical inducers. However, in both HepG2 cells and primary human hepatocytes maximal reporter activation was observed in a construct containing both PBREM and the distal responsive element. In mouse tail-vein injection experiments, a construct containing both the distal responsive element and the proximal PBREM exhibited a strong synergistic expression in phenobarbital-treated mice. These results show that a novel xenobiotic-responsive enhancer module in the distal region of the CYP2B6 promoter (CYP2B6-XREM) together with the PBREM mediates optimal drug-induced expression of CYP2B6.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cytochrome P450 (CYP)1 gene superfamily plays a critical role in the biotransformation of structurally diverse classes of xenobiotics, including drugs, environmental pollutants, and endogenous compounds such as steroid hormones, vitamins, and fatty acids (1). Predominantly expressed in liver, members of the CYP1-3 families exhibit broad substrate specificity and metabolize the majority of administered drugs. Although human CYP2B6 was thought historically to play only a minor role in drug metabolism, more recent estimates suggest that CYP2B6 is involved in the metabolism of nearly 25% of drugs on the market today (2), such as the anticancer drugs cyclophosphamide, ifosfamide (3), and tamoxifen (4), the anti-retrovirals efavirenz and nevirapine (5), the anesthetics ketamine and propofol (6, 7), and the central nervous system active agents mephobarbital, bupropion, and selegiline (9, 10).2 Moreover, recent studies using more selective and specific immunochemical detection methods demonstrate that the average relative abundance of CYP2B6 in human liver ranges from 2 to 10% of the total P450 content compared with an earlier report of 0.2% (11-14). In addition, significant interindividual differences in hepatic CYP2B6 expression, which varies in some studies from 25- to 250-fold, have been reported (11, 12, 15). CYP2B6 has been found to be highly inducible by a series of structurally diverse compounds, such as phenobarbital (PB), rifampicin (RIF), clotrimazole (CLZ), phenytoin (PHY), and carbamazepine (16-18),2 thus contributing to the variability in hepatic CYP2B6 content among the human population. Thus, significant drug-drug interactions could occur by induction of this enzyme in patients subjected to combination drug therapy, such as during chemotherapy and treatment for human immunodeficiency virus (19).

Transcriptional activation of CYP2B genes by xenobiotics is mediated by the interaction of ligand-nuclear-receptor complexes with enhancer sequences that lie upstream from the CYP2B gene proximal promoter. Extensive studies by Negishi and coworkers (20, 21) reveal that the constitutive androstane receptor (CAR; NR1I3) regulates the induction of mouse CYP2B gene expression by PB-like compounds through a 51-bp sequence located between -2339 and -2289 of the CYP2B10 promoter region, termed the phenobarbital-responsive enhancer module (PBREM). Similar elements have been identified in the promoters of rat and human CYP2B genes (22). The definitive role of CAR in the xenobiotic-induced expression of rodent CYP2B was shown in CAR knockout mice, wherein total ablation of CYP2B10 induction by PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) was observed (23, 24).

Recent evidence indicates that various orphan nuclear receptors are involved in the regulation of multiple CYPs by recognizing common response elements containing the half-site AGGTCA separated by 3-6 base pairs (25). Recently, the pregnane X receptor (PXR; NR1I2) has been proposed as a xenobiotic-responsive transcription factor that regulates multiple drug metabolizing enzymes and transporters (26-28). Utilizing electrophoretic mobility gel shift assays, in vitro cell-based reporter gene transfection assays, and P450 induction assays in primary human hepatocytes, several labs have demonstrated that PXR can bind to the NR1 and NR2 sites within the CYP2B6 PBREM and that known PXR ligands can effectively induce CYP2B6 expression in primary human hepatocytes (16, 28).2,3

RIF, which is traditionally known as one of the most potent CYP3A4 inducers in humans, is able to induce CYP2B6 up to 20-fold in primary cultures of human hepatocytes (18).3 Clotrimazole, which is a PXR agonist and possible CAR deactivator, increased CYP2B6 expression and activity up to 20-fold in some human hepatocyte cultures.2 Likewise, the prototypical CYP2B inducer PB caused a 30-70-fold induction of both CYP2B6 mRNA and protein in primary human hepatocytes (16).3 Notably, only small increases (2-4-fold) in reporter gene activity have been observed in transfection assays using constructs containing the PBREM alone (16, 28).3 Thus, the potent induction of CYP2B6 gene expression in primary cultures of human hepatocytes cannot be explained entirely by the relatively modest activation of reporter gene constructs containing the PBREM alone.

More recently, several lines of evidence show that PXR and CAR regulate target genes such as MDR1, CYP3A4, and CYP2C9 via distal enhancer modules to achieve optimal transcriptional activation (27, 30, 31). To determine whether a similar module is involved in the PXR and CAR regulation of CYP2B6, which might explain the discrepancy between drug-induced expression of CYP2B6 in primary hepatocytes and PBREM reporter constructs, we searched for and identified a distal xenobiotic-responsive enhancer module that could specifically bind to PXR and CAR and mediate the transcriptional regulation of CYP2B6. Optimal activation profiles were observed using reporter constructs containing both this novel distal module and the PBREM. Moreover, synergistic effects were demonstrated between these two response modules in in vivo transfection assays conducted in mice. The results from these studies demonstrate that a distal response module in the CYP2B6 promoter synergistically functions with the PBREM to achieve maximal drug-induced gene expression.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Rifampicin, phenobarbital, dexamethasone, clotrimazole, phenytoin, and collagenase type IV were purchased from Sigma. Cell culture media and charcoal-stripped fetal calf serum were obtained from Invitrogen. Effectene transfection reagent was obtained from Qiagen, Inc. (Valencia, CA). Dual-Luciferase Reporter Assay System was from Promega Co. (Madison, WI). Oligonucleotides were purchased from Genosys, Inc. (The Woodlands, TX). All other biological reagents were obtained from commercial suppliers and were either American Chemical Society or molecular biology grade.

Plasmid Constructs-- The reporter gene plasmid containing 1.6-kb fragment of the CYP2B6 promoter region was PCR-amplified by using primers 5'-AGCTAAGGTACCTGTCTGCTCCTCCTGGGTC-3' (forward) and 5'-AGTCTACTCGAGCTGCACCCTGCTGCAGCCT-3' (reverse). This product, which lacked the PBREM, was subcloned into the KpnI-XhoI site of pGL3-basic vector, resulting in a construct termed B-1.6k, and the correct orientation was verified by sequencing. A 1.8-kb segment of the CYP2B6 upstream regulatory region from the transcriptional start codon, which included the proximal region of the CYP2B6 promoter and the PBREM, was inserted into pGL3-basic vector using the same strategy and was termed B-1.6k/PBREM. Amplification of a distal xenobiotic responsive enhancer module (XREM) located at -8.5 kb using primers 5'-CGGGG TACCCTTTCTCCATCCACAAAATGG-3' (forward) and 5'-CCGCTCGAGGATGCTGATTCAGGGAATGGA-3' (reverse) generated a 400-bp PCR product that contained all the potential response elements as indicated in Fig. 2. After sequence confirmation, the 400-bp product containing the distal responsive module was subcloned into the B-1.6k vector linearized by KpnI, creating a construct containing the CYP2B6 proximal promoter (1.6 kb) and the distal element, termed B-1.6/XREM. Utilizing a similar strategy, this 400-bp product was also subcloned into the B-1.6k/PBREM vector to generate a construct containing both the PBREM, and the distal XREM, termed B-1.6k/PB/XREM. pRL-TK was used as an internal control.

Site-directed Mutagenesis-- Site-directed mutagenesis of the PBREM and the distal responsive element was performed within the NR1 and NR3 motifs, respectively. The QuikChangeTM site-directed mutagenesis kit (Stratagene, CA) was used to introduce the mutation according to the recommendation of the manufacturer with the following primers: PBREM-NR1 (mutated bases in the center of the half-site are underlined), 5'-GTAAGAGGTGGAAACTCTGGTTTCCTGACCCTGAAG-3'; XREM-NR3, 5'-GGAAAGATGCCACCATCGGGTTTCCTGACCCCAGGA-3'. The PBREM-XREM double mutation was obtained by introducing the NR3 mutation into the NR1-mutated B-1.6k/PB/XREM vector by using the same mutation primers as described above. All mutated constructs were sequence verified.

Transfection Assays in Hepatoma-derived Cell Lines-- HepG2 and Huh7 cells were cultured in Eagle's minimal essential medium with 100 units/ml penicillin and 100 µg/ml streptomycin and supplemented with 10% fetal bovine serum. Cells were seeded into 24-well plates at 5 × 104 cells/well, cultured at 37 °C overnight, and changed to antibiotic-free Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% charcoal-stripped fetal bovine serum. Transfection was performed using Effectene® transfection reagent following the manufacturer's suggestion. Briefly, the transfection mixes consisted of 100 ng of reporter constructs, 25 ng each of human PXR or human CAR expression plasmid, and 10 ng of pRL-TK as an internal control. Transfected HepG2 cells were exposed for 24 h to RIF (10 µM) or PB (1000 µM), which were selected as prototypical PXR and CAR activators, respectively. Huh7 cells were treated for 24 h with the concentrations of PB, RIF, PHY, or CLZ as indicated under "Results." Cells were harvested, and luciferase activities were measured with the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega).

Human Primary Hepatocyte Cultures and Transfection Assays-- Liver tissues were obtained by qualified medical staff following donor consent and prior approval from the Institutional Review Board at the University of North Carolina at Chapel Hill. Hepatocytes were isolated from human liver specimens by a modification of the two-step collagenase digestion method as previously described (32). Hepatocytes were plated at 3.75 × 105 cells/well into Biocoat® 24-well plates in 0.5 ml Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, antibiotics, insulin, and dexamethasone. After 4 h, cell culture medium was changed to Williams E medium containing 6.25 µg/ml insulin, 6.25 µg/ml transferrin, and 6.25 µg/ml selenium (ITS+), and 0.1 µM dexamethasone. Transfection experiments were performed 24 h later as described above, except in the absence of nuclear receptor expression vectors. After RIF (10 µM) and PB (1000 µM) treatment for 24 h, the cells were harvested, and luciferase activities were measured.

Microsome Preparation and Western Blotting-- Primary human hepatocytes were cultured in the presence of PB (100 and 1000 µM), RIF (1 and 10 µM), PHY (50 µM), or CLZ (10 µM) for 72 h. Microsomal CYP2B6 was detected by Western blotting as described previously (33). Blotting densities were measured using NIH imaging software.

Electrophoretic Mobility Shift Assay-- Electrophoretic mobility shift assays were performed as described by Honkakoski et al. (34). Human PXR, CAR, and RXRalpha proteins were synthesized using the TNT quick-coupled in vitro transcription/translation system (Promega). Probes were labeled with [gamma -32P]dATP and purified by Microspin G-25 columns (Amersham Biosciences). Typically, 10 µl of binding reactions contained 10 mM HEPES (pH 7.6), 0.5 mM dithiothreitol, 15% glycerol, 0.05% Nonidet P-40, 50 mM NaCl, 2 µg of poly(dI-dC), 1 µl of in vitro translated nuclear receptor protein, and 4 × 104 cpm of labeled probe. After incubation at room temperature for 10 min, reaction mixtures were resolved on 5% acrylamide gels in 1× Tris-acetic acid, EDTA buffer at 180 V for 1.5 h. Afterward, gels were dried, and autoradiography was performed at -70 °C for overnight.

In Vivo Gene Transfection Assay-- Mice weighing 23-25 g were quarantined for 1 week before use in temperature- and humidity-controlled rooms with a 12-h light/dark cycle. National Institutes of Health 31 rodent chow and tap water were provided ad libitum. All animal care procedures were in accordance with the National Institutes of Health guidelines. CYP2B6 reporter construct (8 µg) and pRL-SV40 vector (2 µg) (internal control vector) were injected through the tail vein using TransIT in vivo gene delivery system (Mirus, Madison, WI) according to the manufacturer's protocol. PB (100 mg/kg of body weight) or an equal volume of saline (controls) was administered intraperitoneally 3 h after gene delivery. Three PB-treated and 3 control animals were sacrificed 16 h after the administration of each CYP2B6 reporter construct. Livers were homogenized in 5 ml of passive lysate buffer (Promega), and 1 µl of the supernatant obtained by centrifugation at 4 °C was utilized for the dual luciferase assay.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Induction of CYP2B6 Expression and Activation of CYP2B6 PBREM by PXR Ligands-- To investigate the extent of CYP2B6 induction by various PXR ligands, primary human hepatocytes were treated with the prototypical CYP2B inducer PB and known PXR activators, such as RIF, PHY, and CLZ. Fig. 1A shows that CYP2B6 is effectively induced by PB (52-fold), RIF (23-fold), PHY (25-fold), and CLZ (17-fold). These results are in agreement with published observations, which together serve to illustrate that CYP2B6 can be highly induced at both the mRNA and protein levels (15, 16). In contrast to the strong induction of CYP2B6 gene expression observed in primary hepatocytes, Huh7 cells co-transfected with the pGL3- tk-PBREM reporter construct and a human PXR expression plasmid exhibited relatively weak reporter activation by PB (2.9-fold), RIF (3.6-fold), PHY (1.4-fold), and CLZ (2.2-fold) (Fig. 1B).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Induction of CYP2B6 by PXR ligands. A, primary cultures of human hepatocytes were treated with RIF (1 and 10 µM), PB (100 and 1000 µM), PHY (50 µM), and CLZ (10 µM) for 72 h. Cell homogenates (25 µg) were loaded for Western immunoblot and densitometric analysis of CYP2B6 as described under "Experimental Procedures." B, a pGL3-tk-LUC reporter construct containing the human PBREM was co-transfected with a human PXR expression vector into Huh7 cells, which were treated with RIF (10 µM), PB (1000 µM), PHY (50 µM), and CLZ (10 µM) for 24 h. Cell homogenates were prepared, and dual-luciferase activities were measured according to the manufacturer's recommendations. Three independent measures from each treatment were analyzed.

Identification of a Functional Distal PXR/CAR-responsive Element in the CYP2B6 Promoter-- Because of the significant induction of CYP2B6 gene expression in primary hepatocytes and the relatively weak activation of PBREM reporter activity in cell lines by CYP2B6 inducers, we hypothesized that an additional distal PXR/CAR-responsive module existed in the CYP2B6 gene promoter. Utilizing the GCG Wisconsin Package Findpatterns Tool (Accelrys, Princeton, NJ), -10 kb of the CYP2B6 promoter sequence was analyzed for potential PXR/CAR binding sites. A cluster of potential PXR/CAR binding sites was identified at approximately -8.5 kb from the CYP2B6 initiation codon. This cluster included two DR4, one IR6, one ER5, and one DR2 motif. In keeping with the nomenclature utilized to describe the NR1 and NR2 binding sites found within the PBREM, the novel distal motifs were designated as NR3-NR8 as shown in Fig. 2A. Compared with the previously identified CYP2B and CYP3A nuclear receptor response elements, NR3 exhibited the highest sequence homology to the NR1 site in the human PBREM. A single nucleotide shift from T to G was observed at position -8558 (Fig. 2B).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2.   Location and sequence of the CYP2B6 XREM-like module identified through a computer-based search of the -10-kb upstream region of the CYP2B6 gene. Between -8.5 and -8.6 kb a cluster of consensus half-sites were identified. In accordance with the NR1 and NR2 motifs within the PBREM, these novel motifs were termed NR3-NR8 (A). The sequence of NR3 and other known binding motifs from PXR/CAR target genes were compared (B).

To characterize the nuclear receptor binding capacity of these distal elements, electrophoretic mobility shift assays were performed. The results showed that the PXR/RXRalpha heterodimer binds specifically to NR3 and NR8 motifs (Fig. 3A). Competition assays demonstrated that PXR/RXRalpha binding to either motif could be specifically blocked with excess unlabeled probe (Fig. 3A). Likewise, the CAR/RXRalpha heterodimer bound strongly to the NR3 and NR8 motifs (Fig. 3B). As with the PXR/RXRalpha heterodimer, the binding of CAR/RXRalpha to either motif was blocked effectively by excess cold competitor (Fig. 3B). In addition, the CAR/RXRalpha heterodimer bound weakly to the NR7 motif, which was not observed with the PXR/RXRalpha heterodimer. The other potential elements (NR4, NR5, and NR6) did not bind to either the PXR/RXR± or CAR/RXR± heterodimers.


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 3.   PXR/RXR and CAR/RXR heterodimers bind to CYP2B6-XREM motifs. A, PXR/RXR binding to distal elements NR3 to NR8 as defined in Fig. 2. B, CAR/RXR binding to distal elements NR3 to NR8. Oligonucleotides were labeled with [gamma -32P]dATP, nuclear receptors were synthesized in vitro, and electrophoretic mobility shift assays were performed as described under "Experimental Procedures." Unlabeled oligonucleotides were used as cold competitor (CC).

PXR and CAR Activate the Distal and Proximal CYP2B6 Response Elements in HepG2 Cells-- To address the functional relevance of the distal PXR/CAR binding elements and the proximal PBREM, different chimeric reporter constructs were generated as described under "Experimental Procedures." In these studies, the pGL3-basic vector, which lacks an exogenous promoter, was selected as the empty vector to subclone -1.6 kb of the CYP2B6 promoter. All other constructs were built on this basal CYP2B6 promoter construct. HepG2 cells co-transfected with a human PXR expression vector and different CYP2B6 constructs were exposed to solvent alone (0.05% Me2SO) or 10 µM RIF. As indicated in Fig. 4, RIF treatment resulted in a 2-, 10-, 8-, and 20-fold increase in the expression of the B-1.6k, B-1.6k/PBREM, B-1.6k/XREM, and B-1.6k/PB/XREM reporter constructs, respectively. It is clear that both the PBREM and distal PXR/CAR-responsive module can be activated by PXR ligands and that maximum activation was observed in the reporter construct containing both modules.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Maximal activation of the CYP2B6 promoter by rifampicin requires the distal XREM-like module. Different CYP2B6 upstream constructs generated as described under "Experimental Procedures" are shown on the left. HepG2 cells were transiently co-transfected with CYP2B6 reporter vectors and human PXR expression vector except B-1.6kb*, which was transfected with B-1.6kb without PXR. Eighteen hours after transfection, HepG2 cells were treated with RIF (10 µM) or 0.05% Me2SO for 24 h. Dual luciferase activities were measured in cell extracts according to the manufacturer's recommendations. Three independent measures from each treatment were analyzed.

In site-directed mutagenesis studies, B-1.6k/PBREM-NR1mut and B-1.6k/XREM-NR3mut reporter activity increased only 2.5-fold over control in HepG2 cells treated with RIF, whereas no activation was observed in the construct containing the double mutation of NR1 and NR3 (Fig. 4). Mutation of either the NR1 or NR3 motifs alone in the B-1.6k/PB/XREM construct resulted in 8- and 9-fold increases in reporter expression, respectively.

In HepG2 cells co-transfected with different amounts of human CAR vector (0, 10, 50, and 100 ng) and the B-1.6k/PB/XREM reporter, corresponding increases in reporter activity, were observed with increasing amounts of CAR expression (Fig. 5A). As expected, PB treatment did not further increase reporter activity (22). Because of the constitutive activation of CAR in HepG2 cells, similar activation patterns were observed in cells co-transfected with CAR in the absence of inducers as compared with those in cells co-transfected with PXR and treated with RIF (Fig. 5B). Likewise, in HepG2 cells the B-1.6k/PB/XREM reporter containing both the PBREM and XREM exhibited the highest activation levels by constitutively activated CAR (up to 26-fold) compared with the B-1.6k construct.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Transactivation of CYP2B6 promoter modules by human CAR in HepG2 cells. Different CYP2B6 constructs and a human CAR expression vector were co-transfected into HepG2 cells as described under "Experimental Procedures." A, CAR expression vector from 0 to 100 ng was co-transfected with 100 ng of B-1.6k/PB/XREM containing both the PBREM and the distal XREM. Transfected cells were treated with PB (1000 µM) or vehicle alone. B, different CYP2B6 reporter constructs and mutated vectors (100 ng) were co-transfected with 50 ng of CAR expression vector, except one negative control, which was transfected with the B-1.6k vector without CAR, as indicated by B-1.6kb*. Luciferase reporter activities were determined as described under "Experimental Procedures."

Activation of CYP2B6 Promoter Constructs by Endogenous Nuclear Receptors in Primary Human Hepatocytes-- To further evaluate the function of the CYP2B6 enhancer modules, the activities of the different reporter constructs were determined in primary cultures of human hepatocytes in the absence of exogenous nuclear receptor. These experiments were designed to elucidate how endogenous nuclear receptors and other cellular factors within primary human hepatocytes might affect the expression of the various CYP2B6 promoter constructs. As shown in Fig. 6, treatment with 10 µM RIF increased the expression of B-1.6k/PBREM, B-1.6k/XREM, and B-1.6k/PB/XREM by 4-, 4-, and 9-fold, respectively. PB increased the expression of B-1.6k/PBREM, B-1.6k/XREM, and B-1.6k/PB/XREM by 4-, 1.2-, and 7-fold, respectively. These results indicate that the distal XREM-like module could be transactivated by endogenous nuclear receptors, most likely PXR and CAR, in primary human hepatocytes treated with known ligands. As expected, the B-1.6k/PB/XREM construct with both the PBREM and the distal PXR/CAR-responsive module was maximally activated by RIF and PB.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Transactivation of CYP2B6 reporter constructs by endogenous nuclear receptors in primary human hepatocytes. Primary human hepatocytes were cultured in biocoated 24-well plates and transfected with 250 ng of CYP2B6 reporter vectors and 25 ng of pRL-TK internal control vector using Effectene® reagent. After 24 h, cells were treated with RIF (10 µM), PB (1000 µM), or solvent alone (0.05% Me2SO) for an additional 24 h. Luciferase activities were measured according to the manufacturer's recommendations. Triplicate samples were performed for each treatment.

Expression of CYP2B6 Promoter Constructs in Vivo-- Mouse tail-vein gene delivery techniques as described by Zelko et al. (35) were utilized in these studies to examine whether the various CYP2B6 promoter constructs could be transactivated by PB in vivo. Compared with animals receiving vehicle alone, PB administration resulted in significant increases in the expression of CYP2B6 reporter constructs containing the PBREM or the distal XREM-like response module (B-1.6k/PBREM, 9-fold; B-1.6k/XREM, 8-fold) (Fig. 7). Notably, significantly enhanced reporter expression was observed in mice injected with constructs containing both the PBREM and the distal XREM module (B-1.6k/PB/XREM, 40-fold) after PB treatment (Fig. 7). These data indicate that the PBREM and the novel distal PXR/CAR-responsive module are capable of operating synergistically under in vivo conditions.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7.   Expression of various CYP2B6 reporter constructs in mouse liver in vivo. Various CYP2B6 promoter constructs were injected into mice via the tail-vein by using the TransIT In Vivo Gene Delivery System as described under "Experimental Procedures." Three hours after the injection, PB (100 mg/kg of body weight) or saline was administered intraperitoneally. Animals were sacrificed 16 h after the treatment, and dual luciferase assays were performed on liver lysates. For each construct and treatment group, three animals were independently analyzed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A growing body of evidence suggests that human CYP2B6 plays a major role in the clearance of a number of drugs. The fact that it is highly inducible by chemicals, such as phenobarbital, rifampicin, clotrimazole, phenytoin, and carbamazepine, underscores the importance of understanding its involvement in drug-drug interactions in patients subjected to combination drug therapy. In this report, we have demonstrated that CYP2B6 is highly induced in primary cultures of human hepatocytes by PB, RIF, PHY, and CLZ. Our results confirm and expand on those that have been reported by several other laboratories (16, 28).

Although it has been a topic of intense debate for more than a decade, the molecular determinants of xenobiotic-induced expression of the CYP2B genes are only now becoming fully appreciated (2, 16, 34). Induction of CYP2B gene expression is predominantly regulated at the transcriptional level. Most findings to date suggest that induction of CYP2B genes by inducers is mediated by activation of the nuclear receptors CAR and/or PXR through the PBREM located approximately -2 kb upstream from the CYP2B gene transcriptional start site. Several studies demonstrate that CAR and PXR can mutually bind to and activate response elements in the promoter regions of several P450 genes, suggesting that cross-talk occurs between these receptors in the regulation of these genes (16, 28, 36). In contrast to the potent induction of the CYP2B6 gene observed in primary hepatocytes, relatively weak activation of PBREM reporter constructs by prototypical inducers (PB, RIF, PHY, and CLZ) was observed in the present study. In agreement with this observation, Goodwin et al. (16) report that RIF resulted in only a 2.5-fold activation of PBREM reporter constructs in Huh7 cells. Similarly, in CV-1 cells only 3-fold activation of PBREM reporter gene expression was observed by human PXR after RIF treatment (28). The relatively weak response of the PBREM to PXR and CAR ligands or activators in in vitro cell-based transfection assays fails to correspond with the potent induction profiles observed with the same compounds in primary human hepatocyte cultures, suggesting that other cofactors or response elements may be involved.

Recently, several studies demonstrate that distal response elements are located on several PXR and/or CAR target genes, such as CYP3A4, MDR1, and CYP2C9 (27, 30, 31, 37). Considering the discrepancy between the potent induction of CYP2B6 gene in primary hepatocytes and the relatively weak activation of PBREM, we hypothesized that an additional distal nuclear receptor-responsive element might exist that would be functionally involved in the regulation of CYP2B6. After computer analysis of the -10-kb upstream region of the CYP2B6 promoter, a PXR/CAR-responsive element at -8.5 kb was identified that contained several potential nuclear receptor binding motifs, including DR4 and IR6 elements. Notably, a subsequent computer-based search revealed that this cluster of motifs is unique out to -20 kb upstream from the CYP2B6-encoding region. Among these motifs, NR3 and NR8 exhibited the greatest capacity to bind CAR and PXR. Because the NR8 included both the NR3 and an adjacent AGGTCC half-site (Fig. 2), the binding capacity of PXR/RXRalpha and CAR/RXRalpha heterodimers to the NR8 most likely is derived mainly from the NR3 motif. Moreover, the NR3 has the most sequence homology to the NR1 motif within the human PBREM, therefore suggesting that the NR3 element plays a pivotal role in this distal-responsive module in mediating the nuclear receptor response.

Linkage of a 400-bp cluster containing the NR3 and other identified motifs to the CYP2B6 1.6-kb promoter revealed that this XREM-like module could be activated by PXR or CAR to a similar degree as the PBREM after transfection into HepG2 cells. Of importance, maximal activation of the reporter gene was achieved with a construct containing both the proximal PBREM and the distal XREM. These findings indicate that the distal responsive element is involved in PXR/CAR-mediated transactivation of CYP2B6 gene transcription and suggests that optimal gene regulation may require both elements.

Site-directed mutagenesis of the NR3 motif in the B-1.6k/XREM construct reduced reporter activation by CAR or PXR to the level of the B-1.6k construct. This result is significant because it demonstrates that, within the distal response element, NR3 alone appears to be sufficient to confer CAR/PXR-mediated reporter activation. In addition, mutation of NR1 and NR3 individually or simultaneously in the B-1.6k/PB/XREM vector showed that both elements are involved in CAR- and PXR-mediated transactivation of CYP2B6 and that only the double mutation totally eliminated CAR and PXR activation of the reporter gene. These results further demonstrate that NR1 and NR3 in combination contribute predominantly to the overall transcriptional activation of the CYP2B6 gene by drugs and other xenobiotics.

In contrast to transformed cell lines, primary cultures of human hepatocytes represent the most reliable in vitro model for evaluating the xenobiotic-mediated induction of CYP in human liver (29, 34, 38). One possible explanation for this phenomenon is that primary hepatocytes retain most of the endogenous cellular and nuclear cofactors that are essential for normal liver function. The current study is the first report demonstrating that both the PBREM and the distal XREM are activated by endogenous nuclear receptors expressed in a primary human hepatocyte culture system. Notably, maximal activation was observed with the B-1.6k/PB/XREM construct that contains both the proximal PBREM and distal XREM. These results further indicate that the two enhancer modules act cooperatively to confer full xenobiotic-induced expression of the CYP2B6 gene as mediated by endogenous nuclear receptors and cofactors.

To further investigate the role of the XREM in regulating CYP2B6 expression, we determined the activation profiles of different CYP2B6 promoter constructs by PB in vivo by conducting mouse tail-vein gene delivery experiments. Both the PBREM and the distal XREM were activated equivalently by endogenous nuclear receptors after exposure to PB. More importantly, however, a synergistic activation of the CYP2B6 reporter construct containing both the PBREM and distal responsive element was observed. Although these results were conducted in mice, this is the first study to demonstrate that, under in vivo conditions, the distal response element functions more efficiently in conjunction with the proximal PBREM to exert maximal drug-induced transactivation of the CYP2B6 gene.

The observed synergy between the PBREM and distal elements in our studies is in accordance with an earlier report on CYP3A4, in which a distal XREM located -7 kb upstream from the CYP3A4-coding region was found to mediate PXR-dependent induction in cooperation with a proximal ER6 element (30). In addition, Geick et al. (27) recently reported a distal responsive element at -8 kb upstream of the MDR1 gene that is responsible for the PXR-mediated induction of intestinal MDR1 by RIF. In two recent reports on the nuclear receptor regulation of CYP2C9, response elements located at -2 and -3 kb upstream from the CYP2C9-coding region exhibited activation by CAR but not by PXR (31, 37). Notably, RIF is an efficacious inducer of CYP2C9 both in vivo and in primary human hepatocytes (8). With this discrepancy between the induction of CYP2C9 gene expression by PXR activators observed in primary hepatocytes and the lack of response in reporter constructs containing only the proximal 3-kb promoter sequence, it has been proposed that another PXR-responsive element might be present in the distal CYP2C9 promoter region that has not yet been identified (37). Collectively, these observations suggest that the existence and location of these PXR/CAR distal response elements are critical for optimal xenobiotic-responsive induction of drug-metabolizing enzymes and transporters mediated by these nuclear receptors.

In summary, we have identified a novel cluster of response elements located -8.5 kb upstream from the CYP2B6-encoding region that is involved in PXR- and CAR-mediated transcriptional activation of CYP2B6 gene expression. Within this distal cluster of half-sites lies a single DR4 response element (NR3), which is nearly identical to the DR4 element in the human PBREM (NR1) and which bestows most, if not all, of the nuclear receptor-mediated binding and activation properties of the entire module. Moreover, these results demonstrate that the distal XREM-like module operates synergistically with the proximal PBREM for full xenobiotic-induced expression of the CYP2B6 gene.

    ACKNOWLEDGEMENTS

We thank Dr. Binfang Yan (College of Pharmacy, University of Rhode Island) for kindly providing human PXR vector. Human liver tissue was procured with the assistance of Drs. Benjamin Calvo and Kevin Behrns, University of North Carolina at Chapel Hill Hospitals, and Lynn Johnson and Evageline Reynolds of the Lineberger Comprehensive Cancer Center Tissue Procurement Program, University of North Carolina.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants P30 ES10126 and P30 DK34987.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 919-966-9104; Fax: 919-966-0197; E-mail: ed_lecluyse@unc.edu.

Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M212482200

2 S. Faucette, H. Wang, G. Hamilton, S. L., Jolley, D. Gilbert, and E. L. LeCluyse, submitted for publication.

3 Wang, H., Faucette, S. R., Gilbert, D., Jolley, S. L., Sueyoshi, T., Negishi, M., and LeCluyse, E. L. (2003) Drug Metab. Dispos., in press.

    ABBREVIATIONS

The abbreviations used are: CYP, cytochrome P450; PB, phenobarbital; RIF, rifampicin; PHY, phenytoin; CLZ, clotrimazole; PXR, pregnane X receptor; CAR, constitutive androstane receptor; PBREM, phenobarbital responsive enhancer module; XREM, xenobiotic-responsive enhancer module; DR4, direct repeat separated by 4 base pairs; ER6, everted repeat separated by 6 base pairs; kb, kilobase(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Parkinson, A. (2001) in The Basic Science of Poisons (Klaassen, C. D., ed), Vol. 5 , pp. 113-186, McGraw-Hill Inc., New York
2. Xie, W., and Evans, R. M. (2001) J. Biol. Chem. 276, 37739-37742[Free Full Text]
3. Roy, P., Yu, L. J., Crespi, C. L., and Waxman, D. J. (1999) Drug Metab. Dispos. 27, 655-666[Abstract/Free Full Text]
4. Sridar, C., Kent, U. M., Notley, L. M., Gillam, E. M., and Hollenberg, P. F. (2002) J. Pharmacol. Exp. Ther. 301, 945-952[Abstract/Free Full Text]
5. Erickson, D. A., Mather, G., Trager, W. F., Levy, R. H., and Keirns, J. J. (1999) Drug Metab. Dispos. 27, 1488-1495[Abstract/Free Full Text]
6. Court, M. H., Duan, S. X., Hesse, L. M., Venkatakrishnan, K., and Greenblatt, D. J. (2001) Anesthesiology 94, 110-119[Medline] [Order article via Infotrieve]
7. Oda, Y., Hamaoka, N., Hiroi, T., Imaoka, S., Hase, I., Tanaka, K., Funae, Y., Ishizaki, T., and Asada, A. (2001) Br. J. Clin. Pharmacol. 51, 281-285[CrossRef][Medline] [Order article via Infotrieve]
8. Gerbal-Chaloin, S., Pascussi, J. M., Pichard-Garcia, L., Daujat, M., Waechter, F., Fabre, J. M., Carrere, N., and Maurel, P. (2001) Drug Metab. Dispos. 29, 242-251[Abstract/Free Full Text]
9. Kobayashi, K., Abe, S., Nakajima, M., Shimada, N., Tani, M., Chiba, K., and Yamamoto, T. (1999) Drug Metab. Dispos. 27, 1429-1433[Abstract/Free Full Text]
10. Hidestrand, M., Oscarson, M., Salonen, J. S., Nyman, L., Pelkonen, O., Turpeinen, M., and Ingelman-Sundberg, M. (2001) Drug Metab. Dispos. 29, 1480-1484[Abstract/Free Full Text]
11. Code, E. L., Crespi, C. L., Penman, B. W., Gonzalez, F. J., Chang, T. K., and Waxman, D. J. (1997) Drug Metab. Dispos. 25, 985-993[Abstract/Free Full Text]
12. Ekins, S., Vandenbranden, M., Ring, B. J., Gillespie, J. S., Yang, T. J., Gelboin, H. V., and Wrighton, S. A. (1998) J. Pharmacol. Exp. Ther. 286, 1253-1259[Abstract/Free Full Text]
13. Hanna, I. H., Reed, J. R., Guengerich, F. P., and Hollenberg, P. F. (2000) Arch. Biochem. Biophys. 376, 206-216[CrossRef][Medline] [Order article via Infotrieve]
14. Hesse, L. M., Venkatakrishnan, K., Court, M. H., von Moltke, L. L., Duan, S. X., Shader, R. I., and Greenblatt, D. J. (2000) Drug Metab. Dispos. 28, 1176-1183[Abstract/Free Full Text]
15. Gervot, L., Rochat, B., Gautier, J. C., Bohnenstengel, F., Kroemer, H., de Berardinis, V., Martin, H., Beaune, P., and de Waziers, I. (1999) Pharmacogenetics 9, 295-306[Medline] [Order article via Infotrieve]
16. Goodwin, B., Moore, L. B., Stoltz, C. M., McKee, D. D., and Kliewer, S. A. (2001) Mol. Pharmacol. 60, 427-431[Abstract/Free Full Text]
17. Pascussi, J. M., Gerbal-Chaloin, S., Fabre, J. M., Maurel, P., and Vilarem, M. J. (2000) Mol. Pharmacol. 58, 1441-1450[Medline] [Order article via Infotrieve]
18. LeCluyse, E. L. (2001) Eur. J. Pharm. Sci. 13, 343-368[CrossRef][Medline] [Order article via Infotrieve]
19. van Heeswijk, R. P., Veldkamp, A., Mulder, J. W., Meenhorst, P. L., Lange, J. M., Beijnen, J. H., and Hoetelmans, R. M. (2001) Antivir. Ther. 6, 201-229[Medline] [Order article via Infotrieve]
20. Honkakoski, P., and Negishi, M. (1998) J. Biochem. Mol. Toxicol. 12, 3-9[CrossRef][Medline] [Order article via Infotrieve]
21. Kawamoto, T., Sueyoshi, T., Zelko, I., Moore, R., Washburn, K., and Negishi, M. (1999) Mol. Cell. Biol. 19, 6318-6322[Abstract/Free Full Text]
22. Sueyoshi, T., Kawamoto, T., Zelko, I., Honkakoski, P., and Negishi, M. (1999) J. Biol. Chem. 274, 6043-6046[Abstract/Free Full Text]
23. Ueda, A., Hamadeh, H. K., Webb, H. K., Yamamoto, Y., Sueyoshi, T., Afshari, C. A., Lehmann, J. M., and Negishi, M. (2002) Mol. Pharmacol. 61, 1-6[Abstract/Free Full Text]
24. Wei, P., Zhang, J., Egan-Hafley, M., Liang, S., and Moore, D. D. (2000) Nature 407, 920-923[CrossRef][Medline] [Order article via Infotrieve]
25. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., and Chambon, P. (1995) Cell 83, 835-839[Medline] [Order article via Infotrieve]
26. Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Backman, M., Ohlsson, R., Postlind, H., Blomquist, P., and Berkenstam, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12208-12213[Abstract/Free Full Text]
27. Geick, A., Eichelbaum, M., and Burk, O. (2001) J. Biol. Chem. 276, 14581-14587[Abstract/Free Full Text]
28. Xie, W., Barwick, J. L., Simon, C. M., Pierce, A. M., Safe, S., Blumberg, B., Guzelian, P. S., and Evans, R. M. (2000) Genes Dev. 14, 3014-3023[Abstract/Free Full Text]
29. Li, A. P., Maurel, P., Gomez-Lechon, M. J., Cheng, L. C., and Jurima-Romet, M. (1997) Chem. Biol. Interact. 107, 5-16[CrossRef][Medline] [Order article via Infotrieve]
30. Goodwin, B., Hodgson, E., and Liddle, C. (1999) Mol. Pharmacol. 56, 1329-1339[Abstract/Free Full Text]
31. Ferguson, S. S., LeCluyse, E. L., Negishi, M., and Goldstein, J. A. (2002) Mol. Pharmacol. 62, 737-746[Abstract/Free Full Text]
32. Hamilton, G. A., Jolley, S. L., Gilbert, D., Coon, D. J., Barros, S., and LeCluyse, E. L. (2001) Cell Tissue Res. 306, 85-99[CrossRef][Medline] [Order article via Infotrieve]
33. Sahi, J., Hamilton, G., Sinz, M., Barros, S., Huang, S. M., Lesko, L. J., and LeCluyse, E. L. (2000) Xenobiotica 30, 273-284[CrossRef][Medline] [Order article via Infotrieve]
34. Honkakoski, P., Zelko, I., Sueyoshi, T., and Negishi, M. (1998) Mol. Cell. Biol. 18, 5652-5658[Abstract/Free Full Text]
35. Zelko, I., Sueyoshi, T., Kawamoto, T., Moore, R., and Negishi, M. (2001) Mol. Cell. Biol. 21, 2838-2846[Abstract/Free Full Text]
36. Goodwin, B., Hodgson, E., D'Costa, D. J., Robertson, G. R., and Liddle, C. (2002) Mol. Pharmacol. 62, 359-365[Abstract/Free Full Text]
37. Gerbal-Chaloin, S., Daujat, M., Pascussi, J. M., Pichard-Garcia, L., Vilarem, M. J., and Maurel, P. (2002) J. Biol. Chem. 277, 209-217[Abstract/Free Full Text]
38. Maurel, P. (1996) Adv. Drug Deliv. Rev. 22, 105-132[CrossRef]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.