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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: jstegeman@whoi.edu

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: Urs-A.Meyer@unibas.ch

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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McKee, D. D., Oliver, B. B., Willson, T. M., Zetterstrom, R. H., Perlmann, T., and

Lehmann, J. M. (1998) Cell 92(1), 73-82.

14. LeCluyse, E. L. (2001) Chem Biol Interact 134(3), 283-9.

15. Quattrochi, L. C., and Guzelian, P. S. (2001) Drug Metab Dispos 29(5), 615-22.

16. Negishi, M., and Honkakoski, P. (2000) Eur J Pharm Sci 11(4), 259-64.

17. Honkakoski, P., and Negishi, M. (2000) Biochem J 347(Pt 2), 321-37.

18. Kemper, B. (1998) Prog Nucleic Acid Res Mol Biol 61, 23-64

19. Sueyoshi, T., and Negishi, M. (2001) Annu Rev Pharmacol Toxicol 41, 123-43

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13(6), 289-95

21. Gerbal-Chaloin, S., Pascussi, J. M., Pichard-Garcia, L., Daujat, M., Waechter, F.,

Fabre, J. M., Carrere, N., and Maurel, P. (2001) Drug Metab Dispos 29(3), 242-51.

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23. Handschin, C., Podvinec, M., Stockli, J., Hoffmann, K., and Meyer, U. A. (2001) Mol

Endocrinol 15(9), 1571-85.

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Biochem Biophys 373(2), 375-84.

25. Meyer, R. P., Hagemeyer, C. E., Knoth, R., Kurz, G., and Volk, B. (2001) Biochem

Biophys Res Commun 285(1), 32-9.

26. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene

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27. Iniguez-Lluhi, J. A., Lou, D. Y., and Yamamoto, K. R. (1997) J Biol Chem 272(7),

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28. Ioannides, C. (1996) CRC press, Boca Raton, Florida 33431

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30. Kim, J., Min, G., and Kemper, B. (2001) J Biol Chem 276(10), 7559-67.

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Pharmacol 60(4), 681-9.

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D. (1996) Mol Pharmacol 49(1), 63-72.

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18

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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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

60

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

rela

tive

LU

Ca

ctivity

DM

SO

PB

ME

TC

LO

DE

XP

CN

TC

PO

BO

P

RIF

DP

H

C

0

50

100

150

200

250

0

50

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250

300

350

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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

5

6

7

8239bp - DR-4 wt

239bp - DR-4 mut

rela

tive

LU

Cactivity

DMSO RU486 PCN TCPOBOP0

0.5

1.0

1.5

2.0

2.5

3.0239bp - DR-4 wt 239bp - DR-4 mut

DMSO PB MET TCPOBOP

rela

tive

LU

Cactivity

rela

tive

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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