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Vol. 56, Issue 6, 1329-1339, December 1999
Department of Clinical Pharmacology and Storr Liver Unit, University of Sydney at Westmead Hospital, Westmead, Australia
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Summary |
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 Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Cytochrome P-450 3A4 (CYP3A4), the predominant cytochrome P-450
expressed in adult human liver, is subject to transcriptional induction
by a variety of structurally unrelated xenobiotics, including the
antibiotic rifampicin. The molecular mechanisms underlying this
phenomenon are poorly understood. We transfected a human liver-derived
cell line (HepG2) with various CYP3A4-luciferase reporter gene constructs containing a nested set of 5'-deletions of the
CYP3A4 5'-flanking region. Rifampicin-inducible
transcription of the reporter gene was observed only with the longest
construct, which encompassed bases 
13000 to +53 of
CYP3A4 (3-fold induction). The responsive region was
functional regardless of its position or orientation relative to the
proximal promoter of CYP3A4 and was capable of
conferring rifampicin-inducible expression on a heterologous promoter.
Further deletion mutants localized the induction to bases 7836 to
7607. In vitro DNase I footprint analysis of this region revealed
four protected sites (FP1, FP2, FP3, and FP4). Two of these sites, FP3
(bases 7738 to 7715) and FP4 (bases 7698 to 7682), overlapped
binding motifs for the orphan human pregnane X receptor (hPXR).
Cotransfection of responsive constructs with a hPXR expression vector
substantially increased the rifampicin-inducibility to ~50-fold. In
addition, the rifampicin-responsive constructs were strongly activated
by a range of CYP3A inducers. Finally, we demonstrate
cooperativity between elements within the distal enhancer region and
cis-acting elements in the proximal promoter of
CYP3A4. Our results provide evidence for the existence
of a potent enhancer module, 8 kb distal to the transcription start
point, which mediates the transcriptional induction of
CYP3A4 by activators of hPXR.
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Introduction |
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Top
Abstract
 Introduction
 Experimental Procedures
Results
Discussion
References
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Cytochromes
P-450 (P-450) are a superfamily of hemoproteins that play a pivotal
role in the oxidative metabolism of numerous endogenous and exogenous
compounds (Nelson et al., 1996
). The human P-450 3A subfamily contains
three functional members: CYP3A4, CYP3A5, and CYP3A7. CYP3A4 is the
predominant isoform expressed in adult human liver and is reported to
be responsible for the metabolism of more than 60% of therapeutic
drugs (Li et al., 1995). In addition, this enzyme is the primary
catalyst of steroid 6
-hydroxylation (Waxman et al., 1991) and
therefore has a central role in steroid hormone homeostasis. CYP3A4 is
also involved in the bioactivation of anticancer drugs and
environmental procarcinogens, including benzo(a)pyrene and
other dihydrodiol derivatives of polycyclic aromatic hydrocarbons and
carcinogenic mycotoxins (Li et al., 1995). Thus, CYP3A4 is considered a
key enzyme in chemical carcinogenesis in both the liver and
extrahepatic tissues.
CYP3A4 is transcriptionally regulated by a variety of hormones,
including glucocorticoids, growth hormone, and triiodothyronine (Schuetz et al., 1993; Liddle et al., 1998), and xenobiotics such as
phenobarbital, clotrimazole, mifepristone (RU486), and rifampicin (Daujat et al., 1991; Schuetz et al., 1993; Kocarek et al., 1995). Rifampicin, a macrocyclic antibiotic, is known to be one of the most
potent inducers of CYP3A4 expression both in vivo and in cultured hepatocytes (Schuetz et al., 1993; Kocarek et al., 1995; Michalets, 1998). Induction of CYP3A4 expression by
rifampicin and other xenobiotics underlies many reported drug
interactions and is of considerable importance for patients subject to
combination drug therapy such as for HIV/AIDS (Michalets, 1998).
Although the nucleotide sequence of the proximal 5'-flanking region of
CYP3A4 has been reported (Hashimoto et al., 1993), the
molecular mechanisms underlying the transcriptional regulation of this
gene have yet to be elucidated. The proximal promoter of the
CYP3A4 gene (bases 172 to 149) contains two copies of an
AG(G/T)TCA hexamer, the recognition sequence for the nuclear receptor
family of transcription factors (Mangelsdorf et al., 1995). Barwick et
al. (1996) demonstrated that these half-sites, organized as an ER-6
(everted repeat separated by six nucleotides), conferred
rifampicin-responsiveness on heterologous reporter gene constructs when
transfected into rabbit but not rat hepatocytes. These observations led
to the hypothesis that the host cellular environment, rather than the
structure of the gene, is responsible for the well-documented
interspecies differences in CYP3A inducibility (Wrighton et
al., 1985; Kocarek et al., 1995). Importantly, the magnitude of the
rifampicin induction in this study (2- to 4-fold) was considerably less
than would be expected from either the endogenous rabbit
CYP3A6 gene or the human CYP3A4 gene (Daujat et
al., 1991; Kocarek et al., 1995).
Recently, a number of studies have led to the isolation of a human
orphan nuclear receptor, the pregnane X receptor [hPXR (NR1I2)]. This
versatile receptor forms a heterodimer with the 9-cis
retinoic acid receptor [RXR (NR2B1)] and is capable of promoting transcription of heterologous reporter gene constructs containing multimerized copies of the proximal ER-6 element of CYP3A4.
The hPXR is activated by a wide variety of lipophilic compounds,
including CYP3A inducers (Bertilsson et al., 1998; Blumberg
et al., 1998; Lehmann et al., 1998). Interestingly, hPXR and mPXR
(mouse PXR) exhibit divergent activation profiles. Thus, although both
hPXR and mPXR are effectively activated by dexamethasone
t-butylacetate, RU486, corticosterone, and
5-pregnane-3,20-dione, hPXR but not mPXR is activated by rifampicin,
clotrimazole, coumestrol, and diethylstilbestrol. Conversely,
pregnenolone 16
-carbonitrile (PCN), dexamethasone, and
17-hydroxypregnenolone are efficacious activators of mPXR but not of
hPXR (Blumberg et al., 1998; Kliewer et al., 1998; Lehmann et al.,
1998). The pharmacology of mPXR and hPXR activation correlates well
with species-specific CYP3A induction profiles. The
existence of a broad-specificity, low-affinity receptor acting as a
sensor for hormones and xenobiotics and modulating their metabolism by
transcriptionally activating P-450 genes is an attractive model
(Blumberg et al., 1998).
In view of the relatively modest activation by rifampicin of reporter
gene constructs containing the proximal PXR response element (prPXRE)
alone (Barwick et al., 1996; Ogg et al., 1999), we attempted to define
further regulatory regions of the CYP3A4 gene involved in
transcriptional activation by xenobiotics. Here we report the isolation
and characterization of a distal xenobiotic-responsive enhancer module
(XREM) that mediates transactivation of the CYP3A4 gene by
inducers that are hPXR activators. Moreover, we demonstrate cooperativity between response elements in the distal XREM and proximal
promoter regions of CYP3A4. This study clearly establishes that hPXR is the major transactivating factor responsible for the
induction of CYP3A4 by xenobiotics.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
DNA-modifying enzymes, unless otherwise stated,
were obtained from Boehringer-Mannheim Australia (Sydney, Australia).
[
-32P]dATP,
[-32P]dCTP (>3000 Ci/mmol), and Megaprime
random prime DNA labeling kits were obtained from Amersham Pharmacia
Biotech (Buckinghamshire, England). Cell culture media, fetal bovine
serum, and media supplements were obtained from GIBCO BRL (Grand
Island, NY). PCN and RU486 were provided by BIOMOL Research
Laboratories (Plymouth Meeting, PA). Clotrimazole was purchased from
Calbiochem-Novabiochem (San Diego, CA). All other chemicals, including
rifampicin, were obtained from Sigma Chemical Co. (St. Louis, MO).
Oligonucleotides were prepared by Bresatec (Adelaide, Australia).
Isolation of CYP3A4 Genomic Clone.
Two
clones, encompassing bases +53 to 3099 of the CYP3A4
5'-flanking region, were generated from a commercially available system
that facilitates the amplification of unknown DNA adjacent to known DNA
sequence (PromoterFinder; Clontech Laboratories, Palo Alto, CA). These
fragments were radioactively labeled with [-32P]dCTP by random priming and used to
screen a human genomic library contained in the pWE15 cosmid vector
(Stratagene, La Jolla, CA), according to the manufacturer's
instructions. A single positive clone (p3A4-C1) was isolated, and
confirmatory sequencing was performed. This clone was used to generate
all subsequent deletion mutants. The partial nucleotide sequence of
p3A4-C1 (bases 10468 to +906 of CYP3A4) has been deposited
with the GenBank/EMBL/DDBJ databases under accession number
AF185589. Putative regulatory motifs in the XREM region were
identified manually and by screening the TRANSFAC database
(http://transfac.gbf.de/TRANSFAC) with use of the PatSearch and
MatInspector programs (Quandt et al., 1995).
Plasmid Constructs.
Chimeric CYP3A4 luciferase reporter
plasmids were prepared as follows. First, a 1.13-kb fragment of
CYP3A4, encompassing bases 1084 to +53, was amplified by
polymerase chain reaction (PCR) using the oligonucleotides CYP3A4UP1
(5'-CATTGCTGGCTGAGGTGGTT-3'; sense, bases 1084 to 1065) and
CYP3A4GSP2 (5'-catggatccTGTTGCTCTTTGCTGGGCTATGTGC-3'; antisense,
bases +29 to +53), creating a BamHI restriction site at the
3'-end. Construct p3A4-362 was prepared by digesting this 1.13-kb
amplicon with BglII and BamHI and cloning the
resultant 415-bp fragment (bases 362 to +53) into the
BglII site of pGL3-Basic, a promoter-less luciferase
reporter vector (Promega, Madison, WI), destroying the
3'-BglII site. Confirmatory sequencing was performed, and
the fragment was found to be identical with the published sequence
(Hashimoto et al., 1993). Second, a 1.89-kb KpnI/BglII fragment of p3A4-C1 (bases 3195 to
1310) was inserted into KpnI/BglII-digested
p3A4-362. This construct was linearized with BglII, and a
948-bp BglII fragment of p3A4-C1 (bases 1310 to 362) was
inserted, creating p3A4-3195. Third, p3A4-13000 was prepared by cloning
a 10-kb KpnI fragment (bases 13000 to 3195) of p3A4-C1
into p3A4-3195 linearized with KpnI. The remaining clones
illustrated in Fig. 1 were prepared
according to standard techniques (Sambrook et al., 1989) with the
restriction sites indicated. Enhancer deletion mutants were generated
by inserting BamHI or BglII fragments of p3A4-C1
(encompassing bases 12.6 to 6 kb) into the BamHI site
downstream of the luciferase reporter gene in the p3A4-1200 clone (Fig.
3)
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7746 to 7714,
5'-GATCTCAGCTGAAaGcAtTTGCTGACCCTCTGC-3'),
dNR2 mut (bases 7697 to 7665,
5'-GGTGCCCTTGttATCATGTCGGaaCAAGCAGCC-3'), dNR3
mut (bases 7291 to 7258,
5'-AATATATTGTTATaGcAtTATCAAAGCCTTTTCC-3'), and prPXREmut (bases 181 to 144,
5'-CCTCATAGAATATGttCTCAAAGGAGaaCAGTGAGTGG-3'). Mutated constructs were sequenced and verified to be free of
nonspecific base changes.
pCMV, an expression vector containing the -galactosidase cDNA
under the control of human cytomegalovirus promoter and enhancer, was
obtained from Clontech. pGEM3Zf(+) was purchased from Promega. The hPXR
expression vector pSG5-hPXR
ATG, p(ER6)3-tk-Luc
control plasmid (Lehmann et al., 1998), and human 9-cis
retinoic acid receptor- expression vector (pSG5-hRXR) were
generously provided by Steven A. Kliewer (Glaxo Wellcome Research and
Development, Research Triangle Park, NC). The human glucocorticoid
receptor expression vector (hGR) and the
p(GRE)2-tk-Luc control plasmid, which contains
dimerized glucocorticoid response elements (GREs) from the rat tyrosine
aminotransferase gene linked to a minimal thymidine kinase (tk)
promoter and luciferase reporter gene, were a gift from Jan
Carlstedt-Duke (Karolinska Institute, Stockholm, Sweden). All DNA used
in temporary transfection studies was purified using an
anionic-exchange resin (QIAGEN, Clifton Hill, Australia).
Cell Culture and Transfections.
The human hepatocellular
carcinoma cell line HepG2 was obtained from the American Type Culture
Collection (Rockville, MD) and cultured in antibiotic-free Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum.
Murine fibroblast, NIH3T3, and simian kidney (COS7) cells were
maintained under similar conditions. Cells (3 × 105) were inoculated onto 6-well plates (Nunc
A/S, Roskilde, Denmark) 24 h before transfection with the cationic
liposomes
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate or FuGene-6 (Boehringer-Mannheim Australia) according to
manufacturer's instructions. Transfection with 2 µg of luciferase reporter gene constructs, 0.8 µg of pCMV, and 0.1 µg of
expression vector, when included, was allowed to proceed for 5 h
in serum-free medium. The pSG5 expression vector (Stratagene) was used
in control cotransfection experiments. Subsequently, cells were
cultured for an additional 48 to 60 h in fresh medium supplemented
with 10% fetal bovine serum in the presence of various inducers (see figure legends), added as a 1000× stock solution in dimethyl sulfoxide (DMSO). Control cultures received vehicle (0.1% DMSO) alone.
Luciferase activities were determined on cell lysates using a
commercially available assay system (Promega). -Galactosidase assays
were performed as described elsewhere (Foster et al., 1988).
Preparation of Nuclear Extracts and In Vitro DNase I
Footprinting.
Nuclear extracts were prepared from adult male
Wistar rats (220-250 g) according to the method of Gorski et al.
(1986). All buffers were supplemented with 0.4 mM sodium orthovanadate,
1 mM sodium fluoride, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, and 1 mM
dithiothreitol shortly before use. For in vitro DNase I footprint
analysis, a 269-bp fragment encompassing bases 7836 to 7568 of
CYP3A4 was asymmetrically 5'-end-labeled with
[-32P]CTP by PCR as outlined elsewhere
(Krummel et al., 1990). The protein/DNA binding reaction contained the
following components in a volume of 50 µl: 25 mM HEPES, pH 7.6, 60 mM
KCl, 10% glycerol, 0.1 mM EDTA, 2 µg of poly(dI/dC), 5 mM
MgCl2, 1 mM dithiothreitol, and 3 to 25 µg of
nuclear protein extract. The binding reaction was incubated on ice for
10 min before the addition of radiolabeled probe (~12,000 cpm) and
allowed to proceed for am additional 80 min at the same temperature.
DNase I (0.005-0.1 U, DPRF grade; Worthington Biochemical Corporation,
Lakewood, NJ) was added to the binding reaction in 50 µl of 25 mM
HEPES, pH 7.6, 60 mM KCl, 10% glycerol, 5 mM
MgCl2, and 5 mM CaCl2.
After digestion at room temperature for 2 min, samples were processed
as previously described (Lichtsteiner et al., 1987). Purine-specific (G + A) sequence ladders were prepared according to the Maxam-Gilbert
chemical sequencing method (Maxam and Gilbert, 1980).
Electrophoretic Mobility Shift Assay.
Electrophoretic
mobility shift assays (EMSAs) were performed essentially as described
elsewhere (Lehmann et al., 1998). hPXR and hRXR were synthesized
from pSG5-hPXRATG and pSG5-hRXR expression vectors, respectively,
using the TNT rabbit reticulocyte system (Promega). Unprogrammed lysate
was prepared using the pSG5 expression vector (Stratagene). Typically,
binding reactions contained 10 mM HEPES, pH 7.8, 60 mM KCl, 0.2%
Nonidet P-40, 6% glycerol, 2 mM dithiothreitol, 2 µg of poly(dI/dC),
and 1 µl each of synthesized hPXR or hRXR. Control incubations
received unprogrammed lysate alone. Reactions were preincubated on ice
for 10 min before the addition of 32P-labeled
double-stranded oligonucleotide probe (0.2 pmol). Samples were held on
ice for an additional 20 min, and the protein/DNA complexes were
resolved on a pre-electrophoresed 5% polyacrylamide gel in 0.5× TBE
(45 mM Tris-borate, 1 mM EDTA) at room temperature. Gels were dried and
autoradiographed at 70°C for 1 to 2 h. Oligonucleotides used
as probes are indicated in Fig. 6A. Competitor oligonucleotides were
added to the preincubation at 5-, 25-, and 75-fold molar excess.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Functional Analysis of CYP3A4 5'-Flanking Region.
A single
genomic clone (bases 20 kb to +15 kb of CYP3A4,
approximately) was isolated from a cosmid library using two probes corresponding to bases 1084 to +53 and 3099 to 1062. An 11.4-kb BamHI fragment of p3A4-C1 encompassing bases 10468 to +906
of CYP3A4 was subcloned into pGEM-3Zf(+), and the nucleotide
sequence was derived. Nucleotides +14 to +174, corresponding to exon
1, were identical with the reported cDNA sequence (accession
number M18907). Comparison with the previously published sequence of
the CYP3A4 5'-flanking region (bases 1105 to +175)
revealed 5-bp mismatches localized between bases 725 and 714
(Hashimoto et al., 1993). No further differences were observed. We have
not determined the functional significance, if any, of these base changes.
13000 to +53 of
CYP3A4 attached to a luciferase reporter gene were
constructed (Fig. 1) and transiently transfected into HepG2 cells.
Induction of endogenous CYP3A genes in rifampicin-treated
HepG2 cells has been previously demonstrated (Schuetz et al., 1993;
Sumida et al., 1999). In line with observations by others (Hashimoto et
al., 1993), the basal activity of all CYP3A4-luciferase
reporter gene constructs was low (data not shown). Rifampicin treatment
of cells transfected with constructs containing bases 3195 to +53
failed to induce luciferase reporter gene expression (Fig. 1). However, treatment of cells containing the p3A4-13000 construct (bases 13000
to +53) resulted in a 3-fold induction of reporter gene activity (Figs.
1 and 2, A and B). Additional deletion
mutants demonstrated that removal of an XbaI/SpeI
fragment, encompassing bases 7836 to 7493, destroyed the rifampicin
response (Fig. 1).
|
). Moreover, the dose-dependent induction of p3A4-13000 by
rifampicin is comparable to that of endogenous human CYP3A
genes (Schuetz et al., 1996). Finally, the magnitude of the rifampicin
response (3- to 4-fold) is in agreement with that of CYP3A
genes in HepG2 cells (Schuetz et al., 1993; Sumida et al., 1999). Taken
together, these data indicate that activation of
CYP3A4-luciferase reporter gene constructs by rifampicin
closely resembles that of endogenous CYP3A genes and, as
such, represents a valid model for the examination of CYP3A4
transcriptional regulation.
To examine whether the rifampicin-responsive domain exhibited enhancer
properties, fragments of the 5'-flanking region were inserted
downstream of the CYP3A4 promoter proximal and luciferase reporter gene (Fig. 3). Inducible
expression was observed regardless of the position or orientation of
the responsive region relative to the proximal promoter of
CYP3A4 (Fig. 3). Thus, a 3.8-kb BglII fragment,
corresponding to bases 9751 to 6043, imparted
rifampicin-responsiveness on the promoter proximal region (bases 1252
to +53) when inserted in either orientation downstream of the
luciferase reporter gene (Fig. 3). Furthermore, the distal region
(bases 7836 to 7208) was capable of conferring inducibility on a
minimal tk promoter (Fig. 8). Importantly, in this system, the
transcriptional activity of constructs containing the CYP3A4
proximal promoter region alone was not enhanced by rifampicin (Figs. 1,
3, and 4A; see also Fig. 8A).
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7836 to
7797 marginally reduced the rifampicin response, whereas the deletion
of bases 7797 to 7686 removed 70% of the wild-type activity (Fig.
4A). The deletion of bases 7836 to 7607 completely destroyed
rifampicin inducibility (Fig. 4A). These data suggested that the
rifampicin induction was dependent on the integrity of a number of
distinct transcription factor-bindin sites contained within the 7836
to 7607 region of CYP3A4. We have referred to this core
region as an XREM.
DNase I Footprint Analysis of Rifampicin-Responsive region.
DNase I footprint analysis of the responsive region (bases 7836 to
7568) using rat liver nuclear extract revealed two strongly (FP1 and
FP3) and two weakly (FP2 and FP4) protected regions (Figs. 4B and
5). Footprint coordinates on the sense
strand (Fig. 5A) were as follows: FP1, 7811 to 7777; FP2, 7763 to
7740; FP3, 7738 to 7717; and FP4, 7698 to 7682. Footprint
coordinates on the antisense strand (Fig. 5B) were as follows: FP1,
7806 to 7774; FP2, 7754 to 7748; FP3, 7738 to 7715; and
FP4, 7690 to 7682. No other regions were consistently protected on
either the sense or antisense strands. In addition, a number of DNase I-hypersensitive sites were identified. These results are summarized in
Figs. 4B and 5. A similar pattern of nuclease protection was observed
with nuclear extracts prepared from control and rifampicin-treated HepG2 cells (data not shown).
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). A
number of putative cis-acting elements were identified within or overlapping the FP1 region. First, a response element highly
homologous to the recognition sequence for the orphan retinoic acid
receptor-related receptor -1 [ROR1 (NR1F1)] is located between
bases 7783 to 7771 (Giguère et al., 1994). Second, a site
with significant identity to a binding motif in the ornithine transcarbamylase gene that was recognized by both chicken ovalbumin upstream promoter-transcription factors and hepatocyte nuclear factor-4
[COUP-TF/HNF-4 (NR2F1/NR2A1)] is situated between bases 7785 to
7772 (Kimura et al., 1993). Both the ROR1 and COUP-TF/HNF-4 sites
overlap the 5' terminus of FP1 and contain an AGGTCA hexamer, the
recognition sequence for members of the nuclear receptor superfamily (Fig. 4B; Mangelsdorf et al., 1995). We were unable to identify any
potential transcription factor-binding sites within the FP2 domain,
although significant homology with a previously reported CCAAT/enhancer-binding protein- motif was evident (Roman et al., 1990).
Examination of FP3 and FP4 revealed potential binding sites for the
orphan hPXR. Although members of the nuclear receptor family that
heterodimerize with RXR typically bind to repeats of the AGGTCA
hexad (Mangelsdorf et al., 1995), the PXR-RXR complex preferentially
binds the variant AGTTCA half-site (Bertilsson et al., 1998; Blumberg
et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998). Thus, FP3
contained an imperfect DR-3 (direct repeat with a three-nucleotide
spacer) of the AGTTCA hexamer. The structure of this element was almost
identical to the PXRE in the proximal promoter of CYP3A23
(Kliewer et al., 1998). FP4 overlapped an ER-6-like element that was
highly homologous to the previously characterized hPXR-bindin motif in
the proximal promoter of CYP3A4 (bases 172 to 149). The
hPXR-binding motifs encompassed by FP3 and FP4 are nominally dNR1
(distal nuclear receptor-binding element 1) and dNR2, respectively,
whereas the ER-6 motif in the CYP3A4 proximal promoter is
designated prPXRE (proximal PXRE). These elements are illustrated in
Figs. 4B and 6A. Finally, two potential GRE half-sites were evident in this region (Fig. 4B; Jantzen et al.,
1987).
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hPXR-RXR Heterodimers Bind Elements within Responsive
Region.
The ability of the dNRs to bind the hPXR-hRXR
heterodimer was investigated by EMSA (Fig. 6). As reported elsewhere
(Bertilsson et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998),
the prPXRE (ER-6) of CYP3A4 formed a complex with
hPXR-hRXR. dNR1 was capable of binding hPXR-hRXR with high
affinity and effectively competing with prPXRE for binding of the
hPXR-hRXR heterodimer (Fig. 6, B and C). The affinity of dNR1 for
hPXR-hRXR was comparable to that of prPXRE and a PXRE in the
promoter proximal region of the rat CYP3A23 (Fig. 6, B and
C; data not shown; Kliewer et al., 1998). dNR2 was also capable of
forming a complex with hPXR-hRXR, albeit with significantly lower
affinity (Fig. 6B). In addition, dNR2 was only capable of competing
with prPXRE for protein binding at high molar excess (Fig. 6C).
Cotransfection of hPXR with Rifampicin-Responsive Constructs
Substantially Augments Induction.
Recent reports have demonstrated
that hPXR is activated by rifampicin in transfection studies
(Bertilsson et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998).
A heterologous reporter gene construct
[p(ER6)3-tk-Luc] containing multiple copies of
the prPXRE element was capable of conferring rifampicin-responsiveness
on a minimal tk promoter in the presence of hPXR. Furthermore, Kliewer and coworkers demonstrated that rifampicin promotes the association of
hPXR with the steroid receptor coactivator 1 (Lehmann et al., 1998).
Taken together, these studies indicate that rifampicin can serve as a
ligand for hPXR.
; Blumberg
et al., 1998; Lehmann et al., 1998). This observation, in conjunction
with the ability of hPXR-RXR heterodimers to bind elements within
the XREM region, strongly suggested that hPXR may mediate the
rifampicin induction. Therefore, we examined the ability of hPXR to
augment the rifampicin-responsiveness of p3A4-362(7836/7208ins).
Cotransfection of liver-derived HepG2 cells with p3A4-362(7836/7208ins)
and an hPXR expression vector substantially increased the rifampicin
response. Typically, a 40- to 50-fold rifampicin-mediated induction of
luciferase activity was observed in cotransfection experiments (Fig.
7A). This level of induction is similar
to that of the endogenous CYP3A4 gene in primary cultures of
human hepatocytes (Daujat et al., 1991). However, rifampicin treatment
of non-liver-derived simian kidney (COS7) or murine fibroblast (NIH3T3)
cell lines cotransfected with p3A4-362(7836/7208ins) and hPXR resulted
in a modest 3- to 5-fold induction of reporter gene activity (Fig. 7, B
and C), suggesting that other transcription factors, present in HepG2 but not COS7 or NIH3T3 cells, are required for maximal responsiveness. In agreement with earlier experiments (Figs. 1-3 and 4A), cultures of
HepG2 cells transfected with empty expression vector exhibited a 2- to
4-fold induction after treatment. The effect of overexpressed hPXR on
the basal expression of the rifampicin-responsive constructs was
minimal (~1.5- to 2-fold increase in activity). The hPXR-responsive control plasmid p(ER6)3-tk-Luc exhibited a 2- to
4-fold rifampicin activation in the presence of cotransfected hPXR
regardless of cell type (Fig. 7A, inset; data not shown).
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reported that rifampicin was a
nonsteroidal ligand and activator of the hGR. The presence of putative
GREs in the proximal promoter of CYP3A4 (Hashimoto et al.,
1993) and the rifampicin-responsive region (Fig. 4B) led us to examine
the role of hGR in the induction. Rifampicin induction of
p3A4-362(7836/7208ins) was not augmented by cotransfection of an hGR
expression vector. Indeed, overexpression of hGR partially abrogated
the rifampicin response (Fig. 7A; data not shown). The presence of
ligands for both hPXR and hGR, namely rifampicin and dexamethasone (1 nM to 10 µM), did not augment the hPXR-mediated induction of
p3A4-362(7836/7208ins; data not shown). In parallel transfections, a
hGR control plasmid, p(GRE)2-tk-Luc, which
contains dimerized GREs from the tyrosine aminotransferase gene, was
dose-dependently activated by dexamethasone (Fig. 7A). However, in line
with observations by others (Lehmann et al., 1998; Ray et al., 1998;
Jaffuel et al., 1999), we were unable to activate this construct with
rifampicin in the presence of overexpressed hGR, hPXR, or both hPXR and
hGR (Fig. 7A). The proximal promoter construct p3A4-362 was not
activated by rifampicin under any conditions.
Cooperative Interaction of dNRs and prPXRE in Rifampicin
Response.
We next examined the effect of mutation of potential
PXREs in the p3A4-362(7836/7208ins) construct on the rifampicin
response. Thus, mutation of dNR1 reduced the wild-type response by 40 to 50% (Fig. 8A), where the fold
activation of the p3A4-362(7836/7208ins) construct is nominally 100%.
Interestingly, mutation of prPXRE removed ~50% of the wild-type
response. A similar reduction in rifampicin-responsiveness was observed
when the proximal promoter of CYP3A4 was replaced by a
minimal tk promoter (bases 105 to +52), suggesting that there are no
further CYP3A4 promoter-specific elements required for
maximal activity of the XREM region (Fig. 8A). Although prPXRE has no
inherent ability to promote a rifampicin response in the context of the
p3A4-362 construct, it is evidently capable of a cooperative
interaction with elements within the XREM. Surprisingly, mutation of
dNR2 did not reduce rifampicin inducibility. Indeed, disruption of this
element resulted in a significant increase (20-30%) in responsiveness
(Fig. 8A). Furthermore, disruption of both dNR2 and prPXRE (73% of
wild-type activity) resulted in significantly higher rifampicin
responsiveness than mutation of prPXRE alone (54% wild-type activity).
However, mutation of dNR2 in the context of a construct containing the
mutated dNR1 did not enhance induction relative to a construct
harboring a disrupted dNR1 alone. Thus, factors bound at dNR2 exerted a
partially suppressive effect on the activity of dNR1. In vitro, dNR2
was capable of weakly binding hPXR-RXR heterodimers; however, in HepG2 cells, this site may bind additional factors, possibly other members of the nuclear receptor superfamily. The significance of these
latter observations is unclear, but taken together, these data support
the idea of complex cooperative behavior between proteins bound at
distal response elements and elements in the proximal promoter. It is
important to note that the cooperativity between dNRs was retained when
the XREM deletions and mutations were placed upstream of a heterologous
tk promoter (data not shown).
|
7836 to 7493), the deletion of which destroyed inducible reporter gene expression (Fig. 1), did not confer wild-type
rifampicin-responsiveness on either the CYP3A4 proximal
promoter or a minimal tk promoter (Fig. 8B; data not shown) suggested
that the region encompassing bases 7492 to 7208 contained accessory
sites required for maximal activity. Nucleotide sequence analysis of
this region revealed a third putative nuclear receptor-bindin motif
(dNR3; Fig. 8B). This element, an imperfect DR-3 of the AGTTCA hexamer
(bases 7290 and 7270), was capable of weakly binding hPXR-RXR
heterodimers (data not shown). In addition, mutation of this motif
significantly reduced rifampicin responsiveness by 15 to 20% (Fig.
8B).
Rifampicin-Responsive Constructs Are Strongly Activated by a Number
of CYP3A4 Inducers.
In parallel transfections, the
p3A4-362(7836/7208ins) construct was potently activated by a number of
CYP3A inducers, including RU486 (40-fold), clotrimazole (20- to 30-fold), phenobarbital (15-fold), and metyrapone (10- to 15-fold;
Fig. 9). Weak activation by phenytoin
(2-fold) and PCN (3- to 4-fold) was also observed (Fig. 9). In the
absence of exogenously expressed hPXR, these xenobiotics were poor
inducers of p3A4-362(7836/7208ins): maximum induction was observed with
5 µM rifampicin (2- to 4-fold; Figs. 1 and 2; data not shown). The
induction of the XREM region by these and other compounds correlates
well with detailed activation profiles of the hPXR published elsewhere
(Bertilsson et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998).
None of these drugs were able to induce expression of reporter gene
constructs containing the CYP3A4 proximal promoter regions
alone (data not shown).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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CYP3A4, the predominant P-450 expressed in adult human liver, is
known to be transcriptionally regulated by a number of structurally unrelated compounds. Transcriptional activation of this gene is at the
center of many clinically important drug interactions. However, the
mechanisms underlying this induction have remained elusive. Recently, a
number of independent studies have described an orphan nuclear
receptor, hPXR, which interacts with an ER-6 (prPXRE, bases 172 to
149) element in the proximal promoter of CYP3A4. This
receptor is activated by a variety of lipophilic compounds, many of
which, including rifampicin, are CYP3A inducers (Bertilsson
et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998).
In this study, we defined regulatory regions of CYP3A4
involved in its transcriptional activation by rifampicin and other xenobiotics. To dissect functional elements in both proximal and distal
regions, we endeavored to use the proximal CYP3A4 promoter (bases 362 to +53) as a minimal promoter. Here, we report the characterization of a distal enhancer module (XREM) that, in
conjunction with elements in the proximal promoter region, directs the
hPXR-mediated transactivation of CYP3A4.
The XREM region, located ~7800 bp upstream of the CYP3A4
transcription initiation site, is a complex array of transcription factor-binding sites that includes at least two elements (designated dNR1 and dNR2) that are capable of binding hPXR-RXR heterodimers. In
addition, a third putative PXRE (dNR3), located several hundred base
pairs downstream of the core rifampicin-responsive region, appears to
be critical for maximal xenobiotic responsiveness. Two additional
cis-acting elements (designated FP1 and FP2), situated immediately upstream of the hPXR-binding motifs, are critical for full
functionality of the XREM region: deletion of these sites substantially
reduces the rifampicin responsiveness of the XREM region. The precise
nature of protein/DNA interactions at these accessory sites is under
investigation; however, binding motifs for other members of the nuclear
receptor superfamily, notably ROR1 and COUP-TF/HNF-4, were evident
in FP1. Interestingly, a PXRE in the proximal promoter of the rat
CYP3A23 gene is located between functional COUP-TF- and
HNF-4-binding sites (Huss and Kasper et al., 1998; Ogino et al., 1999),
the mutation or deletion of which substantially abrogates PXR-mediated
transactivation (Huss and Kasper et al., 1998).
The ability of hPXR-RXR heterodimers to interact with elements
within the XREM region strongly suggests hPXR mediated rifampicin induction of reporter gene constructs. Indeed, cotransfection of
rifampicin-responsive constructs with a hPXR expression vector increased induction by the drug from 2- to 4-fold to >40-fold, a level
of activation similar to that of the endogenous CYP3A4 gene
in primary cultures of human hepatocytes. The dose- and time-dependent activation of reporter gene constructs, in both the presence and absence of exogenously expressed hPXR, paralleled that of endogenous CYP3A genes (Schuetz et al., 1996; Sumida et al., 1999). The
modest rifampicin induction of these constructs in the absence of
cotransfected PXR is almost certainly due to reduced expression of the
hPXR gene in HepG2 cells. Support for this observation is
provided in a report by Miyata et al. (1995). Thus, binding of nuclear proteins to a probe corresponding to the promoter proximal PXRE from
the rat CYP3A2 gene was substantially lower in extracts
prepared from HepG2 cells than in those isolated from rat liver (Miyata et al., 1995). Interestingly, the rifampicin response was markedly lower in cells of extrahepatic lineage, suggesting that liver-enriched transcription factors, in addition to hPXR, may be required for full
functionality. Indeed, putative binding sites for multiple liver-enriched transcription factors are evident in the
CYP3A4 proximal promoter (Hashimoto et al., 1993), as well
as the XREM region. Identification of the additional liver-specific
factors required for xenobiotic inducibility of these constructs may
provide insights into the developmental and tissue-specific regulation of CYP3A4.
In the present study, we were unable to elicit any rifampicin induction
from CYP3A4-reporter gene constructs containing solely prPXRE. This contradicts an earlier report by Ogg et al. (1999) that
demonstrated that a plasmid containing 1 kb of the 5'-flanking region
of CYP3A4 conferred xenobiotic responsiveness on a reporter gene. Importantly, these studies used the potent cytomegalovirus promoter as a minimal promoter, whereas we have used the native CYP3A4 proximal promoter throughout. However, the isolated
and multimerized prPXRE was capable of mediating activation of
heterologous reporter gene constructs by hPXR. In line with
observations by Hashimoto et al. (1993), luciferase activity from the
p3A4-362 construct was very low, and an attractive hypothesis to
account for this would be the existence of silencer elements in this
region of the CYP3A4 promoter that repress both basal
transcriptional activity and the previously characterized activity of
the isolated prPXRE (Bertilsson et al., 1998; Blumberg et al., 1998;
Lehmann et al., 1998). However, mutation or removal of the prPXRE
reduced the wild-type rifampicin response by ~50%. This indicates
that the prPXRE cooperatively interacts with either the distal nuclear receptor-binding elements themselves or with other elements within the
XREM. Disruption of prPXRE and dNR1 destroyed 80 to 90% of xenobiotic
responsiveness, demonstrating that these two elements are central to
transactivation by ligand-activated hPXR. Although the exact role of
each PXRE remains to be elucidated, it is now clear that there is a
considerable level of complexity to both the XREM and the proximal
promoter regions of CYP3A4. In this respect, the XREM is
analogous to hormone response regions in genes such as the mouse
mammary tumor virus and tyrosine aminotransferase gene, where synergism
between hormone response elements and other transcription factors is
well documented (Jantzen et al., 1987; Tsai and O'Malley et al.,
1994). Similarly, the induction of CYP2B genes by
phenobarbital and other xenobiotics is mediated by distal enhancer
modules that contain multiple nuclear receptor-bindin motifs
(Honkakoski et al., 1998; Stoltz et al., 1998; Sueyoshi et al., 1999).
Finally, we demonstrated that rifampicin-responsive constructs were
potently activated by a number of well-documented CYP3A inducers, including phenobarbital. Recently, Negishi and coworkers reported that transactivation of CYP2B genes by this drug
was mediated by the orphan constitutive androstane receptor
[CAR (NR1I4); Honkakoski et al., 1998; Sueyoshi et al., 1999).
Moreover, the multimerized CYP3A4 prPXRE was shown to be
capable of conferring CAR-RXR-meditated phenobarbital inducibility
on a tk promoter. The ability of CAR-RXR and hPXR-RXR
heterodimers to interact with the same nuclear receptor-binding motif
suggests that interplay between orphan receptors is likely to play a
role in the regulation of CYP3A4 expression.
In summary, a potent xenobiotic-responsive enhancer module controls the transcriptional induction of CYP3A4 by compounds that are activators for the hPXR. Further characterization of this region should provide a framework for understanding the molecular basis of many clinically important drug interactions. Moreover, it is now clear that the transcriptional regulation of P-450 genes by diverse xenochemicals and endogenous compounds, such as steroid hormones, bile acids, cholesterol, and fatty acids, is predominantly mediated by orphan members of the nuclear receptor superfamily. The elucidation of the molecular mechanisms underlying the regulation of P-450 expression should provide valuable insights into the biology of the orphan nuclear receptors.
| |
Acknowledgments |
|---|
We gratefully acknowledge Graham R. Robertson and Geoffrey C. Farrell for their patient advice and support throughout this project.
| |
Footnotes |
|---|
Received July 6, 1999; Accepted August 26, 1999
This work was supported by a grant from the National Health and Medical Research Council (Australia) and the Robert W. Storr bequest to the Medical Foundation, University of Sydney. B.G. was a recipient of the National Health and Medical Research Council (Australia) Dora Lush Biomedical Scholarship.
1
Three independent studies have implicated an orphan
nuclear receptor in CYP3A regulation. The human PXR
(hPXR; Lehmann et al., 1998) and human pregnane activated receptor
(hPAR; Bertilsson et al., 1998) are identical in the derived amino acid
sequence. The human steroid and xenobiotic receptor (hSXR; Blumberg et
al., 1998) contains a single base-pair insertion at position 1225 and a
single base deletion at 1279, relative to hPXR, which results in a
shift in the reading frame for amino acid residues 215-233. However,
hPXR, hPAR, and hSXR almost certainly represent products of the same
gene. In this study, we used the "hPXR" nomenclature.
Send reprint requests to: Dr. Christopher Liddle, Department of Clinical Pharmacology and Storr Liver Unit, University of Sydney at Westmead Hospital, Westmead, NSW 2145, Australia. E-mail: chrisl{at}westgate.wh.usyd.edu.au
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Abbreviations |
|---|
P-450, cytochrome P-450;
PXR, pregnane X
receptor;
RXR, 9-cis retinoic acid receptor-;
PXRE, pregnane X receptor response element;
prPXRE, proximal pregnane X
receptor response element;
HNF, hepatocyte nuclear factor;
EMSA, electrophoretic mobility shift assay;
ROR1, retinoic acid
receptor-related receptor -1;
COUP-TF, chicken ovalbumin upstream
promoter-transcription factor;
PCR, polymerase chain reaction;
PCN, pregnenolone 16-carbonitrile;
tk, herpes simplex virus thymidine
kinase;
hGR, human glucocorticoid receptor;
GRE, glucocorticoid-responsive element;
XREM, xenobiotic-responsive enhancer
module;
DMSO, dimethyl sulfoxide.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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, a novel family of orphan hormone nuclear receptors.
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538-553A (CYP3A2) gene-encoding testosterone 6-hydrxylase.
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S. L. Ripp, J. B. Mills, O. A. Fahmi, K. A. Trevena, J. L. Liras, T. S. Maurer, and S. M. de Morais Use of Immortalized Human Hepatocytes to Predict the Magnitude of Clinical Drug-Drug Interactions Caused by CYP3A4 Induction Drug Metab. Dispos., October 1, 2006; 34(10): 1742 - 1748. [Abstract] [Full Text] [PDF]
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 S. J. Langmade, S. E. Gale, A. Frolov, I. Mohri, K. Suzuki, S. H. Mellon, S. U. Walkley, D. F. Covey, J. E. Schaffer, and D. S. Ory Pregnane X receptor (PXR) activation: A mechanism for neuroprotection in a mouse model of Niemann-Pick C disease PNAS, September 12, 2006; 103(37): 13807 - 13812. [Abstract] [Full Text] [PDF]
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 A. Takeshita, K. Inagaki, J. Igarashi-Migitaka, Y. Ozawa, and N. Koibuchi The endocrine disrupting chemical, diethylhexyl phthalate, activates MDR1 gene expression in human colon cancer LS174T cells. J. Endocrinol., September 1, 2006; 190(3): 897 - 902. [Abstract] [Full Text] [PDF]
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X. Gu, S. Ke, D. Liu, T. Sheng, P. E. Thomas, A. B. Rabson, M. A. Gallo, W. Xie, and Y. Tian Role of NF-{kappa}B in Regulation of PXR-mediated Gene Expression: A MECHANISM FOR THE SUPPRESSION OF CYTOCHROME P-450 3A4 BY PROINFLAMMATORY AGENTS J. Biol. Chem., June 30, 2006; 281(26): 17882 - 17889. [Abstract] [Full Text] [PDF]
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S. R. Faucette, T. Sueyoshi, C. M. Smith, M. Negishi, E. L. LeCluyse, and H. Wang Differential Regulation of Hepatic CYP2B6 and CYP3A4 Genes by Constitutive Androstane Receptor but Not Pregnane X Receptor J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1200 - 1209. [Abstract] [Full Text] [PDF]
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X. Ding, K. Lichti, and J. L. Staudinger The Mycoestrogen Zearalenone Induces CYP3A through Activation of the Pregnane X Receptor Toxicol. Sci., June 1, 2006; 91(2): 448 - 455. [Abstract] [Full Text] [PDF]
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T. Li and J. Y. L. Chiang RIFAMPICIN INDUCTION OF CYP3A4 REQUIRES PREGNANE X RECEPTOR CROSS TALK WITH HEPATOCYTE NUCLEAR FACTOR 4{alpha} AND COACTIVATORS, AND SUPPRESSION OF SMALL HETERODIMER PARTNER GENE EXPRESSION Drug Metab. Dispos., May 1, 2006; 34(5): 756 - 764. [Abstract] [Full Text] [PDF]
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A. Anapolsky, S. Teng, S. Dixit, and M. Piquette-Miller THE ROLE OF PREGNANE X RECEPTOR IN 2-ACETYLAMINOFLUORENE-MEDIATED INDUCTION OF DRUG TRANSPORT AND -METABOLIZING ENZYMES IN MICE Drug Metab. Dispos., March 1, 2006; 34(3): 405 - 409. [Abstract] [Full Text] [PDF]
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S. Aouabdi, G. Gibson, and N. Plant TRANSCRIPTIONAL REGULATION OF THE PXR GENE: IDENTIFICATION AND CHARACTERIZATION OF A FUNCTIONAL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR {alpha} BINDING SITE WITHIN THE PROXIMAL PROMOTER OF PXR Drug Metab. Dispos., January 1, 2006; 34(1): 138 - 144. [Abstract] [Full Text] [PDF]
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D. R. Johnson, C.-W. Li, L.-Y. Chen, J. C. Ghosh, and J. D. Chen Regulation and Binding of Pregnane X Receptor by Nuclear Receptor Corepressor Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) Mol. Pharmacol., January 1, 2006; 69(1): 99 - 108. [Abstract] [Full Text] [PDF]
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S. S. Auerbach, M. A. Stoner, S. Su, and C. J. Omiecinski Retinoid X Receptor-{alpha}-Dependent Transactivation by a Naturally Occurring Structural Variant of Human Constitutive Androstane Receptor (NR1I3) Mol. Pharmacol., November 1, 2005; 68(5): 1239 - 1253. [Abstract] [Full Text] [PDF]
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Y. Chen, G. Kissling, M. Negishi, and J. A. Goldstein The Nuclear Receptors Constitutive Androstane Receptor and Pregnane X Receptor Cross-Talk with Hepatic Nuclear Factor 4{alpha} to Synergistically Activate the Human CYP2C9 Promoter J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1125 - 1133. [Abstract] [Full Text] [PDF]
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S. S. Ferguson, Y. Chen, E. L. LeCluyse, M. Negishi, and J. A. Goldstein Human CYP2C8 Is Transcriptionally Regulated by the Nuclear Receptors Constitutive Androstane Receptor, Pregnane X Receptor, Glucocorticoid Receptor, and Hepatic Nuclear Factor 4{alpha} Mol. Pharmacol., September 1, 2005; 68(3): 747 - 757. [Abstract] [Full Text] [PDF]
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M. A. Mohutsky, D. M. Petullo, and S. A. Wrighton THE USE OF A SUBSTRATE CASSETTE STRATEGY TO IMPROVE THE CAPACITY AND THROUGHPUT OF CYTOCHROME P450 INDUCTION STUDIES IN HUMAN HEPATOCYTES Drug Metab. Dispos., July 1, 2005; 33(7): 920 - 923. [Abstract] [Full Text] [PDF]
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O. Burk, K. A. Arnold, A. K. Nussler, E. Schaeffeler, E. Efimova, B. A. Avery, M. A. Avery, M. F. Fromm, and M. Eichelbaum Antimalarial Artemisinin Drugs Induce Cytochrome P450 and MDR1 Expression by Activation of Xenosensors Pregnane X Receptor and Constitutive Androstane Receptor Mol. Pharmacol., June 1, 2005; 67(6): 1954 - 1965. [Abstract] [Full Text] [PDF]
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C. P. Martinez-Jimenez, M. J. Gomez-Lechon, J. V. Castell, and R. Jover Transcriptional Regulation of the Human Hepatic CYP3A4: Identification of a New Distal Enhancer Region Responsive to CCAAT/Enhancer-Binding Protein {beta} Isoforms (Liver Activating Protein and Liver Inhibitory Protein) Mol. Pharmacol., June 1, 2005; 67(6): 2088 - 2101. [Abstract] [Full Text] [PDF]
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H. Masuyama, N. Suwaki, Y. Tateishi, H. Nakatsukasa, T. Segawa, and Y. Hiramatsu The Pregnane X Receptor Regulates Gene Expression in a Ligand- and Promoter- Selective Fashion Mol. Endocrinol., May 1, 2005; 19(5): 1170 - 1180. [Abstract] [Full Text] [PDF]
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J. E. Chrencik, J. Orans, L. B. Moore, Y. Xue, L. Peng, J. L. Collins, G. B. Wisely, M. H. Lambert, S. A. Kliewer, and M. R. Redinbo Structural Disorder in the Complex of Human Pregnane X Receptor and the Macrolide Antibiotic Rifampicin Mol. Endocrinol., May 1, 2005; 19(5): 1125 - 1134. [Abstract] [Full Text] [PDF]
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C. Cheung, A.-M. Yu, J. M. Ward, K. W. Krausz, T. E. Akiyama, L. Feigenbaum, and F. J. Gonzalez THE CYP2E1-HUMANIZED TRANSGENIC MOUSE: ROLE OF CYP2E1 IN ACETAMINOPHEN HEPATOTOXICITY Drug Metab. Dispos., March 1, 2005; 33(3): 449 - 457. [Abstract] [Full Text] [PDF]
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X. Song, Y. Li, J. Liu, M. Mukundan, and B. Yan Simultaneous Substitution of Phenylalanine-305 and Aspartate-318 of Rat Pregnane X Receptor with the Corresponding Human Residues Abolishes the Ability to Transactivate the CYP3A23 Promoter J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 571 - 582. [Abstract] [Full Text] [PDF]
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X. Ding and J. L. Staudinger Induction of Drug Metabolism by Forskolin: The Role of the Pregnane X Receptor and the Protein Kinase A Signal Transduction Pathway J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 849 - 856. [Abstract] [Full Text] [PDF]
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M.-F. Yueh, M. Kawahara, and J. Raucy HIGH VOLUME BIOASSAYS TO ASSESS CYP3A4-MEDIATED DRUG INTERACTIONS: INDUCTION AND INHIBITION IN A SINGLE CELL LINE Drug Metab. Dispos., January 1, 2005; 33(1): 38 - 48. [Abstract] [Full Text] [PDF]
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E. J. Squires, T. Sueyoshi, and M. Negishi Cytoplasmic Localization of Pregnane X Receptor and Ligand-dependent Nuclear Translocation in Mouse Liver J. Biol. Chem., November 19, 2004; 279(47): 49307 - 49314. [Abstract] [Full Text] [PDF]
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C. A. Vyhlidal, P. K. Rogan, and J. S. Leeder Development and Refinement of Pregnane X Receptor (PXR) DNA Binding Site Model Using Information Theory: INSIGHTS INTO PXR-MEDIATED GENE REGULATION J. Biol. Chem., November 5, 2004; 279(45): 46779 - 46786. [Abstract] [Full Text] [PDF]
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E. T. Williams, M. Leyk, S. A. Wrighton, P. J. A. Davies, D. S. Loose, G. L. Shipley, and H. W. Strobel Estrogen Regulation of the Cytochrome P450 3A Subfamily in Humans J. Pharmacol. Exp. Ther., November 1, 2004; 311(2): 728 - 735. [Abstract] [Full Text] [PDF]
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N. Hariparsad, S. C. Nallani, R. S. Sane, D. J. Buckley, A. R. Buckley, and P. B. Desai Induction of CYP3A4 by Efavirenz in Primary Human Hepatocytes: Comparison With Rifampin and Phenobarbital J. Clin. Pharmacol., November 1, 2004; 44(11): 1273 - 1281. [Abstract] [Full Text] [PDF]
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O. Burk, I. Koch, J. Raucy, E. Hustert, M. Eichelbaum, J. Brockmoller, U. M. Zanger, and L. Wojnowski The Induction of Cytochrome P450 3A5 (CYP3A5) in the Human Liver and Intestine Is Mediated by the Xenobiotic Sensors Pregnane X Receptor (PXR) and Constitutively Activated Receptor (CAR) J. Biol. Chem., September 10, 2004; 279(37): 38379 - 38385. [Abstract] [Full Text] [PDF]
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T. Mitin, L. L. von Moltke, M. H. Court, and D. J. Greenblatt LEVOTHYROXINE UP-REGULATES P-GLYCOPROTEIN INDEPENDENT OF THE PREGNANE X RECEPTOR Drug Metab. Dispos., August 1, 2004; 32(8): 779 - 782. [Abstract] [Full Text] [PDF]
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D. E. Brobst, X. Ding, K. L. Creech, B. Goodwin, B. Kelley, and J. L. Staudinger Guggulsterone Activates Multiple Nuclear Receptors and Induces CYP3A Gene Expression through the Pregnane X Receptor J. Pharmacol. Exp. Ther., August 1, 2004; 310(2): 528 - 535. [Abstract] [Full Text] [PDF]
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Y. Yasunami, H. Hara, T. Iwamura, T. Kataoka, and T. Adachi C-JUN N-TERMINAL KINASE MODULATES 1,25-DIHYDROXYVITAMIN D3-INDUCED CYTOCHROME P450 3A4 GENE EXPRESSION Drug Metab. Dispos., July 1, 2004; 32(7): 685 - 688. [Abstract] [Full Text] [PDF]
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M. Podvinec, C. Handschin, R. Looser, and U. A. Meyer Identification of the xenosensors regulating human 5-aminolevulinate synthase PNAS, June 15, 2004; 101(24): 9127 - 9132. [Abstract] [Full Text] [PDF]
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T. Matsubara, H. J. Kim, M. Miyata, M. Shimada, K. Nagata, and Y. Yamazoe Isolation and Characterization of a New Major Intestinal CYP3A Form, CYP3A62, in the Rat J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1282 - 1290. [Abstract] [Full Text] [PDF]
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L.-S. Wang, B. Zhu, A. M. A. El-Aty, G. Zhou, Z. Li, J. Wu, G.-L. Chen, J. Liu, Z. R. Tang, W. An, et al. The Influence of St. John's Wort on CYP2C19 Activity with Respect to Genotype J. Clin. Pharmacol., June 1, 2004; 44(6): 577 - 581. [Abstract] [Full Text] [PDF]
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V. Bombail, K. Taylor, G. G. Gibson, and N. Plant ROLE OF Sp1, C/EBP{alpha}, HNF3, AND PXR IN THE BASAL- AND XENOBIOTIC-MEDIATED REGULATION OF THE CYP3A4 GENE Drug Metab. Dispos., May 1, 2004; 32(5): 525 - 535. [Abstract] [Full Text] [PDF]
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D. P. Hartley, X. Dai, Y. D. He, E. J. Carlini, B. Wang, S.-e. W. Huskey, R. G. Ulrich, T. H. Rushmore, R. Evers, and D. C. Evans Activators of the Rat Pregnane X Receptor Differentially Modulate Hepatic and Intestinal Gene Expression Mol. Pharmacol., May 1, 2004; 65(5): 1159 - 1171. [Abstract] [Full Text]
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K. Kobayashi, S. Yamagami, T. Higuchi, M. Hosokawa, and K. Chiba KEY STRUCTURAL FEATURES OF LIGANDS FOR ACTIVATION OF HUMAN PREGNANE X RECEPTOR Drug Metab. Dispos., April 1, 2004; 32(4): 468 - 472. [Abstract] [Full Text] [PDF]
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E. G. Schuetz Lessons from the CYP3A4 Promoter Mol. Pharmacol., February 1, 2004; 65(2): 279 - 281. [Full Text] [PDF]
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K. Matsumura, T. Saito, Y. Takahashi, T. Ozeki, K. Kiyotani, M. Fujieda, H. Yamazaki, H. Kunitoh, and T. Kamataki Identification of a Novel Polymorphic Enhancer of the Human CYP3A4 Gene Mol. Pharmacol., February 1, 2004; 65(2): 326 - 334. [Abstract] [Full Text] [PDF]
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Y. Chen, S. S. Ferguson, M. Negishi, and J. A. Goldstein Induction of Human CYP2C9 by Rifampicin, Hyperforin, and Phenobarbital Is Mediated by the Pregnane X Receptor J. Pharmacol. Exp. Ther., February 1, 2004; 308(2): 495 - 501. [Abstract] [Full Text] [PDF]
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X. Song, M. Xie, H. Zhang, Y. Li, K. Sachdeva, and B. Yan THE PREGNANE X RECEPTOR BINDS TO RESPONSE ELEMENTS IN A GENOMIC CONTEXT-DEPENDENT MANNER, AND PXR ACTIVATOR RIFAMPICIN SELECTIVELY ALTERS THE BINDING AMONG TARGET GENES Drug Metab. Dispos., January 1, 2004; 32(1): 35 - 42. [Abstract] [Full Text] [PDF]
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R. G. Tirona, B. F. Leake, L. M. Podust, and R. B. Kim Identification of Amino Acids in Rat Pregnane X Receptor that Determine Species-Specific Activation Mol. Pharmacol., January 1, 2004; 65(1): 36 - 44. [Abstract] [Full Text] [PDF]
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J. C. Ourlin, F. Lasserre, T. Pineau, J. M. Fabre, A. Sa-Cunha, P. Maurel, M.-J. Vilarem, and J. M. Pascussi The Small Heterodimer Partner Interacts with the Pregnane X Receptor and Represses Its Transcriptional Activity Mol. Endocrinol., September 1, 2003; 17(9): 1693 - 1703. [Abstract] [Full Text] [PDF]
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W. Zhang, A. F. Purchio, K. Chen, J. Wu, L. Lu, R. Coffee, P. R. Contag, and D. B. West A TRANSGENIC MOUSE MODEL WITH A LUCIFERASE REPORTER FOR STUDYING IN VIVO TRANSCRIPTIONAL REGULATION OF THE HUMAN CYP3A4 GENE Drug Metab. Dispos., August 1, 2003; 31(8): 1054 - 1064. [Abstract] [Full Text] [PDF]
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Y. Chen, S. S. Ferguson, M. Negishi, and J. A. Goldstein Identification of Constitutive Androstane Receptor and Glucocorticoid Receptor Binding Sites in the CYP2C19 Promoter Mol. Pharmacol., August 1, 2003; 64(2): 316 - 324. [Abstract] [Full Text] [PDF]
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G. R. Robertson, J. Field, B. Goodwin, S. Bierach, M. Tran, A. Lehnert, and C. Liddle Transgenic Mouse Models of Human CYP3A4 Gene Regulation Mol. Pharmacol., July 1, 2003; 64(1): 42 - 50. [Abstract] [Full Text] [PDF]
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Z. Dvorak, M. Modriansky, L. Pichard-Garcia, P. Balaguer, M.-J. Vilarem, J. Ulrichova, P. Maurel, and J.-M. Pascussi Colchicine Down-Regulates Cytochrome P450 2B6, 2C8, 2C9, and 3A4 in Human Hepatocytes by Affecting Their Glucocorticoid Receptor-Mediated Regulation Mol. Pharmacol., July 1, 2003; 64(1): 160 - 169. [Abstract] [Full Text] [PDF]
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J. M. Maglich, D. J. Parks, L. B. Moore, J. L. Collins, B. Goodwin, A. N. Billin, C. A. Stoltz, S. A. Kliewer, M. H. Lambert, T. M. Willson, et al. Identification of a Novel Human Constitutive Androstane Receptor (CAR) Agonist and Its Use in the Identification of CAR Target Genes J. Biol. Chem., May 2, 2003; 278(19): 17277 - 17283. [Abstract] [Full Text] [PDF]
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J. L. Raucy Regulation of CYP3A4 Expression in Human Hepatocytes by Pharmaceuticals and Natural Products Drug Metab. Dispos., May 1, 2003; 31(5): 533 - 539. [Abstract] [Full Text] [PDF]
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S. C. Nallani, B. Goodwin, J. M. Maglich, D. J. Buckley, A. R. Buckley, and P. B. Desai Induction of Cytochrome P450 3A by Paclitaxel in Mice: Pivotal Role of the Nuclear Xenobiotic Receptor, Pregnane X Receptor Drug Metab. Dispos., May 1, 2003; 31(5): 681 - 684. [Abstract] [Full Text] [PDF]
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H. Wang, S. Faucette, T. Sueyoshi, R. Moore, S. Ferguson, M. Negishi, and E. L. LeCluyse A Novel Distal Enhancer Module Regulated by Pregnane X Receptor/Constitutive Androstane Receptor Is Essential for the Maximal Induction of CYP2B6 Gene Expression J. Biol. Chem., April 11, 2003; 278(16): 14146 - 14152. [Abstract] [Full Text] [PDF]
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K. Kawana, T. Ikuta, Y. Kobayashi, O. Gotoh, K. Takeda, and K. Kawajiri Molecular Mechanism of Nuclear Translocation of an Orphan Nuclear Receptor, SXR Mol. Pharmacol., March 1, 2003; 63(3): 524 - 531. [Abstract] [Full Text] [PDF]
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B. Goodwin, K. C. Gauthier, M. Umetani, M. A. Watson, M. I. Lochansky, J. L. Collins, E. Leitersdorf, D. J. Mangelsdorf, S. A. Kliewer, and J. J. Repa Identification of bile acid precursors as endogenous ligands for the nuclear xenobiotic pregnane X receptor PNAS, January 7, 2003; 100(1): 223 - 228. [Abstract] [Full Text] [PDF]
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R. G. Tirona, B. F. Leake, A. W. Wolkoff, and R. B. Kim Human Organic Anion Transporting Polypeptide-C (SLC21A6) Is a Major Determinant of Rifampin-Mediated Pregnane X Receptor Activation J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 223 - 228. [Abstract] [Full Text] [PDF]
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T. A. Kocarek and N. A. Mercer-Haines Squalestatin 1-Inducible Expression of Rat CYP2B: Evidence That an Endogenous Isoprenoid Is an Activator of the Constitutive Androstane Receptor Mol. Pharmacol., November 1, 2002; 62(5): 1177 - 1186. [Abstract] [Full Text] [PDF]
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J. Raucy, L. Warfe, M.-F. Yueh, and S. W. Allen A Cell-Based Reporter Gene Assay for Determining Induction of CYP3A4 in a High-Volume System J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 412 - 423. [Abstract] [Full Text] [PDF]
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W. El-Sankary, V. Bombail, G. G. Gibson, and N. Plant Glucocorticoid-Mediated Induction of CYP3A4 is Decreased by Disruption of a Protein: DNA Interaction Distinct from the Pregnane X Receptor Response Element Drug Metab. Dispos., September 1, 2002; 30(9): 1029 - 1034. [Abstract] [Full Text] [PDF]
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E. Schuetz, L. Lan, K. Yasuda, R. Kim, T. A. Kocarek, J. Schuetz, and S. Strom Development of A Real-Time in Vivo Transcription Assay: Application Reveals Pregnane X Receptor-Mediated Induction of CYP3A4 by Cancer Chemotherapeutic Agents Mol. Pharmacol., September 1, 2002; 62(3): 439 - 445. [Abstract] [Full Text] [PDF]
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A. Takeshita, M. Taguchi, N. Koibuchi, and Y. Ozawa Putative Role of the Orphan Nuclear Receptor SXR (Steroid and Xenobiotic Receptor) in the Mechanism of CYP3A4 Inhibition by Xenobiotics J. Biol. Chem., August 30, 2002; 277(36): 32453 - 32458. [Abstract] [Full Text] [PDF]
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), p3A4-3195 (
),
encompassing base 
) were transfected into HepG2 cells. Cells were treated with the
indicated concentration of rifampicin for 48 h before luciferase
and 
, mutated PXREs. B, identification of
an additional functional PXRE (dNR3) required for maximal rifampicin
responsiveness. The sequence of the putative PXRE is shown, and the
repeated nuclear receptor half-sites within this motif are indicated by
horizontal arrows. The mutated bases are indicated. Transfected HepG2
cells were cultured in the presence of rifampicin (5 µM) for 48 h before harvest and determination of luciferase and 