1, Carmela Gnerre1, John J. Stegeman2 and Urs A. Meyer1§ · show a variety of regulation patterns...
Transcript of 1, Carmela Gnerre1, John J. Stegeman2 and Urs A. Meyer1§ · show a variety of regulation patterns...
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Transcriptional Activation of Cytochrome P-450 CYP2C45 by Drugs is Mediated by the
Chicken Xenobiotic Receptor CXR Interacting with a Phenobarbital-Response
Enhancer Unit
Manuel Baader1, Carmela Gnerre1, John J. Stegeman2 and Urs A. Meyer1§
1Department of Pharmacology/Neurobiology, Biozentrum of the University of Basel,
Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland
2Department of Biology, Woods Hole Oceanographic Institution, MS 32, Woods Hole, MA
02543, USA. E-mail: [email protected]
This work was supported by the Swiss National Science Foundation and by NIH grant P42
ES07381 (John J. Stegeman).
The nucleotide sequences reported in this paper have been submitted to the GenBankTM/EBI
Data Bank with accession numbers …
§To whom correspondence should be addressed. Tel.: ++41-61-267-22-20; Fax: ++41-61-
267-22-08; E-mail: [email protected]
Running title: CYP2C45 Induction
3 The abbreviations used are: CAR, constitutive androstane receptor; CLO, clotrimazole;
CXR, chicken xenobiotic receptor; CYP/P450, cytochrome P-450; DEX, dexamethasone;
DMSO, dimethyl sulfoxide; DR, direct repeat; GRE, glucocorticoid-response element; LMH,
leghorn male hepatoma; LUC, luciferase; MET, metyrapone; NF1, nuclear factor 1; PB,
phenobarbital; PBRU, phenobarbital-response enhancer unit; PXR, pregnane X receptor;
RXR, retinoid X receptor
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on February 26, 2002 as Manuscript M109882200 by guest on February 17, 2020
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Summary
CYP2C enzymes fulfill an important role in xenobiotic metabolism and therefore have
extensively been studied in rodents and humans. However, no CYP2C genes have been
described in avian species to date. In this paper we report the cloning, functional analysis and
regulation of chicken CYP2C45. The sequence shares up to 58% amino acid identity with
CYP2Cs in other species. Over-expression of CYP2C45 in chicken hepatoma cells LMH led
to increased scoparone metabolism. CYP2C45 regulation was studied in LMH cells at the
mRNA level and in reporter gene assays using a construct containing 2.6kb of its 5’-flanking
region. Exposure of LMH cells to phenobarbital or metyrapone led to a 95- or 210-fold
increase in CYP2C45 mRNA and a 140- or 290-fold increase in reporter gene expression,
respectively. A phenobarbital-response enhancer unit (PBRU) of 239bp containing a DR-4
nuclear receptor binding site was identifyed within the 2.6kb fragment. Site-specific mutation
of the DR-4 revealed the requirement of this motif for CYP2C45 induction by drugs. The
chicken xenobiotic receptor CXR interacted with the PBRU in electro mobility-shift and
transactivation assays. Furthermore, the related nuclear receptors mouse PXR and CAR
transactivated this enhancer element, suggesting evolutionary conservation of nuclear
receptor-DNA interactions in CYP2C induction.
Introduction
Cytochromes P-450 (P450, CYP)3 are involved in the oxidative metabolism of numerous
endogenous and exogenous compounds, including steroid hormones, drugs, carcinogens, and
environmental pollutants. To fulfill their detoxifying role they catalyze the metabolism of a
wide spectrum of structurally unrelated substances (1). P450s are often inducible by their
own substrates allowing dynamic adaptation to xenobiotic exposure (2). Together with
CYP3A4, CYP2D6 and CYP1A2, enzymes of the CYP2C subfamily are mainly responsible
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for drug metabolism in human (3) and therefore can cause drug-interactions. Diazepam (4),
ibuprofen (5), phenytoin (6), sildenafil (7) and warfarin (8) are some examples of clinically
used drugs, whose metabolism involves enzymes of the CYP2C subfamily. CYP2C genes
show a variety of regulation patterns including sex-dependent regulation (9), constitutive
expression or transcriptional activation by classical P450 inducers such as phenobarbital
(PB), dexamethasone (DEX) and rifampicine (10).
In the last few years, major advances in understanding the molecular mechanism of P450
induction have been achieved. The constitutive androstane receptor (CAR) has been
identified as a CYP2B activator in mouse and human liver (11) (12). The role of the pregnane
X receptor (PXR) in CYP3A induction has been investigated by several groups (13) (14)
(15). CAR and PXR both bind to their cognate DNA elements as heterodimers with retinoid
X receptor (RXR) and thereby stimulate P450 target gene transcription (16). Two direct,
inverted or everted repeats surrounding a nuclear factor 1 binding site (NF1) have been
described as common features phenobarbital response-enhancer units (PBRU) of CYP2B
genes. Similar structures, but lacking an NF1 site, have been defined as PBRUs in CYP3A
genes. In addition it has been shown, that both CAR and PXR can activate CYP2B and
CYP3A genes thanks to their similar DNA binding preferences (17).
Only little progress has been accomplished in understanding the molecular mechanism of
CYP2C induction. Although human CYP2C 5’-flanking regions have extensively been
analyzed (18), the PB-response has not been associated with any DNA sequences of these
genes to date (19) (20). Recently, the effect of known PXR and CAR activators on CYP2C8,
-2C9, -2C18, and –2C19 mRNA has been analyzed (21). The results are consistent with an
involvement of CAR, PXR and the glucocorticoid receptor in CYP2C8 and CYP2C9 mRNA
induction.
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The chicken xenobiotic receptor CXR was cloned and identified as activator of the chicken
CYP2H1 gene (22). It has activation properties similar to CAR and PXR and also activates
PBRUs of mouse, rat and human P450s (23). Here we report the cloning and characterization
of the avian CYP2C45 gene. Furthermore we describe the identification of a first PBRU in
the 5’-flanking region of a CYP2C gene and the requirement of a DR-4 nuclear receptor
binding site for CXR-mediated induction of CYP2C45.
Experimental Procedures
Primers and Probes. Computer-assisted primer design was performed using the Oligo
Primer Analysis Software verison 5.0 (National Biosciences, USA). Primers were supplied by
Microsynth, CH. TaqMan probes coupled to a 5’ fluorophore (FAM) and a 3’ quencher
(TAMRA) were manufactured by Eurogentec, BE.
Cell Culture and Transfection. Cell culture was carried out as previously described by
Ourlin et al (24). Cells were maintained under serum-free conditions for 5 hours before
transfection or drug exposure. Cells were transiently transfected using the FuGENE 6
transfection reagent (Roche Molecular Biochemicals, CH) according to the supplier’s
protocol. Cells were induced for 16 hours with following drug concentrations: 600µM for PB
and MET, 50µM for DEX, rifampicin, pregnenolone 16α-carbonitrile, phenytoin, 1,4-bis[2-
(3,5-dichloropyridyloxy)]benzene and 10µM for clotrimazole (CLO).
Cloning and Sequencing. Total RNA was isolated from chicken liver tissue using the
peqGOLD RNAPureTM reagent (Axon Lab AG, CH) and subsequently reverse transcribed
using oligo d(14)TN primer and M-MLV Reverse Transcriptase (Life Technologies, CH).
Sequence alignments of fish CYP2 genes were used to design primers in conserved regions
(CYPdeg-fwd 5’-CCNCGNGAYTAYATYGA-3’ and CYPdeg-rev 5’-
AANARRAANARYTCCAT-3’) and a CYP2 related DNA fragment was amplified from a
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chicken cDNA library. Primers CYP-fwd (5’-CCGGGACTATATCGACTGCTTCC-3’) and
CYP-rev (5’-CAGGAAGAGCTCCATGCGCGCC-3’) were designed based on this sequence
and used for PCR amplification of a 550bp fragment from chicken cDNA. A chicken liver
λZAP® cDNA library (Stratagene, NL) was screened using the 32P-random primed labeled
550bp probe (Boehringer Mannheim, D). pBluescript phagemids were in vivo excised from
isolated positive colonies using the ExAssist/SOLR system acording to the manufacturor’s
protocol and analyzed by automated sequencing (ABI 373A, Perkin Elmer, CH).
Primers cod1-fwd (5’-CCGTGCCCACGTGGGAGATGTTGCT-3’ in exon1) and cod396-
rev (5’-GAGAGCAAACCGCCGAAC-3’ in exon3) were used for PCR amplification of a
1.2kb fragment from chicken genomic DNA. Six positive clones resulted from hybridization
of chicken BAC filters (UK HGMP Resource Centre, UK) with the 32P-radiolabelled 1.2kb
genomic DNA probe. BAC clones 25-P8, 86-J8 and 44-H2 were digested with ApaI, NcoI,
NsiI and PstI and further analyzed by southern blotting using the 1.2kb probe. A 3.6kb NsiI
fragment overlapping with exon 1 was subcloned into pGEM®-T Easy (Promega, CH) and
sequenced by primer walking starting with vector specific pBS-fwd (5’-
GTTTTCCCAGTCACGACGTTG-3’) and pBS-rev (5’-
CTATGACCATGATTACGCCAAG-3’) primers.
Protein Expression. LMH cells were transfected with a pCI-CYP2C45 construct or with
empty pCI vector as mentioned above. Cells were harvested in 100mM sodium phosphate
buffer pH 7.4 containing 0.2mM EDTA and 0.5mM DTT after 48 hours and sonicated five
times for 3 seconds on ice with an amplitude of 15 microns. Cell lysates were centrifuged at
9’000g for 10 minutes at 4°C. Supernatants were transferred to fresh tubes and subsequently
centrifuged at 105’000g for 1 hour at 4°C. Microsomal pellets were resuspended in sodium
phosphate buffer and protein concentrations determined using the Protein Assay ESL Kit
(Boehringer Mannheim, CH). Western blotting was performed as described by Ourlin et al
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(24) using a polyclonal goat anti rat CYP2C6 antibody (Daiichi Pure Chemiclas CO, Japan)
and protein G-horseradish peroxidase conjugate (Biorad, CH).
Scoparone Assay. CYP2C45 activities were measured by an assay of differential oxidation
of scoparone. 15µg microsomal proteins were incubated at 37°C for 15 minutes in 100mM
Tris buffer pH 7.6 supplemented with 2mM MgCl2, 80µM scoparone and 7.5mM NADPH.
Metabolites were separated and analyzed by HPLC as described in Meyer et al (25).
TaqMan Real Time PCR. Real time PCR was performed on an ABI PRISMTM 7700
(TaqMan) using the Sequence Detector Software version 1.6.3 (Perkin Elmer, CH).
Computer-assisted design of compatible TaqMan primers and probes was carried out with the
help of the Primer Express Software version 1.0 (Perkin Elmer, CH). 1µg of total RNA were
reverse transcribed as described above and the obtained cDNAs were diluted 1:5 for further
analysis. PCR reactions were performed using TaqMan PCR Core Reagent Kit (Perkin
Elmer, CH). Primer and probe concentrations were optimized as follows: TaqMan-fwd (5’-
CGGTGAAAGAAGCCTTGATTG-3’) 900nM, TaqMan-rev (5’-
GGTCCCCGATAGGCATGTG-3’) 300nM, TaqMan-probe (5’-FAM-
GGCAGCAAACTCATCCGCACGA-TAMRA-3’) 300nM. Levels of GAPDH housekeeping
gene were determined for internal normalization using GAPDH-fwd 5’-
GGTCACGCTCCTGGAAGATAGT-3’, GAPDH-rev 5’-GGGCACTGTCAAGGCTGAGA-
3’ and GAPDH-probe 5’-FAM-TGGCGTGCCCATTGATCACAAGTTT-TAMRA-3’.
Northern Blotting. 20µg of total RNA were subjected to electrophoresis on a formamide-
containing 1% agarose gel. RNAs were transferred to nylon membrane by overnight blotting
in 20xSSC (1x= 150mM NaCl, 15mM sodium citrate). Membranes were crosslinked using
the UV Stratalinker® 2400 (Stratagene, NL). Hybridization was carried out in 50% deionized
formamide, 5X SSC, 5X Denhardt’s solution, 1% SDS and 10% (w/v) dextransulfate. The
same 32P-radiolabelled 550bp cDNA probe as used before for the library screening was
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boiled for 5min in 500µl salmon sperm DNA (10mg/ml) and quickly chilled on ice.
Hybridization was carried out overnight at 45°C. Washes were performed in 2xSSC /1% SDS
at room temperature for 30min and 2xSSC / 1% SDS at 65° for 20min. Membranes were
exposed to X-ray film using intensifying screens for 12-48hr.
Reporter Constructs. A 2.6kb fragment of the 5’-flanking region of the CYP2C45 gene (-7
bp to –2612 bp) containing the homologous promotor was amplified from chicken genomic
DNA using primers flank-2.6kb_fwd 5’-GGAATTCGAACACACTGAGATCATCCTG-3’
and flank-2.6kb_rev 5’-GGAATTCGTGGGCACGAGCTTCTGAG-3’ and was subcloned
into pGL3-Basic reporter vector (Promega, CH). Furthermore, a 2.2kb fragment lacking
372bp of proximal promotor region (amplified with primers flank-2614 5’-
GAACACACTGAGATCATCCTG-3’ and flank-373 5’-TGCCATGTGGGTTTTCTGTTC-
3’) and a putative 239bp PBRU containing a DR-4 nuclear receptor binding site (amplified
with primers flank-162 5’-AATCGGCAGCAGAGAGAC-3’ and flank-380
5’CTTCTGAAAGACCTTGATGTG-3’) were subcloned into pGL3 reporter vector
(Promega, CH) containing the heterologous SV40 promotor (pGL3-SV40, Promega, CH).
The pRSV β-galactosidase vector used for normalization of transfection experiments was
kindly provided by Anastasia Kralli (Biozentrum, University of Basel, CH).
Mutagenesis. Site-directed mutagenesis of the DR-4 element in the 2.2kb and 239bp
fragments was carried out according to the PCR-based method of overlap extension (26)
using primers DR4mut-fwd 5’-AAGCTTTCCACTCGAGGCCCTGGCAATGTCGGAG-3’
and DR4mut-rev 5’-CTCGAGTGGAAAGCTTTGCGTCTCTAAGAACTTC-3’ (altered
nucleotides are indicated in bold). Primers flank-2614 and flank–373 or flank–162 and flank-
380 were used for amplification of mutated overlapping fragments to full-length 2.2kb or
239bp, respectively. Mutated fragments were subcloned into pGL3-SV40 as described.
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Reporter Gene Assay. Transfected and induced cells were harvested using Passive Lysis
Buffer (Promega, CH). Extracts were centrifuged for 3 minutes to pellet cellular debris. LUC
assays were performed on supernatants using Luciferase Assay Kit (Promega, CH) and a
Microlite TLX1 luminometer (Dynatech, CH). Relative β-galactosidase activities were
determined for normalization as described by Iniguez-Lluhi (27).
Electro Mobility-Shift Assay (EMSA). The 239bp EcoRI DNA fragment was 32P-
radiolabelled by 5’ filling-in with Klenow fragment of E.coli DNA polymerase (Boehringer,
CH). CXR and RXR were in vitro synthesized using the TNT transcription/translation-
coupled reticulocyte lysate sytem (Promega, CH) according to the supplier’s protocol. Assay
mixtures contained 10mM Tris pH 8.0, 40mM KCl, 0.05% NP-40, 6% glycerol, 1mM DTT,
0.2mg poly(dI*dC), 2.5µl of in vitro translated products and 25’000 cpm of 32P-radiolabelled
double-stranded DNA probe. The binding reaction was carried out at room temperature for 20
minutes. For supershift assays, antibodies against RXR or CXR were added to the reaction
mixtures. Competition assays were performed with a 100-fold molar excess of unlabeled
double-stranded DNA.
Transcriptional activation assays. CV-1 cells were maintained in DMEM/F-12 medium
supplemented with 10% fetal bovine serum. Before experiments, CV-1 cells were plated in
96-well plates at a density of 60’000 cells per well in DMEM/F12 medium without phenol
red, supplemented with 10% charcoal-stripped FBS. Cells were transiently transfected using
LipofectAMINE reagent (Life technologies) according to the manufacturer’s instructions.
Transfection mixes contained 20 ng of reporter plasmid, 50 ng of β-galactosidase expression
vector, 8 ng expression vector, except for CXR where 1 ng was used, and carrier plasmid.
Twenty-four hours after transfection, the medium was replaced by DMEM/F-12 without
phenol red, supplemented with 10% delipidated, charcoal stripped fetal calf serum (Sigma)
containing the inducers of interest. Cells were then incubated for additional 24 hours and
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harvested using using Passive Lysis Buffer (Promega, CH). Cell extracts were measured as
mentioned in “Reporter Gene Assay”.
Results
Cloning and Sequencing. A 550bp fragment was amplified from chicken cDNA using
primers derived from P450 sequence alignments as described in Experimental Procedures.
This fragment was used as probe to screen a chicken liver λZAP® cDNA library for full
length cDNA. The obtained sequence contained an open reading frame of 1485bp (Fig. 1A)
and was denominated CYP2C45 by David R. Nelson
(http://drnelson.utmem.edu/biblioA.html) based on high sequence identity with CYP2Cs in
other species.
A chicken BAC library was screened to obtain 5’-flanking region sequence information. A
3.6kb fragment was subcloned from a positive BAC clone and further analyzed (Fig. 1B).
Computer-assisted search for putative nuclear receptor binding sites was performed using an
algorithm developed by Michael Podvinec in our laboratory (Podvinec et al, manuscript in
preparation).
Expression and Activity. Immunoblot analysis was performed using an anti-rat CYP2C6
polyclonal antibody cross-reacting with CYP2C45 protein. A CYP2C45-GST fusion protein
had been expressed in BL21 cells to verify this interaction in advance (data not shown).
Microsomes prepared from PB-treated rat livers were used as internal control. Transient
transfection of CYP2C45 full length cDNA in LMH cells led to significant over-expression
of a protein of an estimated molecular mass of 55kDa, which was not detectable in
microsomes of control cells (Fig. 2). In addition, a weak band migrating close to CYP2C45
was visible in transfected and in control cells. Activity of over-expressed CYP2C45 was
measured using an assay of oxidative hydrolysis of scoparone, which had previously been
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used as sensitive indicator to distinguish between different P450 isoforms including CYP2Cs
(25). Weak but significant metabolism of scoparone by over-expressed CYP2C45 in LMH
cells was detected (TABLE 1). Isoscopoletin occured as main metabolite, whereas only low
levels of scopoletin were detected. Small amounts of isoscopoletin were also measured in
control cells.
Regulation of CYP2C45. Relative CYP2C45 mRNA levels were determined by TaqMan
and northern blot analysis. A dose response curve for PB is shown in Fig. 3A. Maximal
induction was obtained with PB concentrations above 600µM. CYP2C45 mRNA of untreated
cells was not detectable on northern blot. MET was the most potent CYP2C45 inducer in our
experimental system, followed by PB, pregnenolone 16α-carbonitrile, DEX, phenytoin and
CLO. Very weak or no induction was detectable after 1,4-bis[2-(3,5-
dichloropyridyloxy)]benzene and rifampicine treatment (Fig. 3B). A similar induction pattern
was obtained by LUC reporter gene assays using a reporter construct containing 2.6kb of
CYP2C45 5’-flanking region including the homologous promotor (Fig. 3C).
Role of DR-4 motif in CYP2C45 Regulation. The role of a DR-4 motif at –2342bp in
CYP2C45 induction was studied by LUC reporter gene assay. Relative LUC activities after
DMSO, PB and MET treatments were measured for the pGL3-SV40 reporter constructs with
following inserts: 2.2kb fragment wildtype, putative 239bp PBRU wildtype, 2.2kb DR-4
mutant, 239bp DR-4 mutant (Fig. 4A). The wildtype 239bp fragment retained almost full
inducibility compared to the 2.2kb fragment, whereas mutation of the DR-4 motif in any of
the fragments abolished induction (Fig. 4B). Physical interaction of CXR with the 239bp
fragment was investigated in electro mobility-shift assays (Fig. 5). Neither CXR, nor RXR
alone shifted the 32P-radiolabelled 239bp fragment. However, a shift was observed when
adding both CXR and RXR to the reaction mixture. This complex was supershifted with an
anti-RXR antibody or disabled adding an anti-CXR antibody. Shift was completely disabled
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when competing with a 100-fold molar excess of unlabelled wildtype DNA. As expected, no
shift was observed when using radiolabelled 239bp DR-4 mutant probe (data not shown).
Transactivation assays were performed to demonstrate not only physical but also functional
interaction between CXR and the 239bp fragment. CV-1 monkey kidney cells were co-
transfected with CXR expression plasmid and LUC reporter constructs containing 239bp
fragments with wildtype or mutant DR-4 motif (Fig. 6A). Treatment with PB or MET led to a
2- or 6-fold increase in reporter gene expression in cells transfected with 239bp DR-4
wildtype construct. No transactivation was observed in cells transfected with 239bp DR-4
mutated construct. Transactivation of the 239bp fragment with the mouse receptors PXR and
CAR were investigated in CV-1 cells. 2-fold PXR-mediated activation of the wildtype
construct was observed with RU486 and pregnenolone 16α-carbonitrile (PCN), whereas no
significant activation was detected with TCPOBOP (Fig. 6B). However, while no activation
was detected in the CAR assay with PB or MET, significant activation of the wildtype
construct was measured with TCPOBOP (Fig. 6C).
Discussion
We report the cloning of a new P450 cDNA in chicken. Comparison of the derived amino
acid sequence with other chicken P450s result in 34-36% identity with CYP1As, 56%
identity with CYP2H1 and 26% identity with CYP3A37. Based on sequence comparisons
with P450s in other species, the cDNA was assigned to the CYP2C subfamily (Fig. 7). It was
denominated CYP2C45 and represents a first member of the CYP2C subfamily cloned in
avian species. Before the discovery of CYP2C45 we had assumed that CYP2H1 may
represent a chicken CYP2C orthologue based on its regulation by drugs (24). However, the
observation that CYP2Cs occur in clusters of highly related genes in other species including
human and rabbit does not support this hypothesis (28).
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We have analyzed transcriptional regulation of CYP2C45 in LMH cells. The LMH cell line is
the first continuously dividing cell line that maintains phenobarbital-type induction of P450s
(29). The basal expression level of CYP2C45 in LMH cells is very low, which means that
neither protein, nor mRNA are detectable in untreated cells (Fig. 2 and 3A). However, a
dose-dependent increase in CYP2C45 mRNA was observed after exposure to increasing PB
concentrations (Fig. 3A). The effect of several prototypical P450 inducers on CYP2C45 was
analyzed both at the mRNA level and in reporter gene assays using a 2.6kb fragment of its 5’-
flanking region (Fig. 3B and C). The results were compared to data obtained from reporter
gene assays with a 264bp PBRU of the CYP2H1 gene (29). Similar induction patterns were
observed, suggesting a conserved mechanism of induction. Indeed, a structure consisting of a
NF1 site and a DR-4 nuclear receptor binding site resembling the CYP2H1 PBRU was
discovered in the 2.6kb fragment. PBRUs of inducible P450 genes in mammals have
extensively been studied and two direct, inverted or everted repeats surrounding a nuclear
factor 1 binding site (NF1) have been described as common features (19) (30). However, in
the case of CYP2H1, a second DR-4 element was only recently detected at a distance of 89bp
from the NF1 site (Michael Podvinec, unpublished data). To further characterize the function
of the putative CYP2C45 PBRU we have cloned a 2.2kb and a 239bp fragment surrounding
the DR-4 and NF1 sites. Both fragments are strongly activated by PB and MET in reporter
gene assays. Site-directed mutagenesis of the DR-4 motif abolished induction in both the
239bp and the 2.2kb fragment (Fig. 4). In contrast, disruption of the NF1 site by site-directed
mutagenesis had no effect on induction (data not shown). We have analyzed the interaction of
the CYP2C45 239bp fragment with CXR, which has been identified as activator of the
CYP2H1 264bp PBRU (22). Physical interaction was investigated in electro mobility-shift
assays, whereas functional interaction was tested in transactivation assays in CV-1 cells. The
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results uniformly demonstrated the requirement of the DR-4 element for induction and the
capability of a CXR-RXR heterodimer to activate the CYP2C45 239bp PBRU.
In mammals, CAR was originally identified as CYP2B activator and PXR as CYP3A
activator. However, overlapping ligand specificities of CAR and PXR and their capability to
activate both CYP2B and CYP3A PBRUs have been demonstrated, for review see (17).
Moreover, interchangeability of nuclear receptors and PBRUs between mouse, rat, human
and chicken has been investigated in our laboratory ((31)). We have investigated the
capability of the mouse receptors PXR and CAR to activate the chicken CYP2C45 239bp
PBRU. In both cases significant transactivation of the wildtype compared to the mutant
construct was detected for some inducers, indicating that both PXR and CAR are able to bind
to and activate the chicken CYP2C45 239bp PBRU. Conclusive, these results give rise to the
hypothesis, that molecular mechanisms of P450 induction are conserved from chicken to
mammals and that the induction of human CYP2C genes might involve the nuclear receptors
CAR and PXR as well as PBRU-like structures.
Surprisingly, DEX has a strong effect on CYP2C45 mRNA but does only modestly activate
the 2.6kb reporter construct. In contrast to CYP2H1 and inducible CYP2B and CYP3A 5’-
flanking regions, no glucocorticoid-response element (GRE) was detected in the 2.2kb
fragment (32) (33) (34). From these observations we suggest that a GRE must be localized
outside the 2.2kb fragment and mediate induction of CYP2C45 by DEX.
In conclusion the analysis of this avian P450 of the CYP2c subfamily indicates that the
induction of CYP2C genes requires the same nuclear receptors and DNA response elements
as the induction of CYP2B and CYP3A genes.
Acknowledgments
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We thank Dr. Margie Oleksiak from John Stegeman’s group for the homology cloning, Dr.
Ralf P. Meyer for helping with the activity assays, Dr. Christoph Handschin and Michael
Podvinec for sequence analysis. We also thank the UK HGMP Resource Center for providing
the Chicken BAC library and the originators Richard Crooijmans et al.
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Figures
Fig. 1. A, full-length cDNA of CYP2C45 was cloned from a chicken liver λZAP® cDNA
library. Translation start (+17) and stop (+1499) codons are highlighted in boldface. Positions
of the first two introns (indicated with bars), were derived form comparison of the cDNA
with the genomic sequence. B, genomic DNA containing a 3.6kb fragment of 5’-flanking
region was obtained from a chicken BAC clone and sequenced by primer walking. The first
two exons were determined form overlap with the cDNA sequence and are shaded. The DR-4
nuclear receptor binding site at position –2342 (surrounded by a box) turned out to be
essential for xenobiotic mediated transcriptional activation.
Fig. 2. CYP2C45 full-length cDNA was subcloned into pCI-vector and over-expressed in
LMH cells for 48 hours (lanes 4-6). Control cells were transfected with empty pCI-vector
(lanes 1-3). 10 µg of microsomal protein were subjected to electrophoresis on a 12%
polyacrilamide gel. A polyclonal antibody generated against rat CYP2C6 was used for
detection. PB induced rat microsomes were added as positive control for the antibody (lane
C).
Fig. 3. A, LMH cells were treated with increasing concentrations of PB (0-1500µM) for 16
hours. CYP2C45 mRNA levels were quantified using the TaqMan real time PCR technology.
Data are represented as relative mRNA levels compared to untreated samples and are
corrected with values measured for GAPDH amplification. Results were confirmed by
northern blot using a 32P-radiolabelled cDNA as probe. B, LMH cells were treated with
various compounds for 16 hours. mRNA levels were quantified as described above. C, LMH
cells were transfected with LUC reporter construct containing 2.6kb of 5’-flanking region and
4 hours later treated for 16 hours with various compounds. Data are represented as relative
LUC activity compared to untreated samples and are corrected with values measured for
empty LUC reporter construct. Abbreviations: DMSO dimethyl sulfoxide, PB phenobarbital,
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19
MET metyrapone, CLO clotrimazole, DEX dexamethasone, PCN pregnenolone 16α-
carbonitrile, DPH phenytoin, TCPOBOP 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, RIF
rifampicine
Fig. 4. A, Schematic picture of the 2.2kb fragment of CYP2C45 5’-flanking region and
localization of the 239bp phenobarbital-response enhancer unit (PBRU). A DR-4 nuclear
receptor binding site and a nuclear factor NF1 site were identified within the 239bp PBRU.
B, LMH cells were transfected with LUC reporter constructs containing the 2.2kb or the
239bp fragment with wildtype or mutated DR-4 element. After 4 hours cells were treated for
16 hours with DMSO, PB or MET. Data are represented as relative LUC activity corrected
with values measured for empty reporter construct. Activity of the 2.2kb construct induced
with MET corresponding to 150-fold was arbitrary set to 100%.
Fig. 5. The 239bp fragment was 32P-radiolabelled and used as probe for electo mobility-shift
assays. In vitro translated CXR and RXR were incubated separately and together with the
probe (lanes 2-4). The shifted CXR-RXR complex was supershifted using an anti RXR
antibody (lane 5). Competition was carried out using a 100-fold excess of cold wildtype DNA
(lane 6). An anti CXR antibody was added to the reaction together with CXR and RXR
protein (lane 7).
Fig. 6. CV-1 cells were transiently co-transfected with CXR, mouse PXR (mPXR) or mouse
CAR (mCAR) expression plasmids and LUC reporter constructs containing either wildtype
or mutated 239bp fragment. Cells were treated for 24 hours with various compounds. Data
represent relative LUC activities compared to DMSO treated samples.
Fig. 7. Phylogeny of human and rat CYP2B and CYP2C amino acid sequences including
chicken CYP2C45. The phylogenic tree was created using the ClustalX 1.8 and TreeView
1.6.1 programs. The scale bar represents 10 substitutions in 100 residues.
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Table 1 Scoparone metabolism of CYP2C45
Isoscopoletin Scopoletin
pCI-CYP2C45 1.77 +/- 0.022 0.07 +/- 0.047
pCI-control 0.05 +/- 0.027 n.d.
Scoparone metabolism of over-expressed CYP2C45 in LMH cells was analyzed by HPLC.
LMH cells were transfected with empty pCI vector as negative controls. Isoscopoletin and
scopoletin were detected as metabolites of over-expressed CYP2C45. Low levels of
isoscopoletin were also measured in control cells. Data are represented as pmol metabolite
per minute and mg microsomal protein and are mean values of three independent transfection
experiments.
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1 CGTGCCCACGTGGGAG TTGCTCCTGGGAGCAGCGAGTGTGGTCCTCCTGGTTTGTGTTGCTTGCCTGCTCTCCATCGTGCAATGGAGAAAAAGGACT
M L L L G A A S V V L L V C V A C L L S I V Q W R K R T
101 GGAAAGGGGAAGATGCCTGAGGGACCAACTCCCCTTCCCATCGTAGGGAACATACTGGAGGTGAAACCAAAGAATTTAGCCAAAACCCTTGAGAAGCTCG
G K G K M P E G P T P L P I V G N I L E V K P K N L A K T L E K L A
201 CTGAGAAATATGGGCCCGTCTTCTCAGTGCAACTGGGTTCAACTCCAGTAGTGGTGCTATCTGGATATGAGGCGGTGAAAGAAGCCTTGATTGATCGTGC
E K Y G P V F S V Q L G S T P V V V L S G Y E A V K E A L I D R A
301 GGATGAGTTTGCTGCCAGAGGACACATGCCTATCGGGGACCGGGCAAACAAAGGATTAGGCATTATTTTCAGCAACAACGAGGGATGGTTACACGTTCGG
D E F A A R G H M P I G D R A N K G L G I I F S N N E G W L H V R
401 CGGTTTGCTCTCAGCACTCTGCGCAACTTTGGGATGGGGAAGAGGAGCATTGAAGAGAGGATCCAGGAGGAAGCTGAGCACTTGCTTGAAGAGATCACAA
R F A L S T L R N F G M G K R S I E E R I Q E E A E H L L E E I T K
501 AAACAAAGAGACTGCCCTTTGACCCAACATTCAAGCTGAGCTGCGCTGTCTCCAACGTCATATGCTCCATTGTCTTTGGGAAGCGATATGACTATAAAGA
T K R L P F D P T F K L S C A V S N V I C S I V F G K R Y D Y K D
601 CAAGAAGTTCCTATCTCTGATGAACAACATGAACAACACATTTGAGATGATGAACTCCCGCTGGGGACAGTTATACCAGATGTTCTCCTACGTTCTGGAT
K K F L S L M N N M N N T F E M M N S R W G Q L Y Q M F S Y V L D
701 TATTTGCCCGGCCCACATAACAATATATTCAAAGAAATTGATGCTGTAAAAGCCTTTGTGGCAGAAGAGGTAAAGCTGCACCAAGCCTCCCTGGATCCCA
Y L P G P H N N I F K E I D A V K A F V A E E V K L H Q A S L D P S
801 GCGCTCCCCAGGATTTCATCGACTGCTTCCTCAGCAAAATGCAGGAGGAAAAAGACAATCCCAAATCACACTTCCACATGACAAACCTGATAACGTCCAC
A P Q D F I D C F L S K M Q E E K D N P K S H F H M T N L I T S T
901 CTTCGACTTGTTCATTGCTGGAACGGAGACAACAAGCACCACCACACGATACGGGCTTCTGCTTCTTCTCAAATATCCCAAGATACAAGAGAAAGTTCAA
F D L F I A G T E T T S T T T R Y G L L L L L K Y P K I Q E K V Q
1001 GAAGAGATTGACCGGGTAGTAGGACGATCACGAAGACCTTGCGTGGCTGACCGGACCCAGATGCCCTACACAGACGCAGTGGTCCACGAAATCCAGCGCT
E E I D R V V G R S R R P C V A D R T Q M P Y T D A V V H E I Q R F
1101 TCATCACTCTCATCCCTACGAGCCTCCCTCATGCTGTGACCAAAGACATCCACTTCAGAGACTACATCATTCCCAAGGGCACCACAGTCATGCCCCTCCT
I T L I P T S L P H A V T K D I H F R D Y I I P K G T T V M P L L
1201 CAGTACTGCACTCTATGACAGCAAGGAGTTTCCAAACCCAACCGAGTTTAATCCTGGACATTTCTTGAACCAGAATGGCACCTTTAGGAAGAGCGACTTC
S T A L Y D S K E F P N P T E F N P G H F L N Q N G T F R K S D F
1301 TTCATTCCCTTCTCAGCAGGGAAACGCATTTGCCCTGGAGAGGGCCTGGCACGCATGGAGATATTCTTACTCCTGACCGCCATCCTGCAGAACTTCACCT
F I P F S A G K R I C P G E G L A R M E I F L L L T A I L Q N F T L
1401 TGAAGCCTGTCATCAGCCCTGAGGAACTCAGCATCACCCCTACACTGAGTGGGACAGGAAATGTTCCTCCCTACTACCAGCTCTGTGCTTTCCCCCGC
K P V I S P E E L S I T P T L S G T G N V P P Y Y Q L C A F P R *
1501 GGGGCACAAAACCTCACTGCTGTGCTCCTCAGCCAGACTGCTCCTTTACACCTCCCCAACTCAAACCAGTGGCAGGAGCGTTGCCCCACCAACCCAAAG
1601 CCTCCACATGACAGCCCGCAGACAAAGTCCCAGGCAGATCAAACCCGGATACTTTGAACACCTCCCTGAACTGCTCCTCCTCACCACAGCGCAGAAGGTA
1701 ATGATGCACCTCACTGCAGTGACATTCTGTGCATGTGCTCCCTGAGCACAGCAGTC
ATG
TG
A
Figure 1a 21
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1 GAGGGCAGCACCCTGCAGGCAGGGCTCTCTTCCCCAGGGTGAGGTCCTGCCTGCTTCACCGCTGGACTCACTGCTGCTGCCTGCAACAATCCCTGCCAGT
101 ATGCTGCACACAGGGTGCTGACAGAAAGCTGCAGCATCCCTCCTGGAGGACAGCTCTCCCCCCAGTTCAGAGGCTGAAGGAAGCCACAGCTTCAGTGCTG
201 GTGAGACCTCTTTGTTGGGACCTCTGCCTGCTGTTGGGGGCCCTTGACAAGAAAACCTTTGACAAGAAGAGGAATTAGGTACCACCATCAATAACTGCTG
301 TGAACCAAAGTGCAAATGGCCCCGTGCACTCTGGGACTGCCTCCAGTGAGAATGCCGTGTGTCTGTCCCGTACCATGCAAGGCATGTGGTACGCTGTGCA
401 GAACAGCTGTGCACAGACATGCAACAATTGACACACCACCCAGGGCCAAAACCTGCCAAAATGCCAAAAGGCACTTGCAGGCAGCAGGGATATCCTCTCC
501 AAGCCTCCCGTGTGAGGGGGGTGCTGGCAGCCCTGAGGGCAGGAGTGGCACTTGCCGCTGACACCTGCTGAACCAAGGTCCAAAAAGAAAAGGAGGAGCA
601 GACAGCAAACCTACAGGAGATTCTACACAGGGGGGACATTTCCCTCAAGCTTGAGTTTTACTTCAGAAGAAAAGAGACCAAGTTACAACACTTGCTAGCC
701 TGTGACTTGACAATCCCAAACACATGTCTCCAATGGAACCAGGAAAAGTGGAAAGAGGGTCTTTGAGCCTCACCTCCCTGGACTTTCAAACAGATGGAGA
801 GAAACAACTAGTCTAGCATACTCGTGGCTTCTGCAAGCAGAAGATAACACTCAGAAAAGCCATGGGCGCTGCTGGGACATGGAAAGGTCTATGGCCTCTG
901 GCAGAAAAAGGTCAGAGCGACTGCACTTGTGAGTTCTCCCTCTTGTAAATAAATTTCTGCGTGCCCGCCAGGGTGGTGGGAGTATTTGGAACACACTGAG
1001 ATCATCCTGATGAAAGCGAGAAATCTTGGAAGTATTTCTAGTTCCAGGCCTTTTGCTAGAATTATGGCACAAAGAGTGAGGCAGCGTTGAGAAGGGAACC
1101 CTGCTGCCCAGGAACAGGGGAAATTCACAGAAAAGCTGGCAGGGATATGAATCGGCAGCAGAGAGACATCGTGTAGCAATACAAACTGCCATGAACTCCT
1201 CTGCAAAGCAAACACCCATGATTTCCACTTTCCCCAACTAGAAGTTCTTAGAGACGCA TCCA GCCCTGGCAATGTCGGAGCTCATGCA
1301 GCAGTTAAGATTAGCATATCAAAACCTTGTCTGAACTTGCTGGCAATTCCGTAATTCGCCAGAGAATCACATCAAGGTCTTTCAGAAGTTAAATGGAAAG
1401 TAGAAAGTTCCAGGCCTTCCAGAGCAGGACTTCCTCACACCACAGCATCAGAGATTGGTTGGATTGAATGGACCCTCAAAGACCATCTAGATCCAACACC
1501 CCTGCCCTGGGAAGGGACACCATCCAATAAACCAGGTTGCTCTAAGTCCCATCCAGCCTGACCTTGAACTCCTCCAGGGATGGGGCATCCATAGCTTCTG
1601 CAGACAGCCTGTTTCTGCACCAAACCACCCTCACTGTGAATACTGTGTTCCTTACGTCTAACCTAACCCTATACCCTTTCAGATTAAAACCATAATGCCC
1701 TGACCTATCTCTACGCACCTTCCACTTCTCTCCTGTGAGCCCTCTTTAAGAACTGGAAGGCTACCGTAAGAGCTCCTTGGAGCCTTCCATTCTCCAGGCT
1801 GAACAAACACAGCTCTCTCAGCCTAACTTCTCAGGAGAGGTGCTTCAGCCATGTCCTTTCACGACCCTCCGCTTTACCTGCTACAAGAAGTCCATGTAAC
1901 TCTTATGCTGGCAGCCGCTGAGCTCCCAGATGCAGAACTGCAGGTTTGGTCTCAGAAGCACAGAGTAGAGTGGCAGAATTACTTCCCTTGACCTGTGGGC
2001 CACATTTCATTTAACGCAAGTGTAGAATACAATTGGATTTTTAAGGCACAGGTCTTCACTGCCAGCTCAAGTCAAGTCTTTCATACAACAGTGCCTCCAA
2101 AACCTACTCCAGAAGGCTGCTCCTTTTGGAGCAGACCTCCTAGTCTCTTCTGGTGTTTGGCATTGCCCCAGACAGGTGCAGGACCTTTAAGTGAGCTTTC
2201 TCCAATGTCCTGAGACCCTGGCACATTCACAACTCAAGCCCTTTCCCCAGAGAGTATCAGCGGCACCCTCAAATTTGACGAGGGAGCACTCAGAAAGCCT
2301 CTTGACATCACTGTCACTTAACCCAAGCAGCCCGCACAAAGAAAGCATTCCCTTCCCAGCCCCGGGGGTAAGGAGAGCTGCAAGCTCCCTTGGCCACAAG
2401 CCAGCAGGCCAGGGTGAACAAAAGGCCTACTCCAAGTCCCTGCAACCAAAGCAAGGGAACCAAGCAACCCCAAGAAGAACACGGCCCCAAACACAGCCAT
2501 GGCCTGCCCTACAACACCCATCCTGCTCTCCCGCCAAGCACCATGAAGTACCTGCTCTTAGCCTGGCCCTTTTAAGCAGTAAAGGCAGCTGGGAATAATA
2601 AGTCCTGATGGTTCCAGGTGGAGCCAACTCTGCAGTTTAACACCTTCTTCTGCTCCCAGCTGCCTCCAAATCCATCACAGTGTGCTTGCACCCGTGCCAG
2701 CAAACAGCCACAGGGCACCTGCTAGCTTCTGTCAAGAGATGGGTCTTGTCCTAACCCAGATCTGTGAGCAGCTCGTTTGTGCACTCCACTCTGGAGCACA
2801 AGATGGGCCTTCCAGCTCTTCCTCTCACCCTGAGTTCTTCCCTCCATGGCAAATCCCACAGTCCTGCCTTCTTTTCCCCACGTTTCCCCCTCTTGAAGCA
2901 ATGATTGCTCATTCCATGGCTTAAGTGCAAAAAGCTGAGTGACAGTGCTCTCTGGTAAAGGAGTCACTGCTTCAAATCCTACACGCAGGCTGCTAACATG
3001 GAAGCACACAAACAGACTCCTGCATGCAATCGATGGATAAGTAGGCAGCTGTGCACACGTGAGAAGAGCTGATTTCCCTTATATACTCCTGGCTCTGGAC
3101 TGCAGCACTGTTTGCATGCAGTGCAACCTTAACCACTGGTGCCAGGAAGACTTCAGGAAGCAGCACAGATGGTTTCCCTCAATGAGAACTGACACAAACC
3201 ATGGTGAAGAACAGAAAACCCACATGGCACCAGTGTATGCGGAACACGCTCGACAAGAGTACATTTCTGGGAGCACCTCAAGTGGAACCCACGGGTGGAA
3301 CCCCACCCATGCTATGGACTCTGTAACAATGAACTTAGAGGGAAATAAGCAGTACTATGAGCAGACATGAAACTTCTTAAACTCTGATGGCCTCTGCAGT
3401 TGTTTGACACAAGGTGTGTCTTTAATCAACAATGCGAGAAGCCCTAATTTCGCAATACAGAGCAGACTCAGAAGTTTGCTTAGGAGTATTGGGTAACTCA
3501 GAAAAACCTGTTGTTTACCTAACAAAGCAGATCCATTCCATATATAAAGGGGCACGGGCAGCCATAACAGTGCACTCAGAAGCTCGTGCCCACGTGGGAG
3601 TTGCTCCTGGGAGCAGCGAGTGGGTCCTCCTGGTTTGGTGCTTGCCTGCTCTCCATCGTGCAATGGAGAAAAAGGACTGGAAAGGGGAAGATGCCTG
3701 AGGGACCAACTCCCCTTCCCATCGTAGGGAACATACTGGAGGTGAAACCAAAGAATTTAGCCAAAACCCTTGAGAAGCTAAGGACTCCCTTCTTTCCTCT
3801 CAGTTTCTGCTGAGGGTGAGGAATGTGNGGGCTGTGTGCACGGGGACGACGTGTGGCTATACCCTGCTGAGGGGCAATGGGCAAGGAGCAGAAAAGCCCT
3901 GGGGATTCTCAGCTGCCCCCCACACTGCTGTCACCCTTGTTTAGGGGCTGATGGCTCGCTGATGTTTGTCTTGAGAGGAACAGGCCTTACTTAACATGGC
4001 AGGTTGGAGCGGCTGCACAGACTGCAGCAGGAGGCAGCAGCCCAATCACTTTTCAACCCCTCCTCTCTACCTGCTCCATGGACACGCTCTCGCTTGCATT
4101 TTGACAGCTCGCTGAGAAATATGGGCCCGTCTTCTCAGTGCAACTGGGTTCAACTCCAGTAGTGGTGCTATCTGGATATGAGGCGGTGAAAGAAGCCTTG
4201 ATTGATCGTGCGGATGAGTTTGCTGCCAGAGGACACATGCCTATCGGGGACCGGGCAAACAAAGGATTAGGCATGTTCACTGACTCAGCCTCCCCTGAGG
4301 AAAAGGGAAGCGCTGAGGACAAGTATGGCAGATGAGGGCTGGAGCAAATG
TGAACT TGAACT
ATG
Figure 1b 22
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51kDa
94kDa
M 1 2 3 4 5 6 C
pCI -
cont
rol
pCI -
CYP2C
45
rat m
icro
som
es
mar
ker
Figure 2 23
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PB [ M]m
0
20
40
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80
100re
lative
mR
NA
levels
2.6kb
1.9kb
A
0 10
50
100
200
400
600
900
1200
1500
Figure 3 24
B
rela
tive
mR
NA
leve
ls
DM
SO
PB
ME
TC
LO
DE
XP
CN
TC
PO
BO
P
RIF
DP
H
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LU
Ca
ctivity
DM
SO
PB
ME
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LO
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RIF
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H
C
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A
DR-4 NF1
-2614 -373
239bp PBRU
CYP2C45 5’-flanking region2.2kb fragment
B
Figure 4 25
0%
25%
50%
75%
100%
125%
rela
tive
LU
Cactivation
2.2kb-pGL3-SV40
239bp-pGL3-SV40
Metyrapone Phenobarbital
wt mut wt mut
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TN
T-m
ock
CX
R
RX
R
CX
R+
RX
R
CX
R+
RX
R+
AB
-RX
R
CX
R+
RX
R+
wt-
com
petito
r
CX
R+
RX
R+
AB
-CX
R
1 2 3 4 5 6 7
free probe
shift
supershift
Figure 5 26
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DMSO PB MET0
1
2
3
4
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6
7
8239bp - DR-4 wt
239bp - DR-4 mut
rela
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DMSO RU486 PCN TCPOBOP0
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1.5
2.0
2.5
3.0239bp - DR-4 wt 239bp - DR-4 mut
DMSO PB MET TCPOBOP
rela
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LU
Cactivity
rela
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LU
Cactivity
A
B
C
Chicken CXR
Mouse PXR
Mouse CAR
0
1.0
2.0
3
4
0.5
1.5
2.5
3.5 239bp - DR-4 wt
239bp - DR-4 mut
Figure 6 27
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CYP2B6
CYP2B21
CYP2B2CYP2B1
CYP2C24
CYP2C11
CYP2C45
CYP2C23
CYP2C7
CYP2C12
CYP2C6
CYP2C19 / 2C9
CYP2C8
CYP2C18
0.1
Figure 7 28
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Manuel Baader, Carmela Gnerre, John J. Stegeman and Urs A. Meyerenhancer unit
the chicken xenobiotic receptor CXR interacting with a phenobarbital-response Transcriptional activation of cytochrome P-450 CYP2C45 by drugs is mediated by
published online February 26, 2002J. Biol. Chem.
10.1074/jbc.M109882200Access the most updated version of this article at doi:
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