ARTICLE |
Correspondence to: Monique Doré, Dépt. de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, CP 5000, St-Hyacinthe, Québec, Canada J2S 7C6. E-mail: doremo@medvet.umontreal.ca
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Summary |
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Cyclo-oxygenase-2 (COX-2) is believed to participate in the repair of gastric ulcer. Like humans, pigs frequently develop gastric ulcers and thus represent an attractive animal model in which to study the repair process of naturally occurring gastric ulcers. However, expression of COX in the pig stomach has not been reported. The objectives of this study were to determine whether COX isoenzymes are expressed in porcine gastric ulcers and to characterize the porcine COX-2 cDNA. Normal stomachs (n=5) and those with gastric ulcers (n=35) were studied by immunohistochemistry and immunoblotting analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to isolate the complete porcine COX-2 cDNA. COX-1 staining was present in normal stomach and in ulcerated areas. No COX-2 was detected in normal stomach, but COX-2 was strongly expressed in the ulcerated area in 28/35 (80%) gastric ulcers (p<0.01). Immunoblotting analysis confirmed the restricted expression of COX-2 in the ulcerated areas. The porcine COX-2 cDNA was shown to code for a 604 amino acid protein that is 89% identical to human COX-2. These results provide the complete primary structure of porcine COX-2 and demonstrate for the first time that the enzyme is induced in naturally occurring porcine gastric ulcers.
(J Histochem Cytochem 50:923933, 2002)
Key Words: cyclo-oxygenase-2, gastric ulcers, pigs, stomach
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Introduction |
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GASTRIC ULCER is a common disease that affects millions of people in the United States each year (
Prostaglandins (PGs) play an important role in protecting the mucosa of the stomach (
COX-1, which is constitutively expressed in the stomach, is believed to be the isoform mostly responsible for the synthesis of gastric PGs (
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Materials and Methods |
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Materials
The citrate phosphate dextrose solution was obtained from Abbott Laboratories (North Chicago, IL). The Vectastain ABC kit was obtained from Vector Laboratories (Burlingame, CA). Diaminobenzidine tetrahydrochloride was purchased from Sigma-Aldrich (Oakville, Ont, Canada). Hybond polyvinylidene difluoride membranes (0.45 µm) were obtained from ICN Pharmaceuticals (Montreal, PQ, Canada). The horseradish peroxidase-labeled donkey anti-rabbit antibodies and the enhanced chemiluminescence (ECL) detection kit were purchased from Amersham Life Sciences (Arlington Heights, IL). Kodak Bio-Max X-ray film was obtained from Eastman Kodak (Rochester, NY). TRIzol total RNA isolation reagent, 1-kb DNA ladder, and synthetic oligonucleotides were obtained from Gibco BRL (Life Technologies; Gaithersburg, MD). Electrophoretic reagents were obtained from Bio-Rad Laboratories (Richmond, CA). The Prime-a-Gene labeling system, the Access RT-PCR System, and pGem-T Easy Vector were purchased from Promega (Madison, WI). The Gene Clean Kit was obtained from Bio 101 (La Jolla, CA). The porcine genomic library was obtained from Clontech (Palo Alto, CA). [-32P]-dCTP was purchased from NEN Life Science Products (Mississauga, Ont, Canada).
Tissue Samples and Platelet Isolation
Thirty-five cases of gastric ulcers from pigs submitted for a necropsy at the Département de Pathologie et Microbiologie of the Faculté de Médecine Vétérinaire (Université de Montréal) were included in the study. All cases were confirmed by examination of hematoxylineosinsaffran-stained sections by a veterinary pathologist. Light microscopic examination of WhartinStarry-stained tissue sections was also done to evaluate the presence of spiral bacteria. Pigs were either brought dead for necropsy or were sacrificed. Five normal stomachs obtained from healthy 6-month-old pigs were used as controls. All tissues studied were fixed in 10% neutral buffered formalin, whereas fresh samples from normal stomachs and from gastric ulcers were frozen at -70C for immunoblotting analysis.
Porcine platelets were isolated from whole blood collected by venipuncture in anticoagulant (citrate phosphate dextrose solution). Platelet-rich plasma was isolated by successive centrifugations of the citrated blood for 3 min at decreasing speeds (700, 650, and 600 x g) as previously described (
Anti-COX Antibodies
Two anti-COX antibodies (antibodies 8223 and MF243) were used. Affinity-purified polyclonal antibody 8223 was raised in rabbits against ovine COX-1, and was shown to be selective for COX-1 in various species (
Immunohistochemistry
Immunohistochemical staining was performed using the Vectastain ABC kit (Vector Laboratories) as previously described (31% positive cells.
Solubilized Cell Extracts and Immunoblotting Analysis
Solubilized cell extracts were prepared as previously described (
Cloning and Characterization of the Porcine COX-2 cDNA
The near-complete porcine COX-2 cDNA was cloned by a combination of three reverse transcription-polymerase chain reactions (RT-PCR) using the Access RT-PCR System (Promega) and following the manufacturer's protocol. Three RT reactions were performed with primers corresponding to the 5'-end, mid-portion, and 3'-end of the transcript (antisense primers 2, 4, and 7; Fig 1). RT reactions were performed at 48C for 45 min on 200 ng of total RNA extracted (TRIzol; Life Technologies) from a pig preovulatory follicle obtained 34 hr after IV administration of human chorionic gonadotropin (hCG; 1500 IU) (
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Genomic library screening was used to characterize two small regions of porcine COX-2 that were not obtained by RT-PCR, i.e., the first 28 bp of the coding region as well as a 120-bp portion in the 5'-untranslated region (UTR), and a short 38-bp gap present between two PCR fragments (Fig 1). A porcine genomic library (Clontech) was screened following the manufacturer's protocol with a 5' 1.5-kb EcoRI fragment of the bovine COX-2 cDNA (-32P]-dCTP using the Prime-a-Gene labeling system (Promega). Two positive clones (clones 2-1 and 3-1) isolated from an initial screen of 450,000 phage plaques were purified through secondary and tertiary screening. Sequencing reactions involving clone 2-1 and antisense primer 5'-GGA CTT GAG TGG TCC AAA CTG-3' and clone 3-1 and antisense primer 5'-CTA TGA CTG CAG CCT TAA ACC-3' were used to characterize the 5'-end of the coding region and the gap region, respectively.
Statistical Analysis
The Fisher's exact test was used to compare the frequency of COX-2 expression between normal stomachs and gastric ulcers. Statistical analyses were performed using the JMP Software (SAS Institute; Cary, NC).
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Results |
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Characteristics of Pigs with Gastric Ulcers
The age of pigs with gastric ulcers ranged from 1 month to 4 years, with a mean of 6.9 ± 1.9 months. All ulcers were localized to the pars esophagea. Of the 35 cases, 23 animals (65.7%) were submitted alive and were sacrificed, 11 pigs (31.4%) were brought dead to the necropsy service, and the information was absent from the necropsy report of one animal. In addition to gastric ulcers, most pigs (n=30) were suffering from other pathological processes, including respiratory or gastrointestinal infections (n=28) and polyserositis (n=5). Examination of WhartinStarry-stained sections to detect the presence of spiral bacteria revealed that bacteria morphologically compatible with Helicobacter spp. were present in the glandular mucosa adjacent to the ulcer in only one sample.
COX Expression in Normal Porcine Stomachs
To determine whether COX-1 and/or COX-2 were expressed under physiological conditions, immunohistochemical staining was performed on the pars esophagea of normal porcine stomachs (n=5). Results showed that some COX-1 was present in blood vessels (in endothelial cells and in smooth muscles of the media) and in fibroblasts in the submucosa in all normal stomachs (100%; Fig 2A and Fig 2B). However, no COX-2 expression was detected in normal porcine stomachs (Fig 2C and Fig 2D).
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COX Expression in Porcine Gastric Ulcers
COX-1-positive cells were found in the ulcer area in 27 of 35 cases (75%) (Fig 3E), while the ulcerated region from eight cases (25%) contained no COX-1-expressing cells. However, in contrast to normal stomachs in which no COX-2 was detected, 28 of the 35 (80%) gastric ulcers expressed COX-2 (p<0.01; Table 1). The extent and intensity of COX-2 staining varied among samples (Table 1). COX-2 immunostaining was predominantly localized in the cytoplasm of elongated fibroblast-like cells that were localized in the granulation tissue proliferating under the ulcerated area (Fig 3A3D).
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Immunoblotting of COX Isoforms in Porcine Gastric Tissues
To characterize each COX isoform, solubilized cell extracts were prepared from normal stomachs, gastric ulcers, and platelets, and proteins were analyzed by Western blotting. When a selective anti-COX-1 antibody was used, a 69,000 Mr band was detected in both normal and ulcerated stomachs (Fig 4A). A band of identical Mr was detected in porcine platelets (Fig 4A) and thus corresponded to porcine COX-1. When a selective anti-COX-2 antibody was used, no signal was detected in normal stomachs but COX immunoreactivity was observed in the two gastric ulcers (Fig 4B). Porcine COX-2 appeared as a 72,00074,000 Mr doublet and a small 62,000 Mr band (Fig 4B) believed to correspond to a proteolytic fragment, as previously observed in other species (
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Cloning and Characterization of Porcine COX-2
The near full-length porcine COX-2 cDNA sequence was obtained from three RT-PCR reactions designed to generate contiguous DNA fragments (Fig 1). The first fragment (clone 1.3) corresponded to the 5'-region of the cDNA and extended from nucleotides 29 to 1334 of the coding region. The second fragment (clone 1.0), which overlapped with the first one, contained the remaining portion of the coding region as well as 541 nucleotides of the 3'-untranslated region. Lastly, the third fragment (clone 1.1) corresponded to the 3'-region of the cDNA, did not overlap with the second fragment, and contained an additional 1.1 kb of 3'-untranslated region. To complete the characterization of the porcine COX-2 cDNA, the remaining portion at the 5'-end of the cDNA and the 38-bp gap between clones 1.0 and 1.1 were obtained from genomic clones. Collectively, the porcine COX-2 cDNA contains a 5'-untranslated region of 120 bp, an open reading frame of 1815 bp (including the stop codon), and a long 3'-untranslated region of 1686 bp containing multiple repeats of the ShawKamen sequence 5'-ATTTA-3' (n=12; Fig 5).
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The amino acid sequence of pig COX-2 was deduced from the coding region of the cDNA, and comparisons were made with the human (
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Discussion |
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This study reports for the first time the induced expression of COX-2 in naturally occurring gastric ulcers in pigs. COX-2-expressing cells were found in the majority (80%) of ulcerated stomachs, principally in the granulation tissue at the margins and underneath the ulcer. In recent years, several reports on the expression of COX-2 in experimentally induced gastric ulcers in laboratory animals have appeared, but COX-2 expression had not yet been documented in naturally occurring gastric ulcers in animals. In contrast to COX-2, COX-1 expression was similar in healthy vs ulcerated stomachs, in agreement with most previous reports (
The implication of COX-2 in gastric ulcer healing has emerged in recent years. Mice and rat models of acetic acid- or ethanol-induced gastric ulcers have been used to document the expression of COX-2 in ulcerated stomachs (
In the present study, COX-2 expression was not observed in normal porcine stomachs. This finding is in keeping with the current dogma that COX-2 is inducible and not constitutively expressed like COX-1, and with reports stating that COX-2 protein is absent from normal gastric tissues (
The cellular localization of COX-2 in ulcerated stomachs was associated with cells that were morphologically compatible with fibroblasts, a finding also in agreement with previous reports (
In humans, H. pylori has been closely associated with chronic gastritis and peptic ulcer disease (
This study is also the first to document the cloning and characterization of the full-length porcine COX-2 cDNA. Comparative analysis showed that the amino acid sequence of porcine COX-2 is very similar to that of other mammalian homologues, being 89%, 88%, 87%, 90%, 91%, 91%, 90%, 88%, 87% to human, rat, mouse, horse, cow, ovine, rabbit, guinea pig, and mink COX-2, respectively (
In summary, our results provide the complete primary structure of porcine COX-2 and demonstrate that the enzyme is expressed in the stomach of the majority of pigs with naturally occurring gastric ulcers, being concentrated in fibroblast-like cells in the granulation tissue proliferating in the ulcerated area. These findings suggest that COX-2 is involved in the tissue repair process during gastric ulceration in pigs, and represent the first demonstration of COX-2 expression in an animal model of naturally occurring gastric ulcers.
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Acknowledgments |
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Supported in part by the Natural Sciences and Engineering Council of Canada Grant 195188 (MD) and the Canadian Institutes for Health Research (CIHR) Grant MT-13190 (JS). JS is the recipient of a CIHR Investigator Award.
We thank Drs Jilly F. Evans and Stacia Kargman (Merck Frosst Centre for Therapeutic Research; Pointe-Claire-Dorval, Québec) for kindly supplying antibody MF243, Dr Bruce Downey and Fabienne Côté for providing porcine follicular tissues, and Dr Khampoune Sayasith and Nadine Bouchard for technical assistance.
Received for publication November 5, 2001; accepted January 23, 2002.
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