From the Department of Biochemistry and Molecular
Biology, University College London, Gower Street, London WC1E 6BT,
United Kingdom and the
School of Biological Sciences, Queen
Mary, University of London, Mile End Road, London E1 4NS, United
Kingdom
Received for publication, July 6, 2000, and in revised form, January 23, 2001
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ABSTRACT |
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The mechanisms by which different classes of
chemicals induce the same cytochrome P450 (CYP) or the same chemical
differentially induces more than one CYP are not well understood. We
show that in primary hepatocytes and in vivo in liver
(transfected by particle-mediated delivery) two orphan nuclear
receptors, constitutive androstane receptor and pregnane X
receptor (PXR1), transactivate a CYP gene via the same
response element in a xenobiotic-specific manner. The constitutive
androstane receptor mediates the barbiturate activation of expression
of CYP2B1 and CYP3A1. PXR1 activates both genes
in response to synthetic steroids. To exert their effect the receptors
bind to the same direct repeat site (DR4) within the phenobarbital
response element of the CYP2B1 promoter and to the same DR3
site in the pregnane X response element of CYP3A1. The
receptors are therefore promiscuous with respect to DNA binding but not
ligand binding. Differences in enhancer half-site spacing may influence
the efficiency of interactions between the receptor and the
transcription machinery and hence form the basis for the differential
induction of CYP2B1 and CYP3A1 in response to barbiturates and
synthetic steroids.
Cytochromes P450 (CYPs)1
are a superfamily of proteins, members of which catalyze the metabolism
of a wide range of endogenous and exogenous chemicals (1). CYP-mediated
detoxification is a key defense mechanism whereby organisms protect
themselves from the potentially harmful effects of foreign hydrophobic
chemicals to which they are exposed. The xenobiotics are converted to
more hydrophilic compounds, which are then more easily excreted (2). Many of these xenobiotics serve as inducers of the particular CYP
required for their metabolism. Induction of CYPs is mediated primarily
at the transcriptional level by the interaction of ligand-nuclear receptor complexes with enhancer sequences that lie upstream of CYP gene promoters (reviewed in Refs. 3 and 4). A single CYP
can be induced to different extents by different classes of chemicals,
and a single chemical can differentially induce more than one CYP. For
instance, CYP2B1 and CYP3A1, members of two different CYP subfamilies,
can both be induced in the liver by the barbiturate phenobarbital (PB)
and by the structurally unrelated synthetic pregnane pregnenolone
16 Here we show that CAR and PXR1 can transactivate the CYP2B1
gene by interacting with the same direct repeat 4 (DR4) element of the
PBRE. Similarly, the receptors transactivate the CYP3A1 gene
by binding to the same DR3 element of the PXRE. In each case, the
receptors transactivate gene expression in a xenobiotic-specific manner; PXR1 is activated by PCN (but not by PB) and CAR by PB (but not
by PCN). The two orphan nuclear receptors thus exhibit promiscuity with
respect to DNA binding but not ligand binding. The significance of the
results for our understanding of the mechanisms that mediate
differential induction of CYPs by xenobiotics is discussed.
Plasmids--
The reporter plasmid (CYP2B1
PBRE)-SV40-luc was constructed by inserting the CYP2B1 PBRE
sequence ( Cell Culture and Transfection--
Primary rat hepatocytes
cultured on Matrigel (1 mg/ml) (Becton Dickinson) (5 × 106 cells/60-mm plate) were transfected using
TfxTM-20 reagent (Promega) according to the supplier's
recommendations. Transfection mixtures (6 ml) contained 2.5 µg of
reporter plasmid, 0.25 µg of control plasmid, and 0.25 µg of an
expression plasmid. Cell extracts were assayed for luciferase
(Dual-Luciferase reporter assay system), chloramphenicol
acetyltransferase (CAT enzyme assay system), or In Vivo Transfection of Liver by Biolistic Particle
Delivery--
Gold particles, 25 mg (1 micron, Bio-Rad), were coated
with 100 µg of DNA. The ratios of (CYP2B1 PBRE)-SV40-luc,
pRL-TK and the expression plasmid (either cDM8-mCAR- Gel Mobility Shift Assays--
Nuclear protein extracts were
prepared from the livers of untreated and PB-treated rats as described
previously (14, 15). CAR, PXR1, and RXR
To assess binding of nuclear receptor/RXR CAR and PXR1 Transactivate CYP2B1 Expression via the PBRE in
Response to PB and PCN, Respectively--
In primary rat hepatocytes,
both PB and PCN increase the expression of a reporter gene under the
control of the CYP2B1 PBRE (Fig.
1A), suggesting that induction
of CYP2B1 by PCN is mediated by the interaction of either CAR or the
ligand's cognate receptor, PXR1, with the PBRE. To investigate this
possibility, we tested the effect of the xenobiotics on the
expression of the CYP2B1 PBRE-reporter construct in
hepatocytes cotransfected with an expression vector for either CAR or
PXR1 (Fig. 1A). Reporter gene expression was constitutively
increased almost 5-fold by CAR but not by PXR1. In cells cotransfected
with a PXR1 expression vector, reporter gene expression was increased
6-fold by PCN but not by PB. In cells cotransfected with a CAR
expression vector, PB increased gene expression more than 3-fold above
the level mediated constitutively by CAR, whereas PCN had no
effect. Therefore induction of CYP2B1 gene expression in
response to PB and PCN is mediated, respectively, by the interaction of
CAR and PXR1 with the PBRE.
We next determined whether CAR and PXR1 bind to the same or
different sites within the PBRE. CYP2B PBREs contain two
DR4 nuclear receptor half-sites, NR1 (5'-TGTACT TTCC TGACCT-3')
and NR2 (5'-TCAACT TTCC TGACCT-3'), separated by a NF-1 (nuclear
factor-1) site (8, 9, 16). PXR1, produced by in vitro
transcription/translation, was found to bind, as a heterodimer with
RXR
Complexes formed between the NR1 site and liver nuclear proteins were
competed by the PXRE of CYP3A1 and were supershifted by
antibodies against PXR1 and RXR
To determine the effect of the receptors on CYP2B1
expression in vivo we performed transfection experiments in
liver (Fig. 1D). A CYP2B1 PBRE-reporter plasmid,
together with expression vectors for either PXR1 or CAR, was introduced
into rat liver by biolistic particle-mediated DNA transfer.
Reporter gene expression was increased constitutively by CAR but not by
PXR1. The CAR-mediated transactivation of reporter gene expression was
increased further in response to PB but not to PCN. Conversely, the
presence of PXR1 stimulated the induction of gene expression by PCN,
whereas it decreased the effect of PB, presumably by competition with endogenous CAR for binding to the PBRE (see Fig.
2F). The results are
qualitatively similar to those obtained in hepatocyte cultures (see
Fig. 1A) and confirm that in vivo both PXR1 and
CAR are able to transactivate gene expression via interaction with the
same CYP2B1 enhancer element (the NR1 of the PBRE) in
response to PCN and PB, respectively. Hence, the regulation of
CYP2B1 expression by xenobiotics involves cross-talk between
ligand-receptor complexes.
We next investigated the relative affinity with which CAR and PXR bind
to the DR4 CYP2B1 NR1 and DR3 CYP3A1 PXRE sites.
Competitive gel shift assays were performed in which constant amounts
of in vitro transcribed and translated CAR and RXR
The ability of CAR and PXR1 to bind to the same response element,
albeit with different affinities, suggests that the receptors will
compete with each other for binding to this site in vivo and
thus exhibit functional cross-talk. This theory was supported by
the finding that CAR-mediated transactivation of a CYP2B1
PBRE-reporter construct in response to PB was inhibited by PXR1 in a
dose-dependent manner (Fig. 2F). Reporter gene
activity was decreased by about 60% when PXR and CAR expression
plasmids were present in equimolar amounts and by as much as 80% by a
5-fold molar excess of PXR over CAR plasmid. As a control for
squelching, hepatocytes were cotransfected with expression vectors for
CAR and RAR, another nuclear receptor that heterodimerizes with RXR CAR and PXR1 Transactivate CYP3A1 Expression via the PXRE in
Response to PB and PCN, Respectively--
In response to PCN, PXR1
stimulates CYP3A1 gene expression by binding to a DR3 PXRE
(5'-TGAACT TCA TGAACT-3') (10, 13). The PXRE of CYP3A1 and
the NR1 of the CYP2B1 PBRE were found to compete in gel
mobility shift assays for binding to rat liver nuclear proteins (Fig.
1C). Furthermore, in vitro synthesized CAR bound
as a heterodimer with RXR The mechanisms by which different classes of chemicals induce the
same CYP or the same chemical differentially induces more than one CYP
are not well understood. Here we show that in primary hepatocytes and
in vivo in the liver two orphan nuclear receptors, CAR and
PXR1, transactivate a CYP gene via the same response element in a xenobiotic-specific manner. CAR mediates barbiturate activation of
expression of CYP2B1 and CYP3A1. PXR1 activates
both genes in response to synthetic steroids. To exert their effect the
receptors bind to the same DR4 site within the PBRE of the
CYP2B1 promoter and to the same DR3 site in the PXRE of
CYP3A1. The receptors are therefore promiscuous with respect
to DNA binding.
In vivo CYP3A1 is less inducible (3-fold) by PB than is
CYP2B1 (50-to 100-fold) (6, 19). Similarly, PCN is a less potent inducer of CYP2B1 (3-fold) than of CYP3A1 (10-to 20-fold) (6). The
deletion of a single base pair from a nuclear receptor half-site spacer
region will alter the displacement (by 3.4 Å) and the rotation angle
(by 36o) between the two half-site complexes (20). Thus,
the dimerization interface between CAR and RXR It has been shown (21) that in HepG2 cells stably transfected with CAR,
the expression of a reporter gene under the control of the human
CYP3A4 PXRE, an everted repeat-6 (ER6) element, was down-regulated by 16 (5 Several models can be proposed to explain how a single CYP can be
induced by different classes of chemicals and how a single chemical can
differentially induce more than one CYP. For instance, CYP
genes may contain multiple xenobiotic response elements, each of which
binds a distinct nuclear receptor. In this model, structurally related
compounds may bind to the same receptor, but different classes of
chemical would interact with different receptors. For example, many
polycyclic aromatic hydrocarbons are able to interact with the aryl
hydrocarbon receptor, whereas several steroids can bind to the PXR1.
Differential induction of CYP genes by the same xenobiotic
may be the result of differences in the sequence or environment of a
particular response element in different genes. In a second model, a
receptor can bind structurally unrelated xenobiotics before interacting
with its cognate response element. For example, chlortrimazole and
TCPOBOP have been shown to bind to human PXR and transactivate gene
expression via a CYP3A4 PXRE (24). Differences in receptor
conformation, as a result of binding different chemicals, may account
for differential induction responses. In this model, differential
induction of CYP genes by the same chemical would result
from binding of the chemical to different receptors, each of which then
binds to its cognate response element and transactivates with a
different efficiency. The results presented in this paper support a
third model in which different receptors interact with the same
response element, i.e. the receptors are promiscuous with
respect to DNA binding but not ligand binding. Differences in the
interface between an orphan receptor and its dimerization partner,
RXR CAR was originally shown to bind to and transactivate gene expression
via DR2 and DR5 retinoic acid response elements (7, 25, 26).
Subsequently, it was found that the receptor could transactivate via a
DR4 element in CYP2B genes (9) and a DR1 peroxisome
proliferator response element in the enoyl-CoA hydratase/3-hydroxy acyl-CoA dehydrogenase gene (27). Here we show that CAR can transactivate CYP gene expression in response to PB via DR4
and DR3 elements. CAR is thus extremely promiscuous with respect to DNA
binding and must therefore have a great flexibility in its interactions
with its heterodimerization partner, RXR PB regulates the transcription of 30 or more genes in the liver (28).
The recent production of a CAR knock-out mouse confirms that this
orphan nuclear receptor mediates the induction of CYP2B by
phenobarbital (29). It will be of interest to determine whether all
PB-inducible genes are activated by CAR and to identify the enhancer
site permutations to which this receptor and others, such as PXR1, can
bind in the promoters of xenobiotic-regulated genes.
The detoxification of xenobiotics is necessary for survival. The
evolution of orphan receptors that exhibit versatility with respect to
the regulatory elements to which they bind in response to xenobiotics
has clear advantages for the ability of organisms to protect themselves
against potentially harmful foreign chemicals by enabling selective
induction of the enzymes required to metabolize such compounds.
However, competition of receptors for the same response element may
lead to adverse drug-drug or xenobiotic-drug interactions.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-carbonitrile (PCN). CYP2B1 is induced to a greater extent by PB
than by PCN (5), whereas CYP3A1 is induced more highly by PCN than by
PB (6). Induction of CYP2B1 in response to PB is mediated by the
interaction of the constitutive androstane receptor (CAR) (7) with a
phenobarbital response element (PBRE) (8, 9), and that of CYP3A1 in
response to PCN by binding of the pregnane X receptor (PXR1) to a
pregnane X response element (PXRE) (10-13). However, no PBRE has been
identified in the flanking regions of the CYP3A1 gene, and a
PXRE has not been identified in the CYP2B1 gene. The
mechanisms of induction of CYP2B1 by PCN and of CYP3A1 by PB are not known.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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2301 to
2142 of the CYP2B1 gene) into the
BglII site of the plasmid pGL3-SV40 promoter (Promega). The
plasmid pRL-TK (Promega), which encodes the Renilla
luciferase gene under the control of the thymidine kinase promoter, was
used as a control vector to normalize the results of transient
transfection assays. The reporter plasmid
(CYP3A1)4-tk-CAT, containing four copies of the
CYP3A1 PXRE, was kindly provided by Dr. S. Kliewer. When
cells were transfected with the chloramphenicol acetyltransferase reporter plasmid, (CYP3A1)4-tk-CAT, the
pSV-
-galactosidase control vector (Promega) was used to
normalize the results of transient transfection assays. Nuclear
receptor expression plasmids for mouse CAR (cDM8-mCAR-
), mouse
retinoid X receptor (RXR) (pSG5-mRXR
), mouse PXR1 (pSG5-mPXR1), and
newt retinoic acid receptor (RAR) (PTL-1) were provided, respectively,
by Drs. D. Moore, P. Chambon, S. Kliewer, and J. Brockes.
-galactosidase
(
-galactosidase enzyme assay system) (all from Promega) activities
according to the supplier's recommendations.
or pSG5-mPXR1)
was 10:1:1. The amount of total DNA/cartridge was 2 µg. Male Harlan Sprague-Dawley rats (200-250 g) were anesthetized and the liver shot
at two different positions in a single lobe. A single dose of PB (100 mg/kg) or PCN (100 mg/kg) was given intraperitoneally as soon as
suturing was complete. 24 h later the liver was removed and
assayed for luciferase activity as described above.
were synthesized in
vitro using a TNT® transcription/translation-coupled
reticulocyte lysate system (Promega) according to the supplier's
recommendations. Gel mobility shift assays contained 10 mM
HEPES (pH 7.6), 0.5 mM dithiothreitol, 15% (v/v) glycerol,
2.5 µg poly(dI·dC), 0.05% (v/v) Nonidet P-40, 50 mM
NaCl, 1.5 µl of in vitro translated products or 5 µg of liver nuclear protein extract, and 3 × 104 cpm of a
32P-labeled double-stranded oligonucleotide. The binding
reaction was performed at room temperature for 20 min. For competition assays, various amounts of unlabeled double-stranded oligonucleotide were included as indicated. The following oligonucleotides, synthesized by Amersham Pharmacia Biotech, were used as probes or competitors: the
CYP2B1 nuclear receptor half-site NR1 (DR4),
5'-TCTGTACTTTCCTGACCTT-3'; the CYP3A1 PXRE (DR3),
5'-AGACAGTTCATGAAGTTCATCT-3'. For supershift assays, rabbit polyclonal
antibodies against RXR
(SC-553x) (2 µg) or PXR1 (SC-7737x) (2 µg) (Santa Cruz Biotechnology, Inc.) were added 15 min after the
start of the binding reaction. Incubation was continued for another 20 min.
heterodimers to DNA
response elements, in vitro transcription/translation
reaction master mixes were prepared, one containing
35S-labeled methionine (RedivueTM
[L-35S]methionine, Amersham Pharmacia
Biotech) and the other, unlabeled methionine. Each master mix was split
into equal batches and the appropriate expression vector added.
Unlabeled and labeled transcription/translations were performed in
parallel. Radiolabeled proteins were assessed by SDS-polyacrylamide gel
electrophoresis and their specific activity determined. Using the
methionine content of each expressed protein, the volumes of unlabeled
reticulocyte lysate mix that would contain equal amounts of CAR, PXR1,
and RXR
were calculated. Gel mobility shift assays were performed
using a 1:1 molar ratio of unlabeled RXR
and either CAR or PXR1 and,
as DNA probe, a radiolabeled double-stranded oligonucleotide specifying
the CYP2B1 NR1 or the CYP3A1 PXRE.
RESULTS
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 1.
PB and PCN induce CYP2B1
expression via the PBRE. A, primary hepatocytes
were transfected with a (CYP2B1 PBRE)-SV40-luc reporter
construct containing one copy of the CYP2B1 PBRE with or
without (No E.P.) expression vectors for CAR or PXR1.
Transfected cells were treated with vehicle (DMSO) alone or
with PB or PCN and then assayed for luciferase activity. *,
significantly different from the corresponding control value,
p < 0.05, (two-tail, paired Student's t
test). B, gel mobility shift assay using as probe the
radiolabeled CYP2B1 NR1 sequence and equimolar amounts of
in vitro synthesized RXR , PXR1, or CAR as indicated.
C, gel mobility shift assays of rat liver nuclear proteins
using as probe a radiolabeled double-stranded oligonucleotide
corresponding to either the CYP2B1 NR1 or the
CYP3A1 PXRE. Assays were done as indicated in the presence
(+) or absence (
) of antibodies against PXR1 (PXR1
Ab) or RXR
(RXR Ab) or of a 100-fold molar excess of
an unlabeled double-stranded oligonucleotide competitor corresponding
to the NR1 site or the PXRE. D, intact rat liver was
transfected with the reporter construct (CYP2B1
PBRE)-SV40-luc with or without (No E.P.) expression plasmids
for CAR or PXR1. Animals were treated with vehicle (DMSO)
alone or with PB or PCN. Luciferase activity was assayed 24 h
post-operative.
, to the NR1 element (Fig. 1B), the same site within
the PBRE to which CAR binds as a RXR
heterodimer (Fig.
1B) (9). Neither CAR nor PXR1 binds to the rat
CYP2B1 NR2 site when heterodimerized with mouse RXR
(17).
However, when heterodimerized with human RXR, CAR can bind to the NR2
site of the mouse Cyp2b10 gene (18).
(Fig. 1C). These results
are similar to those obtained using the CYP3A1 PXRE as probe
(Fig. 1C) and confirm the interaction of PXR1/RXR
heterodimers with the NR1 site of the PBRE.
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Fig. 2.
CAR and PXR1 bind to the same sites within
the promoters of the CYP2B1 and CYP3A1
genes. Gel mobility shift assays of in vitro
synthesized CAR/RXR (A) and PXR/RXR (B) using as
probes radiolabeled double-stranded oligonucleotides corresponding to
CYP2B1 NR1 (A) or CYP3A1 PXRE
(B). Assays were done as indicated in the absence or
presence of increasing amounts of unlabeled double-stranded
oligonucleotide competitors corresponding to the NR1 or PXRE sites.
L represents unprogrammed reticulocyte lysate. The
arrows indicate a nonspecific complex formed between
the probe and proteins present in unprogrammed lysate. C,
gel mobility shift assays using as probes radiolabeled double-stranded
oligonucleotides corresponding to either CYP2B1 NR1 or
CYP3A1 PXRE and increasing amounts of in vitro
synthesized CAR/RXR (C/R) or PXR/RXR (P/R) as indicated. The
arrow indicates a nonspecific complex formed with proteins
in the unprogrammed lysate. D, relative binding affinities
of CAR for NR1 and PXRE (determined from A). E,
relative binding affinities of PXR for PXRE and NR1 (determined from
B). F, primary hepatocytes (5 × 106/60-mm plate) were cotransfected with 2.5 µg of the
reporter construct (CYP2B1 PBRE)-SV40-luc, 0.25 µg of the
control plasmid pRL-TK, 0.25 µg of an expression plasmid for CAR, and
increasing amounts of an expression vector for PXR1 ( ) or RAR
(
). Cells were treated with PB and then assayed for luciferase
activity. The results were plotted as a percentage of CAR-mediated PB
activation of reporter gene expression in the absence of competitor
receptor and were normalized against the expression of the
Renilla luciferase reporter gene cotransfected as an
internal standard.
or PXR
and RXR
were incubated in the presence of unlabeled NR1 or PXRE with
radiolabeled oligonucleotides specifying either the NR1 or PXRE sites.
The CAR·RXR
-NR1 complex was competed more effectively by
NR1 than by PXRE (Fig. 2A), whereas the PXR·RXR
-PXRE
complex was competed more effectively by the PXRE than by NR1 (Fig.
2B). When increasing amounts of in vitro
synthesized CAR/RXR
proteins were incubated with NR1 or PXRE probes,
radiolabeled to the same specific activity, the protein-DNA complex
formed on the NR1 site was more abundant than on the PXRE site (Fig.
2C). Conversely, PXR/RXR
bound more effectively to the
PXRE than to the NR1 site (Fig. 2C). A comparison of
relative binding affinities showed that CAR/RXR
bound to the NR1
site with a 2-fold greater affinity than to the PXRE (Fig. 2D) and that the affinity of PXR/RXR
for PXRE was about
three times that for NR1 (Fig. 2E).
,
and whose cDNA was cloned into the same type of expression vector
as that used for PXR, namely pSG5. RAR decreased reporter gene
expression by ~25% (Fig. 2F). Therefore, although some of
the inhibitory effects of PXR may be due to competition with CAR for
binding to RXR
, the majority most likely is due to competition with
CAR for binding to the NR1 site of the PBRE.
to the PXRE (Fig. 2, A and C), suggesting that PXR1 and CAR may transactivate
CYP3A1 by binding to the same site within the gene's
promoter. To determine whether CAR can mediate PB-induced
transactivation via the PXRE, hepatocytes were cotransfected with a
CYP3A1 PXRE-reporter plasmid and an expression vector for
either CAR or PXR1 (Fig. 3). In cells
cotransfected with CAR, reporter gene expression was induced 10-fold in
response to PB, whereas PCN had little effect. In contrast, in cells
cotransfected with PXR1, gene expression was increased 11-fold by PCN
but not by PB. Thus, as is the case for the CYP2B1 gene,
PXR1 and CAR bind to the same enhancer element within the
CYP3A1 promoter (the PXRE) to activate gene expression in
response to two different xenobiotics, PCN and PB, respectively. Each
receptor can therefore mediate the xenobiotic induction of members of
two different CYP subfamilies by interacting with a different response
element within each of the corresponding genes. The receptors are
therefore promiscuous with respect to DNA binding. However, they are
specific with respect to their xenobiotic activator; PXR1 is activated
by PCN (but not by PB) and CAR by PB (but not PCN).
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Fig. 3.
CAR and PXR1 transactivate via the same
CYP3A1 DNA element. Primary hepatocytes were
transfected with the reporter plasmid
(CYP3A1)4-tk-CAT and the control vector
pSV- -galactosidase with or without (No E.P.) expression
plasmids for CAR or PXR1. Cells were treated with vehicle
(DMSO) alone or with PB or PCN and then assayed for
chloramphenicol acetyltransferase and
-galactosidase activities.
*, p < 0.01; **, p < 0.05, compared with the corresponding control value (two-tail paired
Student's t test).
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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(and between PXR1 and
RXR
) when bound to the DR4 CYP2B1 NR1 will be different
from that of the proteins when bound to the DR3 CYP3A1 PXRE.
In addition, the CAR/RXR
heterodimer binds to the CYP2B1
NR1 more effectively than does the PXR1/RXR
heterodimer, and
PXR1/RXR
binds more effectively to the PXRE than does CAR/RXR
(Fig. 2). Therefore, the length of the half-site spacer in the nuclear
receptor response element may influence both the binding affinity of
the receptor heterodimer and its ability to interact with the
transcriptional machinery and may thus provide a basis for the
differential induction of CYP2B1 and CYP3A1 in response to PB or
PCN.
) androsten-3
-ol (androstenol), an inverse agonist of CAR (22), and restored to its original level by
1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). The
CYP3A4 ER6 PXRE contains a DR4 element similar to the DR4
element of the NR1 site of the PBRE of CYP2B genes. The
discrimination of binding of orphan receptors to direct repeat response
elements is dictated largely by the length of the inter-half-site spacer (23). It is not surprising, therefore, that CAR is able to bind
to the DR4 CYP3A4 PXRE. Our results demonstrate that CAR and
PXR1 can bind to and transactivate via both DR4 and DR3 elements. Furthermore, we show that in primary hepatocytes, pretreatment with an
inverse agonist of CAR is not required to obtain CAR-mediated PB
induction of gene expression via either the CYP2B1 PBRE or the CYP3A1 PXRE. In agreement with our results, Moore
et al. (24) found that the mouse isoform of PXR
transactivates a CYP3A4-reporter gene in response to PCN but
not to PB. These workers also found that neither human nor mouse CAR
was able to transactivate a CYP3A4-reporter gene in response
to PB. However, these experiments were performed in CV-1 cells, which,
unlike primary hepatocytes, do not support PB-induced CAR-mediated
transactivation of gene expression (17).
, because of differences in the half-site spacing of response
elements may influence the efficiency with which the heterodimer
interacts with the transcriptional machinery and hence provide the
basis for the differential induction of CYP genes by the
same chemical. These models are not mutually exclusive, and all three
could be involved in regulating the expression of a particular
CYP gene.
, and with components of the
transcription machinery.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. Moore, S. Kliewer, P. Chambon, and J. Brockes for kindly providing expression or reporter plasmids and Drs. J. Brockes and C. Velloso for help with in vivo transfections.
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FOOTNOTES |
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* This work was supported by Grant 042495 from the Wellcome Trust.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.
§ Recipient of a Wellcome Trust Toxicology Prize studentship.
¶ Recipient of a scholarship from the Royal Thai Government. Present address: Dept. of Clinical Chemistry, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand 50200.
To whom correspondence may be addressed: Dept. of Biochemistry
and Molecular Biology, University College London, Gower Street, London
WC1E 6BT, UK. Tel.: +44-20-7679-2321; Fax: +44-20-7679-7193; E-mail:
e.shephard@ucl.ac.uk.
** To whom correspondence may also be addressed: School of Biological Sciences, Queen Mary, University of London, Mile End Rd., London E1 4NS, UK. Tel.: +44-20-7882-6338; Fax: +44-20-8983-0531; E-mail: I.R.Phillips@qmw.ac.uk.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M005930200
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ABBREVIATIONS |
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The abbreviations used are:
CYP, cytochrome
P450;
PB, phenobarbital;
PCN, pregnenolone 16carbonitrile;
CAR, constitutive androstane receptor;
CAT, chloramphenicol
acetyltransferase;
PBRE, phenobarbital response element;
PXR1, pregnane
X receptor;
PXRE, pregnane X response element;
DR, direct repeat;
RXR
, retinoid X receptor
;
RAR, retinoic acid receptor;
NR, nuclear receptor;
TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene.
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