From the 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
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ABSTRACT |
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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 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 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.
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 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 RXR 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.
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).
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),
To characterize the nuclear receptor binding capacity of these distal
elements, electrophoretic mobility shift assays were performed. The
results showed that the PXR/RXR 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
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.
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.
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.
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 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 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 In summary, we have identified a novel cluster of response elements
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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
proteins were
synthesized using the TNT quick-coupled in vitro
transcription/translation system (Promega). Probes were labeled with
[
-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.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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).
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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).
heterodimer binds specifically to
NR3 and NR8 motifs (Fig. 3A).
Competition assays demonstrated that PXR/RXR
binding to either motif
could be specifically blocked with excess unlabeled probe (Fig.
3A). Likewise, the CAR/RXR
heterodimer bound strongly to
the NR3 and NR8 motifs (Fig. 3B). As with the PXR/RXR
heterodimer, the binding of CAR/RXR
to either motif was blocked
effectively by excess cold competitor (Fig. 3B). In
addition, the CAR/RXR
heterodimer bound weakly to the NR7 motif,
which was not observed with the PXR/RXR
heterodimer. The other
potential elements (NR4, NR5, and NR6) did not bind to either the
PXR/RXR± or CAR/RXR± heterodimers.
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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 [ -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).
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.
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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.
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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."
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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.
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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
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.
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/RXR
and CAR/RXR
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.
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.
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.
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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.
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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.
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ABBREVIATIONS |
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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).
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