Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The cytochrome P-450 (CYP) monooxygenases are present in several ocular tissues, including the cornea (Shichi, 1969, 1975; Das and Shichi, 1981; Kishida et al., 1986). Das and Shichi (1981) were the first to describe CYP-dependent drug-metabolizing activity (aryl hydrocarbon hydroxylase) in bovine corneal epithelial microsomes. Since then, other investigators have shown that there are other CYP-metabolizing activities in bovine and human corneal epithelial microsomes (Schwartzman et al., 1987a,b; Abraham et al., 1987; Matsumoto et al., 1987; Shichi et al., 1996). From the perspective of eicosanoid biology, arachidonic acid-metabolizing CYP activity has been detected in bovine, rabbit, porcine, and human corneal epithelium, with the subsequent production of novel metabolites (Laniado Schwartzman, 1997). The corneal epithelial CYP monooxygenases metabolize arachidonic acid to two major metabolites: 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], a potent inhibitor of Na, K-ATPase activity, and 12(R)-hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE], a vasodilatory, chemotactic, and angiogenic factor. Moreover, these metabolites are formed in response to hypoxic or chemical injury (Conners et al., 1995b, 1997); their formation correlates with the severity of the inflammatory response and inhibition of their formation alleviates inflammation (Conners et al., 1995a). Thus, this metabolic activity represents an inflammatory pathway.

Although the biological activities of these metabolites and their enhanced production with injury to the cornea are well established, our knowledge of the scope of the CYP-catalyzed reactions is still incomplete, as the isoform(s) responsible for their production has yet to be fully characterized or even identified. Several purified CYP isozymes have been shown to metabolize arachidonic acid in a reconstituted system to epoxyeicosatrienoic acids and HETEs (mainly hydroxylation at C₁₆-C₂₀). Among them are CYP1A1, 1A2, 2B1, 2B4, 2C2, 2C11, 2C23, 2E1, 2G1, and 4A1 (Capdevila et al., 1981; Oliw et al., 1982; Falck et al., 1990; Laethem and Koop, 1992; Laethem et al., 1994). To date, no specific CYP isozyme(s) for the formation of 12(R)-HETE in the corneal epithelium has been identified. However, a recent study has demonstrated that human liver CYP2C9 metabolizes arachidonic acid to several metabolites, including 12(R)-HETE (Rifkind et al., 1995; Bylund et al., 1998).

Little is known about CYP isozymes in the corneal epithelium and the factors that may regulate their expression. Matsumoto et al. (1987) have demonstrated the presence of a phenobarbital-inducible CYP in rabbit corneal epithelium. Lin et al. (1993) showed that copper ion induced CYP-derived 12(R)-HETE production in porcine corneal epithelium. Asakura and Shichi (Asakura et al., 1994) demonstrated induced levels of CYP activity in porcine ciliary and corneal epithelial microsomes capable of synthesizing 12(R)-HETE after treatment with 3-methylcholantrene (3-MC) and clofibric acid, suggesting the possibility that a CYP1A1 or 1A1-like isoform is present in the corneal epithelium and that it may be involved in the metabolism of arachidonic acid to 12(R)-HETE/12(R)-HETrE. To that end, it has been suggested that the CYP1A1 can be induced by reactive oxygen species (Paine, 1976; Helferich and Denison, 1991; Kocarek et al., 1993; Okamoto et al., 1993). The demonstrations that reactive oxygen species can affect CYP1A1 expression, together with the finding that CYP1A1 protein is present in the corneal epithelium (Zhao and Shichi, 1995), make this isoform a potential candidate for the synthesis of 12(R)-HETE/12(R)-HETrE in the rabbit inflamed/hypoxic corneal epithelium.

This study was undertaken to characterize the expression of CYP isoforms in rabbit cornea and to attempt the isolation of a CYP isoform whose expression correlates with the hypoxia-induced formation of 12-HETE and 12-HETrE. We demonstrated the presence of several CYP isoforms, including the 3-MC/-naphthoflavone (-NF)-inducible CYP1A1 and clofibrate-inducible CYP4 isoform(s). We further showed that hypoxia results in the induction of a CYP4B1-like isoform and provided evidence to suggest that this isoform may account for the production of the 12-hydroxyeicosanoids in the corneal epithelium in response to hypoxia.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Corneal Organ Culture

Rabbit eyes from 8- to 12-week-old male and female New Zealand White/California White rabbits (1.6-2.5 kg) were obtained from Pel-Freeze Biologicals (Rogers, AR). Fresh eyes were shipped overnight on wet ice in Hanks' balanced salt solution containing 100 µg/ml penicillin G, 100 U/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B (Fungizone; 1× penicillin/streptomycin/amphotericin B; Gibco BRL, Grand Island, NY). The eyes were transferred to a large volume of Dulbecco's modified Eagles medium (DMEM; high glucose) with 2× penicillin/streptomycin/amphotericin B for 10 min at room temperature with occasional stirring (250 ml/30 eyes), after which the eyes were soaked in 200 ml of DMEM with 1× penicillin/streptomycin/amphotericin B. An incision was made 2 to 3 mm outside the limbus of the cornea, and the cornea was removed with the scleral rim. The cornea/scleral rim tissue was then transferred to a plastic Petri dish containing 10 ml of DMEM with 1× penicillin/streptomycin/amphotericin B (15 corneas per plate). The corneas were washed five times with the same medium and each cornea was transferred to a well in a 12-well plate containing 1 ml of DMEM with 0.2% lactalbumin enzymatic hydrolysate and 1× penicillin/streptomycin/amphotericin B. Culture plates then were placed in a 37°C incubator supplied with 5% CO₂/95% air (~20% O₂; normoxia) or in a modular tissue culture chamber (Billups-Rothenburg, DelMar, CA) continuously supplied with 5%CO₂/2% O₂/93% N₂ (hypoxia) bubbled through deionized H₂O into the chamber within a 37°C incubator. Cultures were incubated for 8 to 24 h without a change of medium.

Arachidonic Acid Metabolism

At the end of the culture period, culture plates were placed quickly on wet ice and the epithelium from each cornea was scraped into prechilled 0.1 M potassium phosphate buffer (pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride in a prechilled Petri dish. Pooled epithelial scrapings (4 corneas; ~1 ml) were then collected into an Eppendorf tube and concentrated by centrifugation for 5 min at 3500g at 4°C. Supernatants were removed and pellets were resuspended in 500 µl of cold buffer and homogenized with a glass homogenizer. A total of 10 µl was removed from each resultant homogenate for subsequent protein assay (Bio-Rad, Melville, NY). Homogenates (300 µl; 160-320 µg of protein) were incubated with 0.8 µCi [1-¹⁴C]arachidonic acid (28 µM) in a final volume of 500 µl of potassium phosphate/phenylmethanesulfonyl fluoride buffer (pH 7.4) containing 2.9 U of glucose-6-phosphatase-dehydrogenase, 3.3 mM glucose-6-phosphate, 0.5 mM NADP, and 5 mM MgCl₂. In some experiments, homogenates were incubated with goat anti-rabbit CYP4B1 polyclonal antibody, a gift from Dr. R. Philpot (National Institute of Environmental Health Sciences, Research Triangle Park, NC) or anti-rat CYP4A1 antibody (Gentest, Woburn, MA) or with goat nonimmune serum in a 1:1 protein ratio 15 min before the addition of arachidonic acid and a NADPH-generating system. Incubation was for 1 h at 37°C with constant shaking. Arachidonic acid metabolites were extracted with ethyl acetate and separated by reversed-phase HPLC, and chiral analysis of 12-HETE enantiomers was performed as described (Vafeas et al., 1998).

cDNA Probes

The rabbit CYP1A1 cDNA probe was a 1.46-kilobase (kb) cDNA insert of an AccIII-Bstb 98 I digestion fragment of the pBR 322-based clone (Okino et al., 1985) and was kindly provided by Dr. R. Tukey (University of California, San Diego, CA). The rat CYP4A2 (a 1.7-kb cDNA insert excised by NotI and KpnI digestion of the PCR II plasmid) and CYP4A8 cDNA probes (2-kb cDNA insert of EcoRI digestion) were kindly provided by Dr. Richard Roman (Medical College of Wisconsin, Milwaukee, WI). The rabbit CYP4A6 cDNA probe, a 1.5-kb cDNA probe excised by digestion with XhoI and XbaI (Johnson et al., 1990), was a gift from Dr. Eric F. Johnson (Scripps Institute, San Diego, CA). The rabbit lung CYP4B1 cDNA probe was a 1.7-kb cDNA insert of SpeI and EcoRV digestion (Gasser and Philpot, 1989) and was a gift from Dr. Richard Philpot (National Institute of Environmental Health Sciences). The plasmid containing the rabbit CYP2G1 cDNA probe (a 2.5-kb cDNA insert excised by PstI digestion) was obtained from Dr. X. Ding (University of Michigan, Ann Arbor, MI; Ding et al., 1991). The rabbit CYP2C16 cDNA probe (clone no. 35; Richardson et al., 1995) was a 0.8-kb cDNA insert excised by EcoRI digestion and was a gift from Dr. C. Hassett (University of Washington, Seattle, WA). The human CYP2E1 cDNA probe was a 2.2-kb cDNA insert and was excised by EcoRI digestion (Wang et al., 1996a). The probes were labeled with [-³²P]dCTP with the Random Prime Labeling System (Amersham Pharmacia Biotech, Piscataway, NJ). The 28S rRNA cDNA probes were generated by reverse transcription (RT)-polymerase chain reaction (PCR) from rabbit liver.

RNA Extraction

Total RNA was isolated by the guanidinium thiocyanate-phenol extraction method of Chomczynski and Sacchi (1987) with Trizol reagent (Gibco BRL, Grand Island, NY) according to the protocol of the manufacturer. Tissue samples or cells grown in a monolayer were promptly homogenized in Trizol reagent, were quick-frozen, and were stored at 80°C until use. RNA concentrations were quantified by A₂₆₀. mRNA was purified from total RNA with the Oligotex mRNA Mini kit (Qiagen Inc., Chatsworth, CA), and the concentration was determined as described above.

Northern Blot Analysis

Analysis of the rabbit corneal epithelial RNA was performed by electrophoresis of poly(A)⁺ mRNA on 1.5% denaturing agarose gels containing formaldehyde. Northern blot transfer to Hybond N (Amersham Life Sciences, Buckinghamshire, UK) was achieved by capillary blotting. The integrity of the RNA samples and transfer efficiencies were assessed by rehybridization of membranes with the ³²P-labeled human glyceraldehyde-3-phosphate dehydrogenase cDNA probe (Clontech Laboratories, Inc., San Diego, CA). Membranes were probed with ³²P-labeled CYP cDNA probes in Rapid-Hyb Hybridization Buffer (Amersham Life Sciences, Buckinghamshire, UK). The resulting blots were subjected to autoradiography with reflection autoradiography films (DuPont/NEN, Boston, MA).

PCR Primers

Oligonucleotide primers were synthesized by Gene Link (Thornwood, NY). Primers for CYP1A1 were as follows: forward 5'-GTGGACAGGTTGGACGAGAATG and reverse 5'-TTGGAAGTGTTCACAGCGGG were designed to amplify a 654-base pair (bp) PCR product that covers the sequence of the 3'-flanking region of the 1338-bp coding region; and forward 5'-GGTATTGTCTTGGACCTCTTCGG and reverse 5'-CAACACGGGATGTGGAAAAGG amplify a 1174-bp PCR product containing the sequence belonging to both the coding and the noncoding regions of the rabbit CYP1A1 cDNA. Primers for amplifying a CYP4A sequence were designed based on sequence homology among the rabbit CYP4A gene family including CYP4A4, 4A5, 4A6, and 4A7 and are as follows (numbers in parentheses indicate the corresponding positions in the CYP4A6 cDNA sequence): forward primers 5CRF (92), 5'-TGCTCAAGGCAGCTCAGCTCTA; F5 (454), 5'-TACGACATCCTGAAGCCCTACGTG; 800-F (797), 5'-GATCCAGCAGAGGAAGGCTCAG; and reverse primers 1110-R (1125), 5'-GCACATGGTGGTGTAGGGCATCT; HBR-R (1385), 5'-AATTGYTTCCCRATGCAGTTCC; 1510-R (1528), 5'-GGAGCTTCCTCAGACGCAGGTG; B27-R (1498), 5'-CACAAGACGTGGTTTTTG. The primer HBR-R was modified to recognize the heme-binding domain of all CYP4A isoforms. Primers used for amplification of the 28S rRNA were: forward, 5'-AAACTCTGGTGGAGGTCCGT (1543); reverse, 5'-CTTACCAAAAGTGGCCCACTA (1843).

RT-PCR

cDNA was synthesized with the First-Strand cDNA Synthesis kit (Amersham Pharmacia Biotech) with 100 ng of RNA according to the recommendations of the manufacturer. For the CYP1A1 study, either 1A1-654- or 1A1-1174-specific primer was used in the single reaction. In all other experiments, an oligo(dT) primer was used for first-strand cDNA synthesis according to the recommendation of the manufacturer.

Amplification of CYP1A1 Fragments. Reactions were conducted in a final volume of 100 µl consisting of 50 mM Tris-HCl (pH 9.0; 25°C), 20 mM ammonium sulfate, 1.5 mM magnesium chloride, 200 µM concentrations of each deoxyribonucleotide triphosphate, 3 µl of the first-strand cDNA product, and 100 pmol of each forward and reverse primer. One unit of Thermus Flavus (Tfl) thermostable DNA polymerase (Epicentre Technologies, Madison, WI) was added. The reactions were heated to 94°C for 5 min and then immediately cycled 35 times through a 1-min denaturing step, a 1.5-min annealing step at 55°C, and a 2.5-min extension step at 72°C. After the cycling procedure, a final 10-min elongation step at 72°C was performed. The total liver RNA sample from rabbits treated with 3-MC or -NF was used to generate the RT-PCR-positive control for each of the PCR experiments. To verify the identity of the RT-PCR product, the plasmid containing the full-length rabbit CYP1A1 cDNA (100 ng) was used to generate the PCR products of the expected size and sequence.

Amplification of CYP4A Fragments. The lower reaction mix consisted of 50 mM Tris-HCl (pH 9.0; 25°C), 20 mM ammonium sulfate, 1.5 mM magnesium chloride, 200 µM concentrations of each deoxyribonucleotide triphosphate, and 100 pmol of each forward and reverse primer in a final volume of 50 µl. One AmpliWax PCR Gem 100 (Perkin-Elmer, Oceanport, NJ) was added to each tube. The lower reaction mix was sealed by incubating at 80°C for 8 min and then at 2°C for 2 min. The upper reaction mix was combined in a final volume of 50 µl consisting of 50 mM Tris-HCl (pH 9.0; 25°C), 20 mM ammonium sulfate, and 5 µl of DNA template (either RT first-strand cDNA product or first-round PCR reaction product). One unit of Tfl thermostable DNA polymerase (Epicentre Technologies) was added to each reaction. The upper reaction mix was then applied onto the AmpliWax seal, and reactions were processed as described below. The reactions were heated to 96°C for 2 min and then immediately cycled through a 1-min denaturing step at 96°C, a 1.5-min annealing step at 50°C, and a 3-min extension step at 72°C. Thirty-five cycles were performed for the first-round PCR and 25 cycles for the second-round PCR. After the cycling procedure, a final 10-min elongation step at 72°C was performed. Total RNA obtained from the rabbit kidney was used to generate the RT-PCR-positive control sample for each of the PCR experiments performed. To verify the identity of the RT-PCR product, the rabbit CYP4A6 cDNA-bearing plasmid (100 ng) was used to generate the PCR products of the expected size and sequence.

Amplification of 28S rRNA PCR was done as described for CYP1A1 with 25 to 35 cycles, a 1-min denaturation step at 96°C, a 1.5-min annealing step at 55°C, and a 2-min extension step at 72°C. A final 10-min elongation step at 72°C was performed.

CYP4A6- and CYP4B1-Specific PCR

To differentiate between CYP4A and CYP4B1, the following primers were used: 4A6-F, 5'-ACATGAAGGTGAT-TCTGG and 4A6-R, 5'-ATCAGGGAGATGTCTTGG for amplifying a 264-bp-specific CYP4A6 sequence; and 4B1-F, 5'-TCTCTGGGTTGGACAGTTCATTG and 4B1-R, 5'-TGTCTCCTTTGCCAAACGTACAC for amplifying a 359-bp-specific CYP4B1 sequence. Taq polymerase was obtained from Perkin-Elmer Corp. (Foster City, CA). Reactions were cycled 20 to 30 times through a 1-min denaturing step at 95°C, a 1-min annealing step at 50°C, and a 1-min extension step at 72°C. After the cycling procedure, a final 10-min elongation step at 72°C was performed.

Southern Blot Analysis

Fifteen microliters of the PCR reaction mix was separated through an agarose gel composed of 1.5% low-gelling-point Sea Plaque/1.5% Sea Kem agarose and visualized by ethidium bromide staining. Southern blot transfer to Hybond N (Amersham Life Sciences) was achieved through capillary blotting. To verify the identity of the PCR products, the Southern-blotted membranes were probed with a ³²P-labeled cDNA probe in Rapid-Hyb Hybridization Buffer (Amersham Life Sciences). The resulting blots were subjected to autoradiography with NEF reflection autoradiography films (DuPont/NEN), and the sizes of the products agreed with those expected for the amplified regions of the cDNAs. PCR amplifications and Southern blot analysis were conducted at least twice to verify the reproducibility of the results.

Cloning

PCR products obtained from the second-round PCR were purified with the QIAquick PCR Purification kit (Qiagen) according to the protocol of the manufacturer. The TA Cloning Kit (Invitrogen, Carlsbad, CA) was used for one-step cloning by blunt-end ligation of the PCR product into the pCR2.1 vector. Transformants were isolated by blue-white selection with kanamycin according to the recommendation of the manufacturer. Plasmids from selected clones were purified with the Qiagen Plasmid Mini kit (Qiagen). The presence and the orientation of cloned inserts were confirmed by restriction digestion with EcoRI (Promega, Madison, WI) and by PCR with specific primers.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effect of Hypoxia and CYP Inducers on 12-HETE and 12-HETrE Synthesis. We recently developed a corneal organ culture in which epithelial CYP-derived arachidonate metabolism can be maintained and manipulated within 48 h. Incubation of corneal organ cultures under hypoxic conditions (2% O₂) increased the corneal epithelial synthesis of 12-HETE and 12-HETrE by 2- to 3-fold over the control normoxic conditions. The hypoxia-induced increase was selective to the R-enantiomers of both eicosanoids and was inhibited by clotrimazole and 17-octadecyonic acid, inhibitors of CYP-mediated reactions (Vafeas et al., 1998). Figure 1 compares and contrasts the effect of CYP inducers including 3-MC, -NF, clofibric acid, and phenobarbital to that of hypoxia on the synthesis of 12-HETE. Hypoxia increased 12-HETE by 3-fold. Clofibrate increased the synthesis of 12-HETE by 85% under normoxic conditions; however, it did not cause a significant increase over that of the hypoxia alone (31.29 ± 4.68 and 25.23 ± 2.45 nmol of 12-HETE/mg/h in hypoxia-treated corneas with and without clofibrate, respectively; n = 7; p < .273). Phenobarbital had a similar effect. It did increase 12-HETE synthesis by 50% under normoxic conditions, but it had no additive effect when added under hypoxia. The addition of both 3-MC and -NF under normoxic conditions increased the synthesis of 12-HETE by 2-fold, and no further increase was observed under hypoxic conditions. Similar results were obtained for 12-HETrE (data not shown). The results suggest that CYP isoforms induced by 3-MC and -NF, clofibrate, and phenobarbital may be involved in the increased production of these eicosanoids; however, hypoxia still had the most pronounced effect, and no significant additive effect of these inducers on the hypoxia-induced synthesis seems to occur.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Effect of hypoxia and CYP inducers on 12-HETE synthesis in the corneal epithelium. Corneas were incubated with and without 3-MC (2 µM), -NF (50 µM), clofibrate (CF; 2 µM), and phenobarbital (PB; 2 µM) under normoxic or hypoxic conditions for 24 h. The corneal epithelium was scraped and incubated with arachidonic acid and NADPH as described in Materials and Methods. Results are the mean ± S.E. (n = 4; *p < .05) from normoxia alone;  p < .01 from the corresponding treatment under normoxic conditions.

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Southern blot analysis of RT-PCR products amplified with two sets of CYP1A1-specific primers from hypoxia- and 3-MC+-NF-treated corneal epithelium. Corneas were incubated with and without 3-MC (2 µM) and -NF (50 µM) under hypoxic conditions for 4 and 12 h. The corneal epithelium was scraped and RNA extracted as described in Materials and Methods. PCR products were separated on an agarose gel, transferred to a membrane, and hybridized with the rabbit CYP1A1 cDNA as described in Materials and Methods. A, 654-bp fragment; B, 1174-bp fragment; C, 305-bp fragment amplified with 2BS rRNA-specific primers. Total RNA from -NF/3-MC-treated rabbit and CYP1A1 plasmid (100 ng) were used as controls

CYP mRNA Expression in the Corneal Epithelium. To determine the identity of the hypoxia-induced CYP isoform, mRNA from rabbit corneal organ cultures incubated under normoxic and hypoxic conditions was extracted and hybridized with several CYP cDNA probes from different CYP gene families. We anticipated that by using low-stringency conditions, CYP mRNA, the levels of which are increased by hypoxia, could be detected. The following cDNA probes were used: CYP4A1, CYP4A2, CYP4A8, CYP2G1, CYP2C16, and CYP2E1. The only identifiable signals under these conditions were obtained with the heterologous CYP4A1 cDNA probe. Moreover, the signal corresponding to CYP4A1-like mRNA was slightly stronger in the epithelium from hypoxia-treated corneas (data not shown). On the basis of this and the Northern blot analysis results, we speculated that the enzyme induced by hypoxia may be a member of the CYP4 family.

Cloning and Sequencing of CYP4A PCR Fragments. Primers were selected to take advantage of the high-homology regions between isoforms of the rabbit CYP4A family. Rabbit corneal epithelial mRNA was reverse transcribed and then subjected to first- and second-round PCR. Two PCR fragments were consistently amplified: 1) a 588-bp fragment that was obtained after nested PCR amplification with the 800F-HBRR primer pair; and 2) a 671-bp fragment that was obtained after nested PCR amplification with the F5-1110R primer pair. These two fragments have an overlapping region of about 300 bp, according to the CYP4A family sequences, and they are strongly hybridized to the CYP4A6 cDNA probe. Both the 588-bp and the 671-bp fragments were amplified in the epithelium from control and hypoxia- and clofibrate-treated corneas. We also amplified fragments from the rabbit corneal epithelium cDNA library by using primers targeted to the 5'-region of the CYP4A gene. The fragment flanked by primers 5CRF and 800R was amplified and hybridized with the rabbit CYP4A6 cDNA probe (data not shown). The presence of the CYP4A sequences in the corneal epithelium cDNA library supports the hypothesis that some specific isoform(s) of the CYP4A family is expressed in rabbit corneal epithelial cells and may be involved in synthesis of 12(R)HETE and 12(R)HETrE.

View larger version (77K): [in this window] [in a new window]   Fig. 3.   Partial nucleotide sequence of cDNA encoding the corneal CYP4B1-related isoform and alignment to the known sequence of the rabbit lung CYP4B1. Primer sequences are underlined.

Involvement of a CYP4B Isoform in the Synthesis of 12-HETE and 12-HETrE. With the same primer sets, we amplified the 588- and 671-bp fragments from normoxia-, hypoxia-, and clofibrate-treated corneas. The PCR fragments were hybridized with CYP4A6 and CYP4B1 cDNA probes. The results demonstrated that both the 588-bp and 671-bp PCR fragments readily hybridized to CYP4A6 cDNA without distinguished differences among the different treatments (data not shown). However, major differences were observed when CYP4B1 cDNA was used as the probe for the Southern hybridization. As seen in Fig. 4, the 588-bp PCR fragment did not hybridize to CYP4B1 except for that amplified from clofibrate-treated corneas. However, CYP4B1 readily hybridized to the 671-bp PCR fragment, demonstrating significant differences between the different treatments. Thus, the intensity of the CYP4B1 signals was markedly higher in hypoxia-treated corneas as compared with those from control or clofibrate-treated corneas (Fig. 4). Additional PCR experiments with the F5-HBR-R primer set yielded a longer DNA fragment of 931 bp containing the 671-bp PCR fragment sequence. Southern hybridization of this fragment with the CYP4B1 probe showed a marked increase in hypoxia-treated corneas as well as in clofibrate-treated corneas (Fig. 5), suggesting that a CYP4B1-like mRNA is present in this tissue and that its levels are increased under hypoxic conditions.

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Southern blot analysis of the RT-PCR products amplified from rabbit corneal RNA. Corneas were incubated under normoxic or hypoxic conditions with and without clofibrate (2 µM) for 8 h. The corneal epithelium was scraped and RNA was extracted and reversed transcribed, and two rounds of PCR (except for the 28S rRNA-specific PCR) were performed. Southern blot analysis with the CYP4B1 cDNA probe was performed as described in Materials and Methods. A, 588-bp PCR fragment; B, 671-bp PCR fragment; C, 305-bp 28S rRNA fragment. I, 5CRF-1510R primer pair used in the first round of PCR; II, F5-B27-R primer pair was used in the first round of PCR.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Southern blot analysis of a 931-bp PCR fragment amplified from corneal epithelial mRNA with the F5-HBR-R primer pair and hybridized with the CYP4B1 cDNA probe (A). Corneas were incubated under normoxic or hypoxic conditions with and without clofibrate (2 µM) for 8 h. The corneal epithelium was scraped and RNA extracted. RT-PCR and Southern blot analysis were performed as described in Materials and Methods. B, 28S rRNA 305-bp-specific PCR fragment. RNA from rabbit kidney was used as control.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Southern hybridization of RT-PCR amplification of corneal epithelial mRNA with specific CYP4B1 and CYP4A6 primers. Corneas were incubated under normoxia or hypoxia for 8 h. Total RNA was extracted and RT-PCR performed with CYP4B1 (359-bp fragment)- and CYP4A6-specific (265-bp fragment) primers. The RT-PCR products were hybridized with CYP4A6 (A) and CYP4B1 (B) cDNA probes. C, 28S rRNA 305-bp-specific PCR fragment.

View larger version (56K): [in this window] [in a new window]   Fig. 7.   Time-dependent, hypoxia-induced CYP4B1 expression. Corneas were incubated under hypoxic conditions for 2, 4, and 24 h, and RNA was extracted and amplified with specific primers: A, 671 bp, hybridized with CYP4B1 cDNA probe; B, CYP4B1-specific primers (359 bp, hybridized with a CYP4B1 cDNA probe); and C, 28S rRNA-specific primers (305 bp, hybridized with a 28S rRNA cDNA probe).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effect of anti-rabbit CYP4B1 antibodies on corneal epithelial 12-HETE synthesis. Corneas were incubated with and without phenobarbital (2 µM) or clofibrate (2 µM) under hypoxic conditions for 24 h. The epithelium was scraped, homogenized, and incubated with arachidonic acid and NADPH in the presence and absence of goat anti-rabbit CYP4B1 antibodies (4B1), goat anti-rat CYP4A1 antibody (4A1), or nonimmune serum (NIS) for 1 h as described in Materials and Methods. Results are the mean ± S.E. (n = 4, *p < .05) from the corresponding control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the past few years, we and others have demonstrated that a CYP activity is involved in the synthesis of 12(R)-HETE and 12(R)-HETrE by the corneal epithelium and other tissues (Conners et al., 1997). However, the identity of the CYP isoform or isoforms responsible for this catalytic activity has not been determined. The present study attempted such identification. We herein provide substantial evidence for the involvement of a CYP4B1-like isoform in the hypoxia-induced synthesis of 12(R)-HETE and 12(R)-HETrE in the rabbit corneal epithelium.

The corneal epithelium as a tissue poses a great difficulty when attempting to study molecular expression and cloning of genes. This tissue exhibits rapid cellular turnover, the amount of tissue is limited, and its RNA integrity is questionable. In initial experiments, we tried to avoid these problems by using a freshly isolated tissue; however, quantity was a problem, as was reproducibility of the injury in vivo. One solution was to use cells in culture; however, CYP expression and CYP-dependent enzymatic activities have been shown to diminish with time in culture in many cell types (Maslansy and Williams, 1982; Lin et al., 1995). We have developed a model of corneal organ culture in which hypoxia serves as the injurious stimulus for the generation of 12(R)-HETE and 12(R)-HETrE. In this model, hypoxia induced and maintained CYP-derived 12(R)-HETE and 12(R)-HETrE synthesis for 24 to 48 h. Hypoxia has been considered as the major determinant in increasing the synthesis of these eicosanoids in a model of closed-eye contact lens-induced inflammation in the rabbit eye (Conners et al., 1995b). Thus, the organ culture provided us with a preparation with which we could examine molecular mechanisms underlying the increased synthesis of these 12-hydroxyeicosanoids.

A classical approach to associate enzymatic activity with a specific CYP isoform is the use of inducers. Indeed, Shichi and colleagues demonstrated that treatment of the corneas or the ciliary epithelium with either 3-MC or clofibrate greatly increased the synthesis of 12(R)-HETE in these tissues (Asakura and Shichi, 1992; Asakura et al., 1994). 3-MC is a potent inducer of CYP1A1/1A2, whereas clofibrate induces isoforms of the CYP4A gene family. Based on their findings and the specificity of the inducer, these investigators suggested the involvement of both isoforms in the formation of 12(R)-HETE. Additional experiments with antibodies against these isoforms and competitive substrates provided further evidence for such a claim. In our studies, 3-MC, as well as -NF or a combination of both, increased the synthesis of 12-HETE and 12-HETrE. However, these inducers did not increase the hypoxia-induced 12-HETE and 12-HETrE synthesis. Moreover, although both inducers greatly increased CYP1A1 mRNA in the corneal epithelium, hypoxia failed to induce CYP1A1 mRNA, suggesting that this isoform is not involved in the hypoxia-induced synthesis of these eicosanoids. It has been shown that hyperoxia, but not hypoxia, stimulates CYP1A1 expression in rabbit and sheep lung (Okamoto et al., 1993). Whether oxygen tension is crucial for the expression of this isoform and what mechanisms are responsible for oxygen-regulated CYP expression remain unknown. Another CYP gene family that has been shown to be expressed in ocular tissues is the CYP4A. It is present in the corneal epithelium (Shichi et al., 1996) and its induced expression after clofibrate treatment leads to increased 12(R)-HETE synthesis in pigmented ciliary epithelium (Asakura and Shichi, 1992). Indeed, Northern analysis of corneal epithelial mRNA demonstrated its presence and inducibility in response to hypoxia. However, extensive analysis by PCR techniques failed to support the presence of a hypoxia-induced CYP4A isoform in the corneal epithelium, but instead led to the identification of a 671-bp fragment with a 98.8% sequence homology to the rabbit lung CYP4B1 isoform (Gasser and Philpot, 1989), of which the levels in the corneal epithelium were greatly increased under hypoxic conditions.

The findings of a CYP4B1-like isoform in the corneal epithelium and its increased expression after hypoxia are intriguing. CYP4B1 was first isolated from rabbit lung microsomes (Gasser and Philpot, 1989). Its expression is tissue- and species-specific. In the rabbit, this gene is expressed in the lung and liver and is induced in the liver by phenobarbital. In the rat, CYP4B1 is constitutively expressed in the lung, with little or no expression in the liver regardless of treatment. Bladder mucosa of the rat and the mouse are other tissues where specific expression of CYP4B1 has been detected (Zeldin et al., 1995). CYP4B1 has been cloned from human lung and is expressed in all tumor tissues examined, in sharp contrast with the fact that almost all other CYPs are known to disappear in tumor tissue (Nhamburo et al., 1989). Its specific expression in tumor tissue raises the possibility that hypoxia may be the underlying mechanism for its induction. To date, the function of this isoform in lung or tumor tissue is unknown. It has been reported that, like CYP4A members, CYP4B1 is induced by peroxisomal proliferators such as the hypolipidemic drug and clofibric acid, and it can metabolize fatty acids at the -carbon (Zeldin et al., 1995). However, a specific endogenous substrate or product has not been identified. In our study, we provide substantial evidence that CYP4B1 or a CYP4B1-like isoform may be involved in the hypoxia-induced 12-HETE and 12-HETrE synthesis. First, we showed that its expression is elevated under hypoxic conditions; second, we demonstrated that antibody against CYP4B1 inhibited hypoxia-induced, as well as clofibrate- and phenobarbital-induced, 12-HETE and 12-HETrE synthesis; and third, we showed that lauric acid, which has been shown to be a CYP4B1 substrate, inhibited arachidonic acid conversion to 12-HETE (Zeldin et al., 1995). These results suggest involvement but do not prove that this isoform has the catalytic activity of metabolizing arachidonic acid to these eicosanoids. The latter should be confirmed via cloning of full cDNA sequencing followed by expression of the cDNA and measurement of its catalytic activity with arachidonic acid as the substrate.

Most of the isoforms in the CYP4 gene family are known to catalyze hydroxylation of fatty acid at the -carbon and, to a lesser extent, at the -1 carbon. However, in a recent study, we demonstrated that one of the CYP4 isoforms in the rat kidney, CYP4A2, catalyzed not only /-1 hydroxylation of arachidonic acid but also epoxidation of the 11,12 double bond, to yield 11,12-epoxyeicosatrienoic acid (11,12-EET; Wang et al., 1996b). Little is known about the mechanistic details of the conversion of arachidonic acid into such monohydroxylated metabolites. The mechanism of CYP reactions with olefins typically involves the formation of an epoxide, and several CYP isozymes have been identified as arachidonic acid epoxygenases. Thus, the initial step of oxygenation would be the formation of an 11,12-EET intermediate formed from molecular oxygen and arachidonic acid catalyzed by a specific CYP isozyme(s). The formation of 12(R)-HETE would require direct C-10 hydrogen abstraction, whereas the formation of 12(R)-HETrE would require additional rearrangement of 11,12-EET, either through direct epoxide rearrangement to an isomeric 12-keto-eicosatrienoic acid or through oxidation of 12(R)-HETE to 12-keto-HETE (Murphy et al., 1988). To this end, we have shown that 12(R)-HETrE can be formed not only from arachidonic acid but also from CYP-derived 12(R)-HETE and lipoxygenase-derived 12(S)-HETE via an oxidation/keto reduction activity (Nishimura et al., 1991; Yamamoto et al., 1994). Based on our observation that a CYP4A isoform can catalyze 11,12 epoxidation, it is possible that the hypoxia-induced CYP4B1 in the corneal epithelium catalyzes the first epoxidation reaction. However, it is possible that it oxidizes arachidonic acid to 12(R)-HETE via a lipoxygenase-like reaction without an intermediate epoxide (Capdevila et al., 1986). Such activity has been shown for the human CYP2C9 (Rifkind et al., 1995; Bylund et al., 1998) but not for any of the CYP4 isoforms.

Whether the hypoxia constitutes the factor that directly mediated CYP4B1 transcriptional activation, i.e., via a putative hypoxia-induced factor, needs to be further investigated. However, a different scenario can exist in the corneal epithelium under hypoxic conditions. It is well known that transcriptional activation of members of the CYP4 family by hypolipidemic drugs such as clofibric acid is mediated via activation of peroxisomal proliferator-activated receptors (PPARs). Recent reports suggest that some eicosanoids serve as endogenous ligands of the PPARs (Forman et al., 1995; Yu et al., 1995; Devchand et al., 1996). Yu et al. (1995) showed that cyclooxygenase-derived prostaglandins, including PGD₂, PGA₂, and PGJ₂, bind to PPAR subtypes (, , and ) with different specificities. However, lipoxygenase-derived hydroxy acids, including 8(S)-HETE and leukotriene B₄, have been shown to be potent ligands for the PPAR (Devchand et al., 1996). Hypoxia as well as inflammatory conditions may rapidly induce cyclooxygenase 2, which, in turn, initiates a cascade of cellular processes leading to induction of CYP4B1 via activation of PPARs. The activation of PPAR may constitute the mechanism of induction of CYP4B1 because the exogenous ligands for this nuclear receptor, e.g., clofibrate, also stimulates the formation of 12(R)-HETE and 12(R)-HETrE. Additional studies are needed to elucidate the mechanisms underlying the hypoxia-induced expression of CYP4B1 and its involvement in the synthesis of 12(R)-HETE and 12(R)-HETrE in the corneal epithelium.