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J. Biol. Chem., Vol. 277, Issue 18, 15647-15653, May 3, 2002

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Transcriptional Activation of Cytochrome P450 CYP2C45 by Drugs Is Mediated by the Chicken Xenobiotic Receptor (CXR) Interacting with a Phenobarbital Response Enhancer Unit^*

Manuel Baader, Carmela Gnerre, John J. Stegeman^§, and Urs A. Meyer^¶

From the  Department of Pharmacology/Neurobiology, Biozentrum of the University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland and the ^§ Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543

Received for publication, October 12, 2001, and in revised form, February 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytochromes P450 (CYP)-2C 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. The overexpression of CYP2C45 in chicken hepatoma cells leghorn male hepatoma (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 2.6 kb 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 239 bp containing a DR-4 nuclear receptor binding site was identified within the 2.6-kb 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 electromobility shift and transactivation assays. Furthermore, the related nuclear receptors, mouse PXR and mouse CAR, transactivated this enhancer element, suggesting evolutionary conservation of nuclear receptor-DNA interactions in CYP2C induction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytochromes P450 (CYP)¹ 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, the enzymes of the CYP2C subfamily are mainly responsible 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 (13-15). 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 of 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 with credit 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, CYP2C9, CYP2C18, and CYP2C19 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.

The chicken xenobiotic receptor CXR was cloned and identified as an 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Primers and Probes-- Computer-assisted primer design was performed using the oligo-primer analysis software, version 5.0 (National Biosciences). Primers were supplied by Microsynth. TaqMan probes coupled to a 5'-fluorophore (FAM) and a 3'-quencher (TAMRA) were manufactured by Eurogentec.

Cell Culture and Transfection-- Cell culture was carried out as described previously by Ourlin et al. (24). Cells were maintained under serum-free conditions for 5 h before transfection or drug exposure. Cells were transiently transfected using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Rothrenz, Switzerland) according to the supplier's protocol. Cells were induced for 16 h with following drug concentrations: 600 µM for PB and MET, 50 µM for DEX, rifampicine, pregnenolone 16-carbonitrile, phenytoin, and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, and 10 µM for clotrimazole.

Cloning and Sequencing-- Total RNA was isolated from chicken liver tissue using the peqGOLD RNAPure^TM reagent (Axon Laboratories AG, CH) and subsequently reverse-transcribed using oligo(dT)₁₄N primer and Moloney murine leukemia virus reverse transcriptase (Invitrogen). 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 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 550-bp fragment from chicken cDNA. A chicken liver ZAP® cDNA library (Stratagene) was screened using the ³²P random prime- labeled 550-bp probe (Roche Molecular Biochemicals). pBluescript phagemids were in vivo excised from isolated positive colonies using the ExAssist/SOLR system according to the manufacturer's protocol and analyzed by automated sequencing (ABI 373A, PerkinElmer Life Sciences).

Primers cod1-fwd (5'-CCGTGCCCACGTGGGAGATGTTGCT-3' in exon 1) and cod396-rev (5'-GAGAGCAAACCGCCGAAC-3' in exon 3) were used for PCR amplification of a 1.2-kb fragment from chicken genomic DNA. Six positive clones resulted from hybridization of chicken BAC filters (UK Human Genome Mapping Project Resource Center, United Kingdom) with the ³²P-radiolabeled 1.2-kb 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 1.2-kb probe. A 3.6-kb NsiI fragment overlapping with exon 1 was subcloned into pGEM-T Easy (Promega) 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 100 mM sodium phosphate buffer, pH 7.4, containing 0.2 mM EDTA and 0.5 mM dithiothreitol after 48 h and sonicated five times for 3 s on ice with an amplitude of 15 µm. Cell lysates were centrifuged at 9000 × g for 10 min at 4 °C. Supernatants were transferred to fresh tubes and subsequently centrifuged at 105,000 × g for 1 h at 4 °C. Microsomal pellets were resuspended in sodium phosphate buffer, and protein concentrations were determined using the protein assay ESL kit (Roche Molecular Biochemicals). Western blotting was performed as described by Ourlin et al. (24) using a polyclonal goat anti-rat CYP2C6 antibody (Daiichi Pure Chemicals Co., Tokyo, Japan) and protein G-horseradish peroxidase conjugate (Bio-Rad).

Scoparone Assay-- CYP2C45 activities were measured by an assay of differential oxidation of scoparone. 15 µg of microsomal proteins were incubated at 37 °C for 15 min in 100 mM Tris buffer, pH 7.6, supplemented with 2 mM MgCl₂, 80 µM scoparone, and 7.5 mM NADPH. Metabolites were separated and analyzed by high pressure liquid chromatography as described previously by Meyer et al. (25).

TaqMan Real-time PCR-- Real-time PCR was performed on an ABI PRISM^TM 7700 (TaqMan) using the sequence detector software, version 1.6.3 (PerkinElmer Life Sciences). Computer-assisted design of compatible TaqMan primers and probes was carried out with the help of the primer express software, version 1.0 (PerkinElmer Life Sciences). 1 µg of total RNA was 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 (PerkinElmer Life Sciences). Primer and probe concentrations were optimized as follows: TaqMan-fwd (5'-CGGTGAAAGAAGCCTTGATTG-3') (900 nM), TaqMan-rev (5'-GGTCCCCGATAGGCATGTG-3') (300 nM), and TaqMan-probe (5'-FAM-GGCAGCAAACTCATCCGCACGA-TAMRA-3') (300 nM). The 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-- 20 µg of total RNA were subjected to electrophoresis on a formamide-containing 1% agarose gel. RNAs were transferred to nylon membrane by overnight blotting in 20× SSC (1× = 150 mM NaCl, 15 mM sodium citrate). Membranes were cross-linked using the UV Stratalinker^® 2400 (Stratagene). Hybridization was carried out in 50% deionized formamide, 5× SSC, 5× Denhardt's solution, 1% SDS, and 10% (w/v) dextransulfate. The same ³²P-radiolabeled 550-bp cDNA probe as used before for the library screening was boiled for 5 min in 500 µl of salmon sperm DNA (10 mg/ml) and quickly chilled on ice. Hybridization was carried out overnight at 45 °C. Washes were performed in 2× SSC/1% SDS at room temperature for 30 min and 2× SSC/1% SDS at 65° for 20 min. Membranes were exposed to x-ray film using intensifying screens for 12-48 h.

Reporter Constructs-- A 2.6-kb fragment of the 5'-flanking region of the CYP2C45 gene (from 7 to 2612 bp) containing the homologous promoter 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). Furthermore, a 2.2-kb fragment lacking 372 bp of the proximal promoter region amplified with primers flank-2614 (5'-GAACACACTGAGATCATCCTG-3') and flank-373 (5'-TGCCATGTGGGTTTTCTGTTC-3') and a putative 239-bp 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 containing the heterologous SV40 promoter (pGL3-SV40, Promega). The pRSV -galactosidase vector used for normalization of transfection experiments was kindly provided by Anastasia Kralli (Biozentrum, University of Basel, Basel, Switzerland).

Mutagenesis-- Site-directed mutagenesis of the DR-4 element in the 2.2-kb and 239-bp fragments was carried out according to the PCRbased method of overlap extension (26) using primers DR4mut-fwd (5'-AAGCTTTCCACTCGAGGCCCTGGCAATGTCGGAG-3') and DR4mut-rev (5'-CTCGAGTGGAAAGCTTTGCGTCTCTAAGAACTTC-3') where 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 2.2 kb or 239 bp, respectively. Mutated fragments were subcloned into pGL3-SV40 as described earlier.

Reporter Gene Assay-- Transfected and induced cells were harvested using passive lysis buffer (Promega). Extracts were centrifuged for 3 min to pellet cellular debris. LUC assays were performed on supernatants using a luciferase assay kit (Promega) and a Microlite TLX1 luminometer (Dynatech). Relative -galactosidase activities were determined for normalization as described by Iniguez-Lluhi et al. (27).

Electromobility Shift Mobility Assay-- The 239-bp EcoRI DNA fragment was ³²P-radiolabeled by 5' filling in with Klenow fragment of E. coli DNA polymerase (Roche Molecular Biochemicals). CXR and RXR were in vitro synthesized using the TNT^® transcription/translation-coupled reticulocyte lysate system (Promega) according to the supplier's protocol. Assay mixtures contained 10 mM Tris, pH 8.0, 40 mM KCl, 0.05% Nonidet P-40, 6% glycerol, 1 mM dithiothreitol, 0.2 mg of poly(dI·dC), 2.5 µl of in vitro translated products, and 25,000 cpm of ³²P-radiolabeled double-stranded DNA probe. The binding reaction was carried out at room temperature for 20 min. 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 Dulbecco's modified Eagle's medium/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/well in Dulbecco's modified Eagle's medium/F12 medium without phenol red supplemented with 10% charcoal-stripped FBS. Cells were transiently transfected using LipofectAMINE reagent (Invitrogen) according to the manufacturer's instructions. Transfection mixes contained 20 ng of reporter plasmid, 50 ng of -galactosidase expression vector, 8 ng of expression vector with the exception of CXR where 1 ng was used, and carrier plasmid. 24 h after transfection, the medium was replaced by Dulbecco's modified Eagle's medium/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 an additional 24 h and harvested using passive lysis buffer. Cell extracts were measured as mentioned under "Reporter Gene Assay."

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning and Sequencing-- A 550-bp fragment was amplified from chicken cDNA using primers derived from P450 sequence alignments as described under "Experimental Procedures." This fragment was used as probe to screen a chicken liver ZAP cDNA library for full-length cDNA. The obtained sequence contained an open reading frame of 1485 bp (Fig. 1A) and was denominated CYP2C45 by David R. Nelson (drnelson.utmem.edu/biblioA.html) based on high sequence identity with CYP2Cs in other species.

View larger version (141K): [in this window] [in a new window]   Fig. 1.   A, full-length cDNA of CYP2C45 was cloned from a chicken liver ZAP cDNA library. Translation start (+17) and stop (+1499) codons are highlighted in boldface. Positions of the first two introns, indicated with bars, were derived from a comparison of the cDNA with the genomic sequence. B, genomic DNA containing a 3.6-kb fragment of 5'-flanking region was obtained from a chicken BAC clone and sequenced by primer walking. The first two exons were determined from 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.

A chicken BAC library was screened to obtain 5'-flanking region sequence information. A 3.6-kb fragment was subcloned from a positive BAC clone and further analyzed (Fig. 1B). A computer-assisted search for putative nuclear receptor binding sites was performed using an algorithm developed by Michael Podvinec in our laboratory.²

Expression and Activity-- Immunoblot analysis was performed using an anti-rat CYP2C6 polyclonal antibody cross-reacting with CYP2C45 protein. A CYP2C45-glutathione S-transferase 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 overexpression of a protein of an estimated molecular mass of 55 kDa, 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. The activity of overexpressed CYP2C45 was measured using an assay of oxidative hydrolysis of scoparone, which had previously been used as a sensitive indicator to distinguish among different P450 isoforms including CYP2Cs (25). Weak but significant metabolism of scoparone by overexpressed CYP2C45 in LMH cells was detected (Table I). Isoscopoletin occurred as a main metabolite, whereas only low levels of scopoletin were detected. Small amounts of isoscopoletin were also measured in control cells.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   CYP2C45 full-length cDNA was subcloned into pCI vector and overexpressed in LMH cells for 48 h (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% polyacrylamide 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).

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 600 µM. 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 16-carbonitrile, DEX, phenytoin, and clotrimazole. 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 2.6 kb of CYP2C45 5'-flanking region including the homologous promoter (Fig. 3C).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   A, LMH cells were treated with increasing concentrations of PB (0-1500 µM) for 16 h. CYP2C45 mRNA levels were quantified using the TaqMan real time PCR technology. Data are represented as relative mRNA levels compared with untreated samples and are corrected with values measured for GAPDH amplification. Results were confirmed by Northern blot using a 32P-radiolabeled cDNA as probe. B, LMH cells were treated with various compounds for 16 h. mRNA levels were quantified as described above. C, LMH cells were transfected with LUC reporter construct containing 2.6 kb of 5'-flanking region, and 4 h later cells were treated for 16 h with various compounds. Data are represented as relative LUC activity compared with untreated samples and are corrected with values measured for empty LUC reporter construct. DMSO, dimethyl sulfoxide; CLO, clotrimazole; DPH, phenytoin; RIF, rifampicine

Role of DR-4 Motif in CYP2C45 Regulation-- The role of a DR-4 motif at 2342 bp in CYP2C45 induction was studied by LUC reporter gene assay. Relative LUC activities after Me₂SO, PB, and MET treatments were measured for the pGL3-SV40 reporter constructs with following inserts: 2.2-kb fragment wild type, putative 239-bp PBRU wild type, 2.2-kb DR-4 mutant, and 239-bp DR-4 mutant (Fig. 4A). The wild type 239-bp fragment retained almost full inducibility compared with the 2.2-kb fragment, whereas the mutation of the DR-4 motif in any of the fragments abolished induction (Fig. 4B). Physical interaction of CXR with the 239-bp fragment was investigated in electromobility shift assays (Fig. 5). Neither CXR nor RXR alone shifted the ³²P-radiolabeled 239-bp 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 by adding an anti-CXR antibody. The shift was completely disabled when competing with a 100-fold molar excess of unlabeled wild type DNA. As expected, no shift was observed when using radiolabeled 239-bp DR-4 mutant probe (data not shown). Transactivation assays were performed to demonstrate not only physical but also functional interaction between CXR and the 239-bp fragment. CV-1 monkey kidney cells were co-transfected with CXR expression plasmid and LUC reporter constructs containing 239-bp fragments with wild type 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 239-bp DR-4 wild type construct. No transactivation was observed in cells transfected with 239-bp DR-4-mutated construct. The transactivation of the 239-bp fragment with the mouse receptors PXR and CAR was investigated in CV-1 cells. A 2-fold PXR-mediated activation of the wild type construct was observed with RU486 and pregnenolone 16-carbonitrile, whereas no significant activation was detected with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (Fig. 6B). However, although no activation was detected in the CAR assay with PB or MET, significant activation of the wild type construct was measured with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (Fig. 6C).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   A, schematic picture of the 2.2-kb fragment of CYP2C45 5'-flanking region and localization of the 239-bp PBRU. A DR-4 nuclear receptor binding site and a nuclear factor NF1 site were identified within the 239-bp PBRU. B, LMH cells were transfected with LUC reporter constructs containing the 2.2-kb or the 239-bp fragment with wild type or mutated DR-4 element. After 4 h, cells were treated for 16 h with Me2SO, PB, or MET. Data are represented as relative LUC activity corrected with values measured for empty reporter construct. Activity of the 2.2-kb construct induced with MET corresponding to 150-fold was arbitrarily set to 100%.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   The 239-bp fragment was 32P-radiolabeled and used as probe for electromobility 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 wild type DNA (lane 6). An anti-CXR antibody was added to the reaction together with CXR and RXR protein (lane 7).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   CV-1 cells were transiently co-transfected with CXR, mouse PXR, or mouse CAR expression plasmids and LUC reporter constructs containing either wild type or mutated 239-bp fragment. Cells were treated for 24 h with various compounds. Data represent relative LUC activities compared with Me2SO (DMSO)-treated samples.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report the cloning of a new P450 cDNA in chicken. A 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 it 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).

View larger version (15K): [in this window] [in a new window]   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.

We have analyzed the 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 is detectable in untreated cells (Figs. 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 2.6-kb fragment of its 5'-flanking region (Fig. 3, B and C). The results were compared with data obtained from reporter gene assays with a 264-bp 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 2.6-kb fragment. PBRUs of inducible P450 genes in mammals have extensively been studied, and two direct, inverted or everted repeats surrounding 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 89 bp from the NF1 site.² To further characterize the function of the putative CYP2C45 PBRU, we have cloned a 2.2 and 239-bp 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 the induction in both the 239- and 2.2-kb fragment (Fig. 4). In contrast, a 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 239-bp fragment with CXR, which has been identified as an activator of the CYP2H1 264-bp PBRU (22). The physical interaction was investigated in electromobility shift assays, whereas the functional interaction was tested in transactivation assays in CV-1 cells. The results uniformly demonstrated the requirement of the DR-4 element for induction and the capability of a CXR-RXR heterodimer to activate the CYP2C45 239-bp PBRU.

In mammals, CAR was originally identified as CYP2B activator, and PXR was identified 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 Ref. 17). Moreover, the 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 239-bp PBRU. In both cases, significant transactivation of the wild type compared with the mutant construct was detected for some inducers, indicating that both PXR and CAR are able to bind to and activate the chicken CYP2C45 239-bp PBRU. Conclusively, these results give rise to the hypothesis that the 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 2.6-kb reporter construct. In contrast to CYP2H1 and inducible CYP2B and CYP3A 5'-flanking regions, no glucocorticoid response element was detected in the 2.2-kb fragment (32-34). From these observations we suggest that a glucocorticoid response element must be localized outside of the 2.2-kb fragment and mediate the 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.

	ACKNOWLEDGEMENTS

We thank Dr. Margie Oleksiak from John Stegeman's group for the homology cloning, Dr. Ralf P. Meyer for helping with the activity assays, and 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, R. Crooijmans, J. Vrebalov, R. J. Dijkhof, J. J. Van der Poel, and M. A. Groenen.

	FOOTNOTES

^* This work was supported by the Swiss National Science Foundation and by National Institutes of Health Grant P42 ES07381 (J. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) .

^¶ 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.

Published, JBC Papers in Press, February 26, 2002, DOI 10.1074/jbc.M109882200

² Podvinec, M., Kaufmann, M. R., Handschin, C., and Meyer, U. A. (2002) Mol. Endocrinol., in press.

	ABBREVIATIONS

The abbreviations used are: CYP/P450, cytochrome P450; CAR, constitutive androstane receptor; CXR, chicken xenobiotic receptor; DEX, dexamethasone; DR, direct repeat; 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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