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INFLAMMATION AND IMMUNOPHARMACOLOGY

Cloning and Characterization of Cyclooxygenase-1b (Putative Cyclooxygenase-3) in Rat

Departments of Physiology and Pharmacology (J.A.S., B.K., D.W.B.) and Pathology (G.S.S.), Wake Forest University Health Sciences, Winston-Salem, North Carolina; and Department of Neuroscience (J.A.H.), University of Connecticut Health Center, Farmington, Connecticut

Received for publication October 19, 2004
Accepted January 12, 2005.

	Abstract

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A splice variant of cyclooxygenase-1 (COX-1), COX-1b (previously termed as COX-3), has been identified in canine tissues as an acetaminophen-sensitive isoform, but the sequence of COX-1b mRNA and the encoded protein are not known in rats. We cloned and sequenced rat COX-1b mRNA from cerebral endothelial cells. Sequence analysis indicated that the 98-base pair intron-1 of COX-1 gene remains unprocessed in the COX-1b mRNA, causing a frameshift mutation and a 127-amino acid open reading frame with no sequence similarity with known cyclooxygenases. Transient and permanent transfection of COS-7 cells with a vector containing the rat COX-1b cDNA resulted in synthesis of a protein of the expected size. We generated an affinity-purified polyclonal antibody against the rat COX-1b protein. Western blot analysis of rat tissues using this antibody demonstrated the likely existence of rat COX-1b protein in vivo with the highest expression in heart, kidney, and neuronal tissues. Our results on both stable and on transiently transfected COS-7 cells suggest that rat COX-1b does not have cyclooxygenase activity and does not have any effect on the inhibition of prostaglandin production by acetaminophen. Because this protein has a completely different amino acid sequence than COX-1 and COX-2 and it does not have cyclooxygenase activity, we suggest a name cyclooxygenase variant protein to distinguish it from the known prostaglandin-synthesizing cyclooxygenase isoforms.

Recently, COX-1b mRNA has been detected in tissues from rat (Kis et al., 2003b, 2004a), mouse (Shaftel et al., 2003), and human (Chandrasekharan et al., 2002; Dinchuk et al., 2003). In particular, we have shown that COX-1b mRNA is relatively abundant in cultured cerebral endothelium (Kis et al., 2003b), as well as in freshly harvested cerebral microvessels (Kis et al., 2004a) of rat, and that these preparations are unusually sensitive to the inhibition of prostaglandin synthesis by acetaminophen (Kis et al., 2004b). Retention of intron-1, however, which is 98 bp in rat (GenBank NW_047653.1) and mouse (GenBank NT_039206 [GenBank] .2), and 94 bp in human (GenBank NT_017568) should lead to a shift in the reading frame and to the synthesis of a protein completely unlike COX-1 and with questionable cyclooxygenase activity. Although not yet tested, it is possible that factors such as a different initiation site related to the insertion of intron-1 or alternative downstream splicing will restore the reading frame so that a fully functioning cyclooxygenase variant is produced.

This study had five purposes. They are: first, to determine whether the entire intron-1 is inserted into the COX-1b mRNA in rat, we used primary cultures of rat cerebral endothelial cells (CECs) as well as freshly harvested cerebral microvessels because they show abundant expression of COX-1b mRNA (Kis et al., 2003b, 2004a); second, to determine the complete sequence of the rat COX-1b mRNA and to predict the amino acid sequence of the protein likely to be produced; third, to transfect COS-7 cells with a rat COX-1b cDNA construct to determine whether it will lead to the synthesis of the predicted protein; fourth, to generate an antibody against the rat COX-1b mRNA-encoded protein and to demonstrate the existence of this protein in vivo in rat tissues; and fifth, to examine the cyclooxygenase activity of the predicted protein and its sensitivity to acetaminophen and also its interaction with COX-2.

We show here that rat COX-1b mRNA retains the entire intron-1 from the parent COX-1 gene. Retention of the 98-bp intron-1 in rat causes a frameshift resulting in a predicted 127-amino acid protein with no COX-1 sequence similarity. Transfection of COS-7 cells with the rat COX-1b cDNA resulted in the detection of a protein of the predicted size. Generation of an antibody against the rat COX-1b mRNA-encoded protein provided evidence for the existence of this protein in vivo. Our experiments also demonstrated that the protein encoded by rat COX-1b does not have cyclooxygenase activity and has no influence on the sensitivity of COX-2 to acetaminophen.

	Materials and Methods

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Culturing Rat Cerebral Endothelial Cells. Wistar rats were obtained from Harlan (Indianapolis, IN). All animal experiments were approved by the Animal Care and Use Committee of Wake Forest University Health Sciences. Primary rat CECs were isolated and cultured in collagen type IV and fibronectin-coated 35-mm dishes as described previously (Kis et al., 1999). Culture medium consisted of Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine plasmaderived serum (Animal Technologies Inc., Tyler, TX), 2 mM glutamine, 1 ng/ml basic fibroblast growth factor, 50 µg/ml endothelial cell growth supplement (BD Biosciences, Bedford, MA), 100 µg/ml heparin, 5 µg/ml vitamin C, and antibiotics. Confluent cultures (4–5th day in vitro) consisted of more than 95% rat CECs verified by positive immunohistochemistry for von Willebrand factor and negative immunochemistry for glial fibrillary acidic protein and -smooth muscle actin.

Isolation of Rat Cerebral Microvessels. Halothane anesthetized male Wistar rats (body weight 200 ± 20 g) were transcardially perfused with chilled saline containing 1000 U/l heparin. Cerebral cortices were freed from larger vessels, pial membranes, and myelin and then were finely minced and incubated in DMEM containing collagenase (200 U/ml; Worthington Biochemicals, Lakewood, NJ) and DNase (30 U/ml; Sigma-Aldrich, St. Louis, MO) at 37°C for 2 h. After incubation, the digested tissue was triturated, mixed with 20% bovine serum albumin in DMEM, and centrifuged at 1000g for 20 min. The pellet containing the microvessels was washed twice with phosphate-buffered saline and stored on dry ice until total RNA isolation. After further processing, this preparation leads to viable CECs that can be used for culturing (Kis et al., 1999).

RNA Isolations and RT-PCR. Total RNA was isolated from the samples by SV Total RNA isolation system (Promega, Madison, WI). The poly(A) mRNA fraction was isolated by PolyATract mRNA isolation system (Promega). RT-PCR experiments were carried out as described previously (Kis et al., 2003b). The design of the specific primers for rat COX-1b detection was based on the hypothesis that the rat COX-1b mRNA is identical to the full-length form of COX-1, except that it retains intron-1, as in the dog. The sense primer (5'-CAGAGTCATGAGTCGTGAG; 1376773–1376791 bp of GenBank NW_047653.1) was designed to bind to the 5' end of the putative rat COX-1b including the start codon of the COX-1 gene and part of intron-1. The antisense primer (5'-AGAGGGCAGAATGCGAGTAT; 501–520 bp of GenBank S67721 [GenBank] ) binds to exon 5 of the rat COX-1 gene. The expected length of the RT-PCR product was 573 base pairs. Our COX-1b primer set distinguishes between COX-1 and COX-1b, and it also distinguishes between COX-1b and "partial" cyclooxygenases (PCOX-1a and PCOX-1b) because our antisense primer binds to exon 5, which is lacking in PCOXs (Chandrasekharan et al., 2002).

Cloning and Sequencing of the Rat COX-1b mRNA. Amplified RT-PCR product was separated on a 3% NuSieve agarose gel (Cambrex, Rockland, ME) and purified using Wizard SV gel clean up system (Promega). To amplify and accurately sequence both ends of the COX-1b fragment, the purified RT-PCR product was ligated into pGEM-T Easy Vector (Promega) and transformed into Escherichia coli (JM109; Promega). Automated DNA sequencing of the COX-1b construct was performed on an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA).

Expression of the Protein Encoded by Rat COX-1b mRNA in COS-7 Cells. To examine whether the COX-1b mRNA expresses the predicted protein, we amplified the sequence of the COX-1b inserted into the pGEM-T Easy Vector. The sense primer (5'-TAATACGACTCACTATAGGG) was designed to bind to the T7 promoter. The antisense primer (5'-CGGTCTAGAGCAGGAAAT) was designed to bind to the 3' end of the COX-1b sequence. The 3' primer lacked the COX-1b stop codon and contained an XbaI restriction site for creation of a C-terminal 3x FLAG-tagged form of COX-1b. For this purpose, the PCR product was digested with NotI and XbaI and inserted into NotI-XbaI-digested p3XFLAG-CMV-14 expression vector (Sigma-Aldrich). For transient transfection, COS-7 cells were cultured in 24-well plates and were transfected with the COX-1b-FLAG construct (0.5 µg plasmid/well) by TransFast transfection reagent (Promega). Twenty-four hours after transfection, proteins were isolated and subjected to Western blot analysis using antibody against the FLAG epitope. COS-7 cells transfected with p3XFLAG-CMV-7-BAP plasmid were used as a positive control. For stable transfection, COS-7 cells were cultured in 60-mm dish and were transfected with the COX-1b-FLAG (5 µg of plasmid/dish) by Trans-Fast transfection reagent (Promega). One day after transfection, the culture medium was supplemented with 500 µg/ml G418 (Geneticin; Invitrogen). Surviving colonies were trypsinized in cloning rings and transferred to 12-well plates for further propagation in the presence of the selective medium. Proteins were isolated from the third generations of clones of stable transfected cells and were subjected to Western blot analysis using anti-FLAG monoclonal antibody.

Production of COX-1b Protein in E. coli. Primers (sense primer, 5'-CCCCATATGAGTCGTGAGGTATAC; antisense primer, 5'-ACAGGATCCTGTCAGCAGGA) containing NdeI and BamHI restriction sites were used to amplify rat COX-1b cDNA. The PCR product was inserted into the NdeI and BamHI restriction sites of the pET-14b expression vector (Novagen, Madison, WI) and transformed into BL21(DE3)pLysS-competent E. coli (Promega). Cells were harvested and His-tagged COX-1b protein was extracted using Ni-nitrilotriacetic acid His-Bind purification kit (Novagen) according to the manufacturer's instructions. This purified protein was used as a positive control in Western blots using our anti-rat COX-1b antibody (see below).

Cotransfection of COS-7 Cells with COX-2 and COX-1b. The rat COX-2 cDNA in pcDNA3.1 vector was constructed as described previously (Vidwans et al., 2001). The rat COX-1b expression vector was created by digesting the pGEM-T Easy Vector containing the rat COX-1b with NotI and SpeI, and the purified product was inserted using NotI and XbaI restriction sites into pcDNA3.1 vector. The resulting plasmid DNAs (pcDNA3.1-COX-2 and pcDNA3.1-COX-1b) were amplified in E. coli and purified for transfection with a DNA purification system (Promega). COS-7 in 60-mm dishes were transfected at 50% confluence with empty pcDNA3.1 plasmid (10 µg/dish), pcDNA3.1-COX-2 plus empty pcDNA3.1 plasmids (5 µg/dish from each), pcDNA3.1-COX-1b plus empty pcDNA3.1 plasmids (5 µg/dish from each), or with pcDNA3.1-COX-2 plus pcDNA3.1-COX-1b plasmids (5 µg/dish from each) using TransFast transfection reagent (Promega). One day after the transfection, the cells were split into 12-well plates for further experiments. The expression of COX-2 and COX-1b proteins was assessed by Western blot analysis.

Production of Polyclonal Anti-COX-1b Antibodies. Peptides corresponding to the N-terminal two to 17 amino acids of rat COX-1b mRNA-encoded protein (SRESDPSGAPTRPGIR), as predicted by cDNA sequences, were synthesized and coupled to keyhole limpet hemocyanin. The peptide-keyhole limpet hemocyanin complexes were injected into New Zealand White rabbits four times at 3-week intervals. The serum of the rabbit was collected 77 days after the first injection. The resulting polyclonal antibodies were then affinity purified using the above-mentioned peptide immobilized on Affi-Gel column (Bio-Rad, Hercules, CA).

Subcellular Fractionation. Confluent cultures of rat CECs were washed two times with phosphate-buffered saline. Then, the cells were scraped from tissue culture dishes and collected in microcentrifuge tubes. Cells were recovered by centrifugation for 5 min at 1000g, resuspended in ice-cold hypotonic buffer (10 mM HEPES, pH 7.4, 1 µg/ml aprotinin, 50 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin), incubated on ice for 15 min, and then homogenized with a 2-ml Dounce homogenizer. The homogenate was adjusted to 250 mM sucrose using a 2 M stock and centrifuged at 700g for 10 min. The postnuclear supernatant was transferred into a fresh microcentrifuge tube and subjected to an additional round of centrifugation at 700g to ensure complete removal of nuclei. The supernatant from the second 700g spin was then centrifuged at 120,000g for 1 h using a Beckman TL-100 ultracentrifuge (Beckman Coulter, Fullerton, CA). The resulting pellet consisted of the total postnuclear membrane fraction, and the supernatant contained the cytosol.

Western Blotting. Proteins were isolated from samples using Nonidet P-40 lysis buffer supplemented with proteinase inhibitors (1 µg/ml aprotinin, 50 µg/ml phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin) as described previously (Kis et al., 2003a). Western blot membranes were prepared (Kis et al., 2003a), and the blots were incubated with monoclonal anti-FLAG (1:2000; Sigma-Aldrich), polyclonal anti-COX-2 (1:5000; Cayman Chemical, Ann Arbor, MI), or polyclonal anti-COX-1b antibodies (1:2000) overnight at 4°C. The membranes were then washed three times in Tris-buffered saline with 0.1% Tween 20 and then incubated for 1 h in the blocking buffer with anti-rabbit IgG (1:50,000; Jackson ImmunoResearch Laboratories, West Grove, PA) or anti-mouse IgG (1:5000; Jackson ImmunoResearch Laboratories) conjugated to horseradish peroxidase. The final reaction products were visualized using enhanced chemiluminescence (SuperSignal West Pico; Pierce Chemical, Rockford, IL) and recorded on X-ray film.

Immunocytochemistry. Primary cultures of rat CECs were cultured on glass coverslips. Cultures were washed with phosphate-buffered saline, fixed in 3.7% formaldehyde, and permeabilized with 0.1% Triton X-100. The rat CECs were incubated with polyclonal rabbit anti-rat COX-1b primary antibody (1:200) at room temperature for 60 min and then incubated with fluorescein isothiocyanate-labeled anti-rabbit (1:100; Vector Laboratories, Burlingame, CA) secondary antibody at room temperature for 30 min. Confocal images of cellular fluorescence were acquired on an LSM 510 laser scanning microscope (Carl Zeiss, Jena, Germany).

Prostaglandin E₂ (PGE₂) Measurement from Transfected Cells. COX-1b p3XFLAG-CMV-14 stably transfected COS-7 cells were cultured in 12-well plates. Confluent cultures were washed twice with 37°C DMEM, and then the cells were incubated at 37°C for 15 min in DMEM containing 30 µM arachidonic acid with or without 100 µM acetaminophen. After the incubation, media were collected and stored at –60°C until assayed.

COX-2 and/or COX-1b transiently transfected COS-7 cells were cultured in 12-well plates. Confluent cultures were washed twice with 37°C Earl's balanced salt solution (143.6 mM Na⁺, 5.4 mM K⁺, 1.8 mM Ca²⁺, 0.8 mM Mg²⁺, 125.3 mM Cl^–, 26.2 mM , 1.1 mM , 0.8 mM , and 5.5 mM glucose). The cells were then incubated for 30 min in Earl's balanced salt solution containing 15 µM arachidonic acid, 2 mM L-glutamine with or without acetaminophen. After the incubation, media were collected and stored at –60°C until assayed. PGE₂ concentrations in media were measured with a specific ELISA (Oxford Biomedical Research, Oxford, MI).

	Results

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Using mRNA from cultured rat CECs and isolated brain microvessels, a COX-1b PCR product of 573 bp was generated. The sequence showed 100% identity with the respective exonic parts and the entire 98-bp-long intron-1 of the rat COX-1 gene located on chromosome 3 (Fig. 1). Sequence analysis revealed that the rat COX-1b mRNA encodes a predicted 127-amino acid protein with a molecular mass of approximately 13 kDa (Fig. 1). COX-1b protein did not show homology to any existing database entries. The mRNA sequences of rat COX-1b isolated from CECs and from cerebral capillaries have been deposited in the GenBank with accession numbers AY523672 [GenBank] and AY523673 [GenBank] , respectively. RT-PCR experiments demonstrated the presence of the COX-1b sequence in the poly(A) RNA fraction isolated from CECs, suggesting that the COX-1b sequence is polyadenylated, which is a necessary step during the formation of mature mRNA (Fig. 2).

View larger version (44K): [in this window] [in a new window]   Fig. 1. Sequence of the rat COX-1 (partial) and COX-1b mRNA and their translation into amino acid sequences. Total RNA was isolated from primary cultures of rat CECs and from rat cerebral microvessels. COX-1b was detected by RT-PCR using specific primers. The PCR product was ligated into pGEM-T Easy Vector and transformed into E. coli to amplify and accurately sequence both strands of the rat COX-1b cDNA. Automated DNA sequencing of the COX-1b construct was performed on an ABI Prism 377 DNA sequencer.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Detection of rat COX-1b in poly(A) RNA isolated from rat cerebral endothelial cells. Representative gel electrophoresis of RT-PCR product for COX-1b detected in total RNA and poly(A) RNA fractions isolated from primary cultures of rat CECs. The expected molecular weight of the PCR product was 573 bp. M, molecular weight marker.

When we transiently transfected COS-7 cells with the p3XFLAG-CMV-14 vector containing the cDNA of COX-1b, we detected a clear protein band by Western blot analysis using the anti-FLAG antibody. The COX-1b/FLAG fusion protein was detected at a molecular mass of about 17 kDa (Fig. 3A), which compares favorably with the calculated molecular mass of 16.6 kDa for the COX-1b/FLAG fusion protein. As a positive control, COS-7 cells were transfected with the p3XFLAG-CMV-7-BAP plasmid, and they showed the expression of the bacterial alkaline phosphatase/FLAG fusion protein (calculated molecular mass of 69 kDa). We did not detect specific bands in untransfected cells (Fig. 3A).

View larger version (31K): [in this window] [in a new window]   Fig. 3. COX-1b protein expression in COS-7 cells. A, COS-7 cells were transiently transfected with the COX-1b p3XFLAG-CMV-14 vector containing the cDNA of rat COX-1b (lanes 1–4) or with p3XFLAG-CMV-7-BAP vector (lanes 5–8). Protein expression was demonstrated by Western blot analysis using anti-FLAG antibody. In the lysate of COS-7 cells transfected with the COX-1b p3XFLAG-CMV-14 vector, we detected COVAP/FLAG fusion protein at a molecular weight of about 17 kDa (lanes 1–4). The cells transfected with the p3XFLAG-CMV-7-BAP plasmid expressed the bacterial alkaline phosphatase (BAP)/FLAG fusion protein (lanes 5–8). We could not detect specific bands in untransfected cells (lanes 9–12). The numbers on the left indicate the masses and the positions of the molecular weight markers in kilodaltons. B, COS-7 cells were transfected with COX-1b p3XFLAG-CMV-14 vector, and stable transfected clones were selected by culturing the cells in Geneticin-containing medium. Protein expression was demonstrated by Western blot analysis using anti-FLAG antibody. This panel shows that three of the five selected Geneticin-resistant clones expressed the COVAP/FLAG fusion protein. C, proteins isolated from clones 2 and 3 of the COX-1b p3XFLAG-CMV-14 stably transfected COS-7 cells (see B) were subjected to Western blot analysis using anti-COX-1b polyclonal antibody. 2, clone 2; 3, clone 3.

During our attempt to establish clones of COS-7 cells stably transfected with COX-1b p3XFLAG-CMV-14 vector, we successfully subcultured five Geneticin-resistant colonies. Western blot analysis using anti-FLAG antibody detected bands of COX-1b/FLAG fusion protein in three of the five stable transfected clones (Fig. 3B).

To demonstrate the existence of COX-1b protein in rat tissues, we generated affinity-purified polyclonal antibodies against the rat COX-1b protein. The antibody recognized the purified COX-1b protein produced in E. coli (Figs. 4 and 5). In addition to the COX-1b protein, other bands were also visualized in our Western blots. These additional bands are easily distinguished from COX-1b, and most of them also occurred when incubation with the primary antibody was omitted (Fig. 5). However, in neuronal tissue samples there is a strong band at 60 kDa, which is related to the primary antibody (Figs. 4 and 5). This protein, which is recognized by the anti-COX-1b antibody, seems to be expressed primarily in neurons (Fig. 6).

View larger version (52K): [in this window] [in a new window]   Fig. 4. COX-1b expression in rat tissues. Proteins isolated from rat tissues were subjected to Western blot analysis with (A) or without (B) polyclonal anti-rat COX-1b antibody. The numbers on the left indicate the masses and the positions of the molecular weight markers in kilodaltons. 1, heart; 2, liver; 3, lung; 4, aorta; 5, spleen; 6, pancreas; 7, kidney; 8, muscle; 9, brainstem; 10, cerebellum; 11, cortex; and 12, spinal cord.

View larger version (65K): [in this window] [in a new window]   Fig. 5. COX-1b expression in rat neural tissues. Proteins isolated from different parts of the rat central nervous system were subjected to Western blot analysis using polyclonal anti-rat COX-1b antibody. Lanes 9 to 11 show the result when incubation with the primary antibody was omitted. S, COX-1b standard produced in E. coli; 1, 5, and 9, brainstem; 2, 6, and 10, cerebellum; 3, 7, and 11, cortex; 4 and 8, spinal cord.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Comparison of the expression of the 64-kDa protein in neurons, cerebral capillaries, and cerebral endothelial cells. Proteins isolated from primary cultures of rat CECs, rat cerebral microvessels, and primary cultures of rat cortical neurons were subjected to Western blot analysis using polyclonal anti-rat COX-1b antibody.

We checked the expression of COX-1b protein in 12 different rat tissue samples. COX-1b was expressed in all tissues we examined; the expression was the strongest in the heart, kidney, and neuronal tissues (Figs. 4 and 5). After subcellular fractionation of cultured rat CECs, we observed COX-1b protein predominantly in the cytosolic fraction, with weak staining in the nuclear fraction and no detectable signal in postnuclear membranes (Fig. 7A). Our immunocytochemistry experiments on primary cultures of rat CECs showed a slightly punctate staining pattern distributed throughout the cytoplasm (Fig. 7B). When the incubation with the primary anti-COX-1b antibody was omitted during the immunocytochemistry, no staining was visible.

View larger version (82K): [in this window] [in a new window]   Fig. 7. Intracellular localization of COX-1b protein in cultured rat cerebral endothelial cells. A, proteins isolated from subcellular fractions of cultured rat CECs and were subjected to Western blot analysis using polyclonal anti-rat COX-1b antibody. 1, nuclear fraction; 2, membrane fraction; 3, cytosolic fraction; and S, COX-1b standard produced in E. coli. B, immunostaining of primary cultures of rat CECs using anti-rat COX-1b antibody.

To determine the cyclooxygenase activity of the COX-1b protein, first we used COS-7 cells that were stable transfected with the COX-1b/FLAG fusion protein. We measured the PGE₂ production in COX-1b-positive (clone 2 on Fig. 3B) and COX-1b-negative (clone 3 on Fig. 3B) stably transfected COS-7 cells in the presence of exogenous arachidonic acid. We could not detect a significant difference between the PGE₂ production in COX-1b-positive and COX-1b-negative COS-7 cells (Fig. 8). Acetaminophen did not have a significant effect on the PGE₂ production in either cell line (Fig. 8). In a second series of experiments, we transiently transfected COS-7 cells with native COX-1b (without FLAG epitope), with murine COX-2, and cotransfection with both COX-2 and COX-1b. Western blot analysis demonstrated the expression of the respective proteins in the transfected cells (Fig. 9A). The PGE₂ production of COX-1b-transfected cells was not different compared with the empty vector-transfected control cells (Fig. 9B). COX-2 transfection resulted in a 3-fold increase in PGE₂ production, which was slightly inhibited by a low concentration of acetaminophen. The COX-2/COX-1b-cotransfected cells showed a similar PGE₂ production profile as the COX-2 only transfected cells (Fig. 9B).

View larger version (30K): [in this window] [in a new window]   Fig. 8. PGE2 production in COX-1b-stably transfected COS-7cells. Rat COX-1b-expressing (clone 2; see Fig. 3) and -nonexpressing (clone 3; see Fig. 3) stable-transfected COS-7 cells cultured in 12-well plates were incubated at 37°C for 15 min in DMEM containing 30 µM arachidonic acid with or without 100 µM acetaminophen. PGE2 concentrations in media were measured with ELISA.

View larger version (34K): [in this window] [in a new window]   Fig. 9. PGE2 production in COX-2 and/or COX-1b transiently transfected COS-7 cells. COS-7 cells were transfected with the empty pcDNA3.1 vector, pcDNA3.1 vector containing rat COX-2 cDNA, pcDNA3.1 vector containing rat COX-1b cDNA, or cotransfected with the COX-2- and the COX-1b-containing vectors. A, expression of COX-2 and COX-1b protein in transfected cells. B, PGE2 production in the transfected cells. COS-7 cells cultured in 12-well plates were incubated at 37°C for 30 min in Earl's balanced salt solution containing 10 µM arachidonic acid with or without 100 µM acetaminophen. PGE2 concentrations in media were measured by ELISA.

	Discussion

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There are five major findings of our present study. 1) This is the first study that describes the entire sequence of the rat COX-1b mRNA. 2) We demonstrated that the rat COX-1b mRNA encodes a protein that has a completely different amino acid sequence than the known cyclooxygenases. 3) We generated an antibody against the rat COX-1b protein that is suitable for Western blot analysis. 4) We demonstrated that COX-1b protein probably exists in vivo in multiple rat tissues. 5) COX-1b apparently does not have cyclooxygenase activity.

COX-1b (originally named as COX-3) was identified in canine tissues as the long-sought brain-specific cyclooxygenase enzyme that is the target of acetaminophen (Chandrasekharan et al., 2002), thus potentially solving the mystery of the mechanism of action of this drug (Botting, 2000). Although COX-1b as a COX-1 splice variant was demonstrated in rats, mice, and humans, there was a serious question as to whether COX-1b mRNA in these species also encodes an acetaminophen-sensitive cyclooxygenase as in canines, because in these species the retention of the entire intron-1, which is 98 bp in rat and mouse and 94 bp in human, would shift the original reading frame of COX-1 (Dinchuk et al., 2003; Schwab et al., 2003). Hypothetically, a different initiation site or an alternative downstream splicing might restore the original COX-1 reading frame resulting in the synthesis of a peptide with homology to COX-1, except the N-terminal end, which would be encoded by the inserted intron-1. To address this hypothesis, it was necessary to clone and sequence the entire rat COX-1b mRNA. We cloned and sequenced a 573-bp-long COX-1b mRNA fragment that showed that the entire 98-bp-long intron-1 is inserted into the rat COX-1b mRNA (Fig. 1). We considered all three reading frame possibilities based upon the sequence of the detected 573-bp-long COX-1b mRNA fragment. First, maintaining the original start codon for COX-1 mRNA defines an open reading frame with a stop codon at base 382 (Fig. 1). Because the available sequence of the 5' end is very limited, we cannot rule out the possibility of a more proximal translation initiation site for the COX-1b mRNA. The other two possible reading frames, however, indicate premature stop codons at bp 2 or 9 (Fig. 1). Therefore, even if present, a more proximal translation initiation site than known for COX-1 could not save the original COX-1 reading frame.

How the COX-1 variant mRNA transcript avoids the nonsense-mediated decay pathway, which specifically targets mRNAs containing premature stop codons for degradation (Hentze and Kulozik, 1999; Lynch and Kewalramani, 2003), is currently not known. An mRNA is targeted by nonsense-mediated decay by the presence of a cis-acting downstream element appropriately located within 200 nucleotides downstream of the stop codon (Ruiz-Echevarria and Peltz, 1996; Ruiz-Echevarria et al., 1998). Conversely, mRNA containing premature stop codons can avoid nonsense-mediated decay if they possess a cis-acting stabilizer element (Ruiz-Echevarria et al., 2001). Thus, it is possible that the COX-1 variant mRNA lacks a properly positioned downstream element or carries a stabilizer element.

We detected the COX-1b sequence with RT-PCR in poly(A) RNA samples isolated from rat CECs, which suggested that the COX-1b sequence may exist in a form of mature mRNA that makes it highly possible that this sequence is translated into a protein. According to our sequence analysis, the only possible protein that could be translated from the rat COX-1b mRNA is a 127-amino acid protein that has a completely different amino acid sequence than the known cyclooxygenases. Initially we did not have an antibody against this hypothetical COX-1b protein, and therefore, we inserted the rat COX-1b cDNA into the p3XFLAG-CMV-14 vector proximal to the sequence of the FLAG protein. When we transfected COS-7 cells with this construct, we detected the COX-1b/FLAG fusion protein with anti-FLAG antibody in Western blot analysis at the expected molecular mass. This was the first evidence that the rat COX-1b mRNA contains an open reading frame and can be translated into a protein. We have also generated clones of COS-7 cells that were stable-transfected with the COX-1b p3XFLAG-CMV-14 vector. Western blot analysis demonstrated the synthesis of the COX-1b/FLAG fusion protein in these stable transfected COS-7 cells.

To explore the possible expression of COX-1b in rat tissues samples, we generated an anti-rat COX-1b antibody. Western blot analysis demonstrated that the antibody specifically binds to the purified rat COX-1b protein produced in E. coli (Fig. 5). We tested the antibody further on protein samples isolated from COS-7 cells that were transfected with the COX-1b p3XFLAG-CMV-14 vector (Fig. 3C). This experiment also proved that our anti-COX-1b antibody was specific and sensitive enough to be used in Western blot analysis. Using this antibody, we detected COX-1b protein in 12 different rat tissue samples, demonstrating that COX-1b protein may be synthesized and may exist in vivo. The expression was strongest in the heart, kidney, and neuronal tissues (Figs. 4 and 5). Among the neuronal tissues, it seems that the COX-1b protein expression is the highest in spinal cord, which correlates well with our previous RT-PCR data that demonstrated the highest COX-1b mRNA expression in the spinal cord among the neuronal tissues we studied (Kis et al., 2004a).

Besides the band of the COX-1b protein, some other bands were also visualized in our Western blots. These additional bands are easily distinguished from the band of COX-1b according to their molecular mass, and most of them are a result of nonspecific binding of the anti-rabbit secondary antibody to rat proteins (Figs. 4 and 5). The nonspecific binding was especially strong in the spleen samples, presumably due to the high immunocompetent cells/antibody content of the spleen. In neuronal tissue samples an additional protein was recognized by the rat anti-COX-1b antibody. Our search of protein databases revealed that the hapten peptide, which was used to induce antibody production (amino acids 2–17 of the rat COX-1b), shows homology to the sequence of an unnamed protein with a molecular mass of 64 kDa(GenBank XP_227405 [GenBank] .2). Therefore, our polyclonal antibody might also bind to this 64-kDa protein, which would be at the right location on the blot to account for this additional band. This protein is highly abundant in cultured rat cortical neurons (Fig. 6), which can explain why we detected the strong additional band in neuronal tissues by Western blot analysis.

COX-1b does not show homology to any protein sequences of published databases. It has a very basic character with the estimated pI value of 12.40. In addition to the abundance of basic amino acids, the protein is also very proline-rich (11.81% proline), arguing against the formation of extensive -helical domains and for the possible formation of -turns and perhaps the formation of antiparallel -sheets. Hydrophobicity analysis by the method of Kyte and Doolittle (1982) did not reveal the existence of transmembrane domains; however, the protein displayed an unusual periodicity, perhaps giving rise to an amphipathic character responsible for peripheral association with intracellular membrane surfaces. This may explain COX-1b's punctate distribution in CECs. Other motifs, including a possible protein kinase C phosphorylation site and cysteine-rich domains, were also noted in the amino acid sequence. The analysis did not identify a signal peptide in the molecule, suggesting that COX-1b is not a secretory protein.

Subcellular fractionation of cultured CECs (Fig. 7A) and rat tissues (data not shown) followed by Western blot analysis suggests that COX-1b is a cytosolic protein because it was detected predominantly in the cytosolic fraction of the preparations. Immunostaining of primary cultures of rat CECs confirmed this result, showing that COX-1b immunoreactivity is not localized to specific intracellular structures, but rather, it manifested as a punctate pattern distributed throughout the cytoplasm (Fig. 7B).

The rat COX-1b has an amino acid sequence completely different from COX-1 or COX-2, which makes it unlikely that COX-1b is involved in prostaglandin production. We used stable and transiently transfected COS-7 cells to determine the cyclooxygenase activity of COX-1b. COS-7 cells were chosen specifically because Vidwans et al. (2001) showed that these cells have little endogenous cyclooxygenase activity, but when they were transfected with COX-2 construct, their PGE₂ production increased severalfold. Our current findings support these results. Our results on both stable and transiently transfected cells suggest that rat COX-1b does not have cyclooxygenase activity. Moreover, COX-1b did not have any effect on the inhibition of COX-2 by acetaminophen. To support our findings, Warner et al. (2004) demonstrated in a variety of rat tissues that the production of prostanoids is dependent on the two known isoforms of cyclooxygenase COX-1 and COX-2 and that there is no evidence for the involvement of a particular acetaminophen-sensitive COX-1b (named COX-3) isoform. On the other hand, our results make it unlikely that the acetaminophen-induced hypothermia is the consequence of the inhibition of COX-1b as suggested by Ayoub et al. (2004).

We have also sequenced the mouse COX-1b mRNA (Gen-Bank AY547265 [GenBank] ), which shows 93% homology to its rat counterpart, suggesting that the mouse COX-1b mRNA encoded protein has a similar amino acid sequence as in the rat. However, if the whole 94-bp intron-1 is retained in the human COX-1b, it will lead to the synthesis of a completely different protein with no similarity to the rat COX-1b.

Our experiments have led us to the discovery of a new protein that is transcribed from COX-1b mRNA, an alternative splice variant of the COX-1 gene. Because this protein has a completely different amino acid sequence than COX-1 and COX-2, and it does not have cyclooxygenase activity, we suggest the name cyclooxygenase variant protein (COVAP) to distinguish it from the known prostaglandin-synthesizing cyclooxygenase isoforms.

	Acknowledgements

 
We thank Dr. Sandra J. Hewett and Nancy Busija for critical reading of the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health Grants HL-30260, HL-66074, HL-65380, and DK-62372 and AHA Bugher Foundation Award 0270114N (to D.W.B.).

doi:10.1124/jpet.104.079533.

ABBREVIATIONS: COX, cyclooxygenase; bp, base pair; CEC, cerebral endothelial cell; DMEM, Dulbecco's modified Eagle's medium; RT-PCR, reverse transcription-polymerase chain reaction; PCOX, partial cyclooxygenase; PCR, polymerase chain reaction; PGE₂, prostaglandin E₂; ELISA, enzyme-linked immunosorbent assay; COVAP, cyclooxygenase variant protein.

¹ These authors contributed equally to this work.

Address correspondence to: Dr. Bela Kis, Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Medical Center Blvd., Winston-Salem, NC 27157. E-mail: bkis{at}wfubmc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

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