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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Division of Clinical Pharmacology and Experimental Therapeutics, Children's Mercy Hospital and Clinics, Kansas City, Missouri (A.G., D.W.B., R.G., R.E.P., C.A.V., J.S.L.); Department of Medicinal Chemistry, University of Washington, Seattle, Washington (R.A.T.); and Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (D.C.Z.)
Received June 8, 2006; accepted July 21, 2006.
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).
There is also evidence that CYP2J2 plays a role in carcinogenesis. Recent investigations have suggested that up-regulation of this enzyme, probably through a c-Jun-responsive module, promotes the neoplastic phenotype of carcinoma cells and may be involved in the pathogenesis of a variety of human cancers (Jiang et al., 2005; Marden and Murray, 2005). However, CYP2J2 can also metabolize a number of xenobiotics, including the antihistamine drugs ebastine (Hashizume et al., 2002) and astemizole (Matsumoto et al., 2002), contributing to their presystemic elimination in the small intestine. Taken together, CYP2J2 emerges as a crucial enzyme in a number of physiological functions.
The human CYP2J2 cDNA was first cloned and characterized by Wu et al. (1996). In vitro expression demonstrated that it metabolized arachidonic acid predominantly via olefin epoxidation to EETs. Immunoblotting studies revealed that it was primarily expressed in heart, and, to a lesser extent, in liver, ileum, jejunum, colon, and kidney. Furthermore, inter-individual variation in the expression of CYP2J2 mRNA in heart and liver was remarkably low. The expression of CYP2J2 in human tissues was subsequently investigated in more detail, demonstrating that the enzyme was abundantly expressed in several tissues (Enayetallah et al., 2004). The CYP2J2 gene was eventually cloned and sequenced and was found to consist of nine exons similar to most other members of the human CYP2 family (King et al., 2002). Currently, the Cytochrome P450 Nomenclature Committee has defined nine allelic CYP2J2 variants (http://www.cypalleles.ki.se/cyp2j2.htm). In vitro expression studies indicated reduced metabolic activity toward both arachidonic acid and linoleic acid for CYP2J2*2, *3, and *6, and toward arachidonic acid for CYP2J2*4, whereas the CYP2J2*5 variant did not seem to have altered activity (King et al., 2002). CYP2J2*7 involves a G>T substitution in the regulatory region at position –76 (–50 of transcription start), which causes the loss of a Sp1 transcription factor binding site. Consequently, this leads to lower amounts of CYP2J2 protein and reduced levels of circulating CYP2J2 epoxygenase metabolites in vivo (King et al., 2002; Spiecker et al., 2004). In addition, two novel variants, CYP2J2*8 and *9, have recently been discovered in a Korean population, and in vitro expressed CYP2J2*8 was associated with severely compromised activity toward astemizole O-demethylation and ebastine hydroxylation (Lee et al., 2005). The limited allele frequency data available suggest that the majority of reduced-function alleles are rare and that the occurrence of some alleles may be restricted to certain populations [e.g., CYP2J2*3–*6 were only detected in individuals of African descent (King et al., 2002) and CYP2J2*8 and *9 were only detected in Koreans (Lee et al., 2005)]. The sole variant that seems to be present in subjects of all ethnicities is CYP2J2*7, which has been found at frequencies between 0.026 and 0.18 (King et al., 2002; Lee et al., 2005; Wang et al., 2006).
Most studies on CYP2J2 have been performed with adult subjects or adult-derived tissues. Because CYP2J2 seems to play an important role in EET formation, has a wide tissue distribution, and is highly conserved (i.e., low degree of polymorphic expression), one may speculate that it also carries out critical functions during human development. However, to our knowledge, there is limited information about prenatal CYP2J2 gene expression and function. To gain more insight into CYP2J2 expression during human development, we performed quantitative PCR and immunoblot analyses on a large panel of liver samples genotyped for CYP2J2 and compared expression levels to that in other tissues. We also detected and characterized alternative splice variants and resequenced selected samples with high and low CYP2J2 protein levels.
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). The post mortem interval was <6 h for all samples. Genomic DNA (n = 376) was derived from liver tissue (n = 76) or discarded or anticoagulated blood obtained for routine clinical management of hospitalized patients (Caucasians, n = 128; African Americans, n = 128; American Asians, n = 44). Ethnicity was self-reported or retrieved from data sheets accompanying the tissues. The protocol for sample collection and use was approved by the University of Missouri-Kansas City Pediatric Health Sciences Review Board.
DNA Isolation and Genotyping for CYP2J2. DNA was isolated using DNeasy Tissue or QIAamp Blood DNA kits (QIAGEN, Valencia, CA). Genotyping was performed by PCR-RFLP. Assays were designed de novo using GenBank accession number AF272142 [GenBank] as reference sequence. Details, including primer sequences and PCR reaction conditions, are given in Table 1. PCR amplifications were performed with JumpStart REDTaq DNA polymerase (Sigma-Aldrich, St. Louis, MO) at 1.5 mM MgCl2 with the buffer provided or at optimized MgCl2 concentrations (Table 1). Fragment lengths, restriction enzymes, and restriction patterns for respective assays are shown in Table 2. Restriction enzymes and the 100-bp DNA ladder were purchased from New England Biolabs (Beverly, MA). Digested PCR fragments were separated on 3% agarose gels containing Synergel (Diversified Biotech, Boston, MA).
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RNA Isolation and Quantitative Real-Time-PCR Analysis. Total RNA was extracted from frozen tissue (20–35 mg) according to the QIAGEN RNeasy protocol with an on-column DNase I treatment. RNA quality was ensured by agarose gel electrophoresis, and quantity was measured spectrophotometrically. QRT-PCR was performed with the QuantiTect SYBR Green one-step reverse transcription-PCR kit from QIAGEN on a DNA Engine Opticon 2 instrument (MJ Research, Watertown, MA). Each 12-µl reaction contained 15 ng of RNA and was carried out in triplicate. Primers and annealing temperatures are given in Table 1. Serial dilutions of a CYP2J2 amplicon served as standard to generate a calibration curve ranging from 102 to 107 molecules. Transcript numbers were calculated from log-linear regression analysis of the calibration curve. Data were normalized to 18S rRNA, which was measured with the TaqMan Ribosomal RNA Control Reagent kit (Applied Biosystems, Foster City, CA).
Immunodetection of CYP2J2 Protein. Microsomal fractions were prepared from prenatal and postnatal liver as described previously (Leeder et al., 2005). Microsomal samples were heated to 80°C for 10 min. Microsomal proteins (4 µg/lane) were separated using the Novex NuPAGE System [10-well 4–12% gradient gels, XCell Sure-Lock Mini-Cell apparatus, lithium dodecyl sulfate sample buffer, sample reducing agent, antioxidant, and 3-(N-morpholino)propanesulfonic acid SDS running buffer; Invitrogen, Carlsbad, CA] or the Criterion XT System [18-well 10% Criterion XT gels, Criterion Cell apparatus, XT sample buffer, reducing agent, and 3-(N-morpholino)propanesulfonic acid running buffer; Bio-Rad, Hercules, CA]. Proteins were separated at 150 or 180 V for 3 h when using the Novex and Criterion systems, respectively. Proteins were transferred to Hybond-C Extra nitrocellulose (Amersham Biosciences, Piscataway, NJ) using a Bio-Rad semidry transfer unit with transfer buffer consisting of 48 mM Tris, 39 mM glycine, 20% methanol, and 0.0375% SDS at pH 9.2. Membranes were blocked overnight at room temperature on a rocker in 10 mM Tris, 150 mM NaCl, and 0.2% Tween 20, pH 8 (TNT), containing 4% skim milk powder (M-TNT). Blocked membranes were incubated for 2 h with CYP2J2 antibodies designated as anti-CYP2J2rec (Wu et al., 1996) or anti-CYP2J2pep3 (Seubert et al., 2004) diluted 1:50,000-fold in M-TNT and alternately washed in TNT and H2O (5 min x 3 TNT/H2O wash cycles). The anti-CYP2J2rec antibody was made against a partially purified preparation of human CYP2J2 protein and has been shown to cross-react with CYP2J proteins of multiple species, but not with non-CYP2J isoforms. The anti-CYP2J2pep3 antibody was raised against a peptide common to all CYP2J isoforms (for details, see King et al., 2002). To detect bound antibody, blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:50,000) (Amersham Biosciences) in M-TNT for 30 min and then alternately washed in TNT and H2O (5 min x 3 TNT/H2O wash cycles), incubated with ECL Advance chemiluminescence reagents according to the manufacturer's protocol (Amersham Biosciences), and exposed to Hyperfilm ECL. Films were scanned using a flat-bed scanner. Heterologously expressed CYP2J2 protein (BD Gentest, San Jose, CA) was present on each membrane as a positive control. Each sample was run twice to ensure reproducibility.
cDNA Synthesis. Reverse transcription was performed on 2 µgof total RNA with an oligo(dT) primer using the QIAGEN Omniscript reverse transcriptase kit. Reaction volumes were 20 µl. Subsequent CYP2J2 transcript-specific PCR reactions (full length or partial) were carried out on 0.4 µl of cDNA in 8-µl reactions (Table 1).
Amplification, Cloning, and Characterization of CYP2J2 Transcripts. Full-length CYP2J2 cDNA was amplified in a long-range PCR reaction to allow generation of fragments shorter and longer than the expected wild-type 1.57-kb transcript, should such transcripts exist. Subsequently, cDNA from two samples (one prenatal testis sample and one postnatal liver sample) containing potential variant transcripts was cloned with the pCR-XL-TOPO cloning kit designed for efficient cloning of larger amplicons (Invitrogen) and screened for insert length. Clones with inserts deviating in size from that of correctly spliced mRNA were further analyzed by DNA sequencing with DYEnamic ET dye terminator chemistry and a MegaBACE 500 capillary sequencer (Amersham Biosciences).
To detect differentially spliced variants in panels of cDNAs from various tissues, we designed primers amplifying the affected regions. The reaction for splice variants SV2 and SV3 generated three fragments identifying correctly spliced, exon 7A-deleted and exon 7B-retained transcripts, respectively, whereas the reaction for SV4 yielded two PCR products corresponding to correctly spliced and exon 2-deleted transcripts, respectively. Primers and other reaction details are given in Table 1.
CYP2J2 Gene Resequencing. Four long-range PCR products ranging from 5.3 to 8.7 kb and covering all exons as well as flanking intronic and the 3'- and 5'-untranslated regions were generated (Table 1). The fragments were treated with Exo/SAP-IT (USB, Cleveland, OH) and subsequently sequenced with DYEnamic ET dye terminator chemistry and a MegaBACE 500 capillary sequencer using appropriately spaced CYP2J2 primers to cover all exons, at least 100 bp of flanking intronic, 550 bp upstream and 375 bp of downstream sequence, respectively.
Automated Splice Site Analysis. The strengths of splice sites and splice enhancer binding sites were determined by automated analysis available at https://splice.cmh.edu using models based on the April 2003 release of the Human Genome Sequence (Build 33). According to the models, donor and acceptor splice sites are defined by an information content (Ri value) >0 bit (Rogan et al., 1998; Nalla and Rogan, 2005).
In Vitro Expression and Characterization of CYP2J2*10. The CYP2J2 cDNA was expressed in the bacterial expression vector PCW+ (source pCWhum3A4HT; Guryev et al., 2001) after truncation of the N terminus and addition of a hexahistidine tag to the C terminus to facilitate purification (wild-type construct, CYP2J2.1). The P115L amino acid change was introduced into the wild-type construct using the primers 5'-CCGCCCCGTGACCCTTATGCGAGAACATA-3' and 3'-TATGTTCTCGCATAAGGGTCACGGGGCGG-5' (the changed nucleotide is underlined) with a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The integrity of the variant construct was confirmed by DNA sequencing. Protein expression was performed as described previously (Cheesman et al., 2003) and also in the presence of chaperone proteins (GroES-GroEL) (Mitsuda and Iwasaki, 2006). Expressed CYP2J2 protein was affinity purified using a nickel-nitrilotriacetic acid agarose column (QIAGEN), and eluted fractions were dialyzed against 100 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol before storage in 15 to 20 µM aliquots at –80°C. Heme content in these preparations was determined using the pyridine hemochromogen assay of sodium dithionite reduced heme (Smith, 1975). Details of the bacterial expression and purification systems will be described elsewhere (J. P. Jones, M. Fallon, M. Cheesman; D. C. Zeldin, and R. A. Totah, manuscript in preparation).
Statistical Analyses. Analyses were performed with the SPSS version 12.0 software package for Windows (SPSS Inc., Chicago, IL). A normal distribution was established by Q-Q-Plot for the number of transcripts/ng total RNA. Univariate analyses of variance were performed using log-transformed values for number of transcripts per nanogram of total RNA. To assess whether expression levels differed among tissues, a mixed model analysis with a Bonferroni adjustment for multiple comparisons was performed. Linear regression analysis was performed with Microsoft Excel 2002. A p value <0.05 was considered statistically significant.
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Variability in CYP2J2 Gene Transcription. Expression of CYP2J2 mRNA is reported as transcripts per nanogram of total RNA corrected for the expression of 18S rRNA as recommended by Koch et al. (2002) and as applied previously to fetal liver CYP3A expression (Leeder et al., 2005). As shown in Fig. 1A, expression varied 127-fold in the panel of human fetal liver samples (n = 58). Mean ± S.D. (range) expression was 1351 ± 717 transcripts/ng total RNA (23–2957 transcripts/ng total RNA). A subset of four samples had markedly reduced levels of CYP2J2 mRNA, of which three samples (CMM311, CMM32, and CMM361) subsequently were found to be devoid of immunoreactive protein, whereas the fourth sample, CMM1153, did reveal protein. These four samples were significantly different compared with the remaining 54 samples (p = 0.001). These samples have been previously reported to contain extremely low levels of CYP3A7 mRNA, immunoreactive protein, and catalytic activity (Leeder et al., 2005). In the subset of 54 samples, mRNA expression was normally distributed, and the level of expression was not affected by age, either estimated gestational age (r2 = 0.047) or postnatal age (r2 = 0.0003). When excluded from the analysis, variability in CYP2J2 mRNA expression decreased from 127- to 8-fold (n = 54). Furthermore, there was no statistically significant difference between the pre- and postnatal groups regardless of whether the four samples described above were included (p = 0.86) or excluded (p = 0.17) from the analysis. In the 18 postnatal samples (ranging from 4 days to 18 years of age), mRNA expression varied only 19-fold (mean ± S.D., 1482 ± 1229 transcripts/ng total RNA; range, 253-4728 transcripts/ng total RNA). The lowest transcript number was observed in a sample from a 16-day-old infant that was homozygous for the CYP2J2*7 allele. We also noted that this sample exhibited some RNA degradation, and it was not conclusive whether the low transcription level was due to the genotype or RNA quality. Exclusion of this sample decreased the variability to 15-fold; mean ± S.D. (range) expression was 1554 ± 1227 transcripts/ng total RNA (326–4728 transcripts/ng total RNA). Genotype, i.e., the presence of the CYP2J2*7 allele, did not affect the number of transcripts/ng total RNA. As shown in Fig. 2, there was no statistically significant difference between the two genotype groups in either the fetal (p = 0.37) or postnatal group (p = 0.67).
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Variability in CYP2J2 Immunoreactive Protein. Immunoreactive protein content was assessed in 51 prenatal and 18 postnatal liver microsomal samples. Representative immunoblots illustrating the range of immunoreactive CYP2J2 content in samples ranging from 11.1 weeks of gestation to 17 years of life are shown in Fig. 3A. Of note, the polyclonal anti-CYP2J2rec antibody recognized two predominant immunoreactive proteins in the postnatal samples and in one prenatal sample (CMM1153; 32 weeks EGA). Upon close inspection, some samples revealed an additional minor band. This band pattern has been observed previously in adult liver microsomes but not in heart microsomes (Wu et al., 1996). In contrast, the lower molecular mass protein was not observed in all other fetal liver samples. The CYP2J2pep3 anti-peptide antibody consistently recognized two proteins in the fetal samples, whereas the postnatal pattern consisted of multiple bands of variable intensities (Fig. 2B). Of note, sample CMM1153 again exhibited a pattern resembling that observed in postnatal microsomes. These data suggested the presence of novel CYP2J2 isoform(s) or alternatively, the presence of an unrelated protein with an epitope(s) recognized by both CYP2J2 antibodies used.
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Considerable variability in protein content was also observed among the panel of prenatal samples. This finding is summarized in Fig. 4. The amount of immunoreactive protein ranged from "low" (including nondetectable) to "intermediate" and "high" (by visual inspection, categories used for descriptive purposes only). The low protein bands approximated 0.006 pmol of CYP2J2 protein per microgram of microsomal protein, whereas the strongest bands in the high-protein category were between 0.012 and 0.02 pmol of CYP2J2 protein per microgram of microsomal protein. No relationship between transcript number and the amount of immunoreactive protein was apparent across the panel of 51 samples (data not shown).
CYP2J2 Splice Variants and Automated Splice Site Analysis. Because mRNA expression seemed to be comparable in prenatal and postnatal liver and immunoreactive protein expression patterns were variable, we explored the possibility that developmentally regulated alternative splicing may lead to reduced expression of the canonical transcript and thus, translation into CYP2J2 protein in prenatal liver. Therefore, full-length cDNA was amplified from a panel of tissue samples. The canonical transcript and amplification products that were smaller and larger than the correctly spliced transcript were detected in all samples, implying the presence of alternative splice products (Fig. 5A). Cloning and sequencing the cDNA from two tissue samples (prenatal testis and postnatal liver) with appreciable levels of the variant transcripts revealed three novel splice variants (Fig. 5B). Two variants, SV2 and SV4, had deletions of exon 2 and exon 7, respectively, whereas SV3 retained a cryptic exon. To discriminate the canonical exon 7 from the cryptic exon 7, they have been designated as exons 7A and 7B, respectively. Both exon deletions and the retention cause frame shifts and introduce premature stop codons.
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Splice variants SV2 and SV3 were minor transcripts and difficult to visualize by gel electrophoresis, but they seemed to be present in all tissues tested (data not shown). In contrast, PCR amplification specific for the exon 2 deletion variant (SV4) did not reveal any visible product in a panel of tissues including the two subjects from which it was originally cloned (CMM684 and CMM1409), suggesting that it constitutes only a rare variant (data not shown).
The strengths of the splice donor and acceptor sites were calculated with the information theory-based splice site analysis tool that we have successfully applied to the analysis of other P450 splice sites (Rogan et al., 2003; Gaedigk et al., 2005) (Table 3). Each canonical donor and acceptor splice site contained at least 3.7 bits of information. The exon 2 and exon 7A deletions could not be explained by the presence of weak splice acceptor or donor sites. However, the observed splice junctions of the cryptic exon 7B corresponded to strong 9.0-bit and 5.2-bit acceptor and donor sites, respectively, as predicted by splice site analysis.
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The variability in fetal liver CYP2J2 protein expression patterns was unlikely due to SV2, SV3, and SV4, because these splice variants were minor and rather ubiquitously present across tissues. Therefore, we searched for additional splice variants, which may have escaped our initial cloning/screening experiments. However, amplification of cDNA covering exons 2 through 9 and exons 2 through 7A did not reveal any additional splice products in samples classified as high (n = 3), intermediate (n = 2), and low (n = 3) protein expression. The samples used for this analysis corresponded to those shown in Fig. 4.
Discovery and Characterization of the Novel CYP2J2*10 Allele. A nonsynonymous C>T SNP was discovered at AF272142 [GenBank] g.16810 (cDNA position 344) when selected DNA samples with low levels of immunoreactive protein were resequenced. This SNP was detected in a single subject of unknown ethnicity (CMM1059) and was confirmed by sequencing multiple PCR-generated templates as well as by PCR-RFLP genotyping. Subsequent genotyping of the tissue DNA panel (n = 76 including the carrier) and an additional set of DNAs including 128 Caucasians, 128 African Americans, and 44 American Asians (total of 600 alleles) did not reveal any other carrier, making this allele a rare event. The novel allele was designated as CYP2J2*10 by the Cytochrome P450 Nomenclature Committee at http://www.cypalleles.ki.se/cyp2j2.htm.
To assess the functional consequence of the P115L amino acid substitution, CYP2J2.10 protein was expressed in the presence and absence of chaperone proteins GroEL/GroES using a bacterial expression system. Based on protein content, expression with the chaperones yielded 432 and 345 nM CYP2J2 wild-type and CYP2J2.10 protein, respectively. SDS-polyacrylamide gel electrophoresis analysis with the CYP2J2pep3 antibody showed a protein band at 57 kDa that was identical in size to that derived from the wild-type cDNA and both expressed proteins. However, CYP2J2.10 seemed to be devoid of heme as judged by the lack of a P450 COdifference spectrum at 450 nm and the presence of a large absorbance peak at 280 nm (data not shown). Moreover, the ratio of free to protein-bound heme revealed only trace amounts of heme in the CYP2J2.10 preparation, whereas heme was readily incorporated into wild-type protein (ratios of 0.87 and 0.02 for wild type and CYP2J2.10, respectively). These results strongly indicate that P115L affects proper heme binding or protein folding. Therefore, CYP2J2.10 protein activity is likely severely compromised. Preliminary results using terfenadine as a probe substrate for CYP2J2 activity corroborate our findings and further support our conclusion that CYP2J2.10 protein activity is likely severely compromised (J. P. Jones, M. Fallon, M. Cheesman, D. C. Zeldin, and R. A. Totah, manuscript in preparation). An alignment of CYP2 family members, including human, rat, and rabbit sequences, showed that Pro115 is located within the substrate recognition site-1 that precedes the formation of the B'-helix, but it does not seem to be highly conserved. Currently, however, it remains speculative whether Pro115 is important for protein folding and/or heme binding.
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; Matsumoto et al., 2002). Although CYP3A4 and CYP2D6 are not expressed at appreciable amounts in fetal liver, their contribution to fetal ebastine and astemizole metabolism may be negligible. However, CYP3A5 and CYP3A7, which are present in fetal liver (Leeder et al., 2005), and CYP4F family members, which probably are also expressed prenatally, given their importance in fatty acid epoxide inactivation (Le Quere et al., 2004), may contribute to the overall activity measured toward ebastine and astemizole and therefore render these probes nonspecific. Efforts to overcome these challenges are currently underway.
We also detected different pre- and postnatal protein expression patterns. In the postnatal liver samples, the anti-CYP2J2 antibody recognized two or three distinct bands, which is similar to the observations of Wu et al. (1996) in adult liver using the same antibody. Interestingly, a predominant immunoreactive protein was detected in the fetal liver tissue samples, similar to the results reported by Wu et al. (1996) in adult heart. These authors speculated that adult liver contains additional CYP2J2 isoforms and/or other proteins that share immunochemical determinants with CYP2J2, but their nature remains elusive. Regardless, it seems that protein expression in adult heart resembles that in fetal liver and that there is a distinct pattern in postnatal liver. Differential protein patterns between pre- and postnatal liver were also observed with a second antibody, anti-CYP2J2pep3, providing further evidence that these developmental differences are real. In contrast, Gu et al. (2000) found only a single immunoreactive band in fetal olfactory mucosa (n = 8) and liver (n = 6) as well as in their single adult control sample using an anti-rat Cyp2J4 antibody. We can only speculate at this point whether the anti-rat Cyp2J4 antibody is indeed more specific compared with the antibodies used in this study and others (Wu et al., 1996; Yu et al., 2000; King et al., 2002; Jiang et al., 2005) or fails to detect certain CYP2J2 postnatal liver-specific isoforms. Clearly, further studies are required to determine whether the various proteins recognized by the CYP2J2 antibodies are indeed isoforms and if so, to characterize their functional properties in a developmental context.
The four samples with low CYP2J2 transcript numbers (Fig. 1A) were derived from fetuses with severe congenital anomalies. These samples also presented with low CYP3A7 mRNA expression, protein content, and activity. As described and discussed in detail by Leeder et al. (2005), these observations could not be explained by poor sample quality. Noteworthy is sample CMM1153, which demonstrated a pattern of immunoreactivity unlike the other three samples (Figs. 1A and 3) but very similar to that observed in postnatal samples. This unique presentation of a "fetal" sample may be explained by compromised viability due to porencephaly or other unknown factors.
To explain the variability in protein content, the liver panel was genotyped for CYP2J2*2 through *7 by PCR-RFLP. The only variant found was the more common CYP2J2*7 allele, which could not explain the variable amounts of protein. Therefore, we performed gene resequencing on eight subjects with high, intermediate, and low protein levels, but no sequence variations explaining the variable amounts of protein were revealed. Only a single novel SNP at position AF272142
[GenBank]
g.16810 was discovered in one subject of unknown ethnicity. Subsequent genotyping of the liver panel and additional Caucasian, African-American, and Asian samples totaling 600 chromosomes, did not reveal any additional carriers of this novel allele termed CYP2J2*10. The failure to detect any additional novel SNPs within the coding and 100 bp of intronic sequences is consistent with previous studies that discovered few sequence variations at rather low frequencies (King et al., 2002; Spiecker et al., 2004; Lee et al., 2005). However, the presence of a SNP(s) located deep within an intron may explain low levels of protein. Such a scenario is the main cause of CYP3A5 poor metabolism, where a SNP in intron 3 creates a cryptic splice site that leads to a premature stop codon and nonfunctional truncated protein (Kuehl et al., 2001). Because resequencing focused on exons, flanking intron portions and 500 bp of promoter sequence, SNPs outside these regions eluded discovery in this investigation.
Gene reporter studies have demonstrated that the loss of a SP1 binding site on the CYP2J2*7 allele causes a reduction in transcription level (Spiecker et al., 2004), but the transcript number/ng total RNA was not significantly different between our homozygous and heterozygous groups, respectively (Fig. 2). At this point, we can only speculate whether the functional allele compensates for the reduced expression of CYP2J2*7 in liver tissue, whether there are additional splice variants that are initiated from a different promoter or whether other factors are at play. Albeit significantly lower concentrations of EET metabolites in plasma have been attributed to the presence of the CYP2J2*7 allele (Spiecker et al., 2004), this in vivo phenotype reflects EET metabolism of all tissues where CYP2J2 is expressed. Clearly, additional studies on hepatic CYP2J2 activity are warranted.
To assess whether C>T (P115L) in CYP2J2*10 conveys any functional consequences, CYP2J2.10 protein was expressed in vitro in a bacterial system (J. P. Jones, M. Fallon, M. Cheesman, D. C. Zeldin, and R. A. Totah, manuscript in preparation). Because the majority of apoprotein was devoid of heme, one may speculate that P115L interferes with heme incorporation in vivo and thereby compromises the amount of functional protein. CYP2J2*10 was found in a composite heterozygote with CYP2J2*7, which could explain the low level of immunoreactive protein in this particular sample. Yet, given the rarity of this allelic variant, it did not explain the observed variability and likely does not play a significant role in overall interindividual variation in CYP2J2 levels.
Next, in an effort to detect any aberrant mRNA splice variants, cDNA was amplified from the same subjects (representing high, intermediate, and low protein) in whom the CYP2J2 gene was resequenced. Each revealed a major product corresponding to the correctly spliced mRNA and two minor products. Subsequent analysis on full-length cDNA on a set of tissues from one pre- and one postnatal subject did not reveal any additional tissue-specific splice products. These findings are also in agreement with the automated splice site analysis shown in Table 3 that calculated relatively high bit values for almost all canonical splice sites. None of the three alternate splice variants that were ultimately identified (Fig. 5B) encode functional protein due to frame shifts introduced by deletion and retention events. Again, this set of experiments failed to find any explanation for the variability in protein expression. There is no evidence in the literature suggesting that CYP2J2 transcripts include alternative exons at the 5' end (i.e., other canonical exon(s) 1), but it is conceivable that alternate first exon(s) exist. Such exons were, however, not captured by using primers directed to the known exon 1 sequence. Further characterization of splice events affecting the 5' ends may ultimately discover additional splice variants that could account for the variable amounts of protein detected. Possibly, such variants may also encode the immunoreactive proteins identified in postnatal and adult liver, but not fetal liver and adult heart.
In summary, we provide an initial characterization of CYP2J2 mRNA and protein expression during human development. Although mRNA expression seems to be tightly regulated, immunoreactive protein varies considerably. Furthermore, resequencing of the gene and characterization of splice variants did not provide any explanation for the variability seen on the protein level. Future investigations will be directed toward the development of specific probe substrates to permit assessment of CYP2J2 activity in liver microsomal preparations and more detailed characterizations of mRNA splicing in the 5' and 3' ends of CYP2J2 transcripts.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: 20-HETE, 20-hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; P450, cytochrome P450; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; bp, base pair(s); QRT, quantitative real-time; SV, splice variant; kb, kilobase(s); EGA, estimated gestational age; SNP, single-nucleotide polymorphism; TNT, 10 mM Tris, 150 mM NaCl, and 0.2% Tween 20, pH 8; M-TNT, TNT containing 4% skim milk powder.
Address correspondence to: Dr. Andrea Gaedigk, Children's Mercy Hospital, Division of Clinical Pharmacology, 2401 Gillham Rd., Kansas City, MO 64108. E-mail: agaedigk{at}cmh.edu
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