Selective Cyclooxygenase-2 Blocker Delays Healing of Esophageal Ulcers in Rats and Inhibits Ulceration-Triggered c-Met/Hepatocyte Growth Factor Receptor Induction and Extracellular Signal-Regulated Kinase 2 Activation
2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64918-8
ISSN1525-2191
AutoresDolgor Baatar, Michael K. Jones, Rama Pai, Hirofumi Kawanaka, Imre Szabó, Woo Sung Moon, Seigo Kitano, Andrzej S. Tarnawski,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoNonsteroidal anti-inflammatory drugs, both nonselective and cyclooxygenase-2 (COX-2) selective, delay gastric ulcer healing. Whether they affect esophageal ulcer healing remains unexplored. We studied the effects of the COX-2 selective inhibitor, celecoxib, on esophageal ulcer healing as well as on the cellular and molecular events involved in the healing process. Esophageal ulcers were induced in rats by focal application of acetic acid. Rats with esophageal ulcers were treated intragastrically with either celecoxib (10 mg/kg, once daily) or vehicle for 2 or 4 days. Esophageal ulceration triggered increases in: esophageal epithelial cell proliferation; expression of COX-2 (but not COX-1); hepatocyte growth factor (HGF) and its receptor, c-Met; and activation of extracellular signal-regulated kinase 2 (ERK2). Treatment with celecoxib significantly delayed esophageal ulcer healing and suppressed ulceration-triggered increases in esophageal epithelial cell proliferation, c-Met mRNA and protein expression, and ERK2 activity. In an ex vivo organ-culture system, exogenous HGF significantly increased ERK2 phosphorylation levels in esophageal mucosa. A structural analog of celecoxib, SC-236, completely prevented this effect. These findings indicate that celecoxib delays esophageal ulcer healing by reducing ulceration-induced esophageal epithelial cell proliferation. These actions are associated with, and likely mediated by, down-regulation of the HGF/c-Met-ERK2 signaling pathway. Nonsteroidal anti-inflammatory drugs, both nonselective and cyclooxygenase-2 (COX-2) selective, delay gastric ulcer healing. Whether they affect esophageal ulcer healing remains unexplored. We studied the effects of the COX-2 selective inhibitor, celecoxib, on esophageal ulcer healing as well as on the cellular and molecular events involved in the healing process. Esophageal ulcers were induced in rats by focal application of acetic acid. Rats with esophageal ulcers were treated intragastrically with either celecoxib (10 mg/kg, once daily) or vehicle for 2 or 4 days. Esophageal ulceration triggered increases in: esophageal epithelial cell proliferation; expression of COX-2 (but not COX-1); hepatocyte growth factor (HGF) and its receptor, c-Met; and activation of extracellular signal-regulated kinase 2 (ERK2). Treatment with celecoxib significantly delayed esophageal ulcer healing and suppressed ulceration-triggered increases in esophageal epithelial cell proliferation, c-Met mRNA and protein expression, and ERK2 activity. In an ex vivo organ-culture system, exogenous HGF significantly increased ERK2 phosphorylation levels in esophageal mucosa. A structural analog of celecoxib, SC-236, completely prevented this effect. These findings indicate that celecoxib delays esophageal ulcer healing by reducing ulceration-induced esophageal epithelial cell proliferation. These actions are associated with, and likely mediated by, down-regulation of the HGF/c-Met-ERK2 signaling pathway. Nonsteroidal anti-inflammatory drugs (NSAIDs) delay healing of experimental gastric ulcers by arresting epithelial proliferation in the ulcer margin, interfering with re-epithelialization, and inhibiting angiogenesis in the granulation tissue.1Wang JY Yamasaki S Takeuchi K Okabe S Delayed healing of acetic acid-induced gastric ulcers in rats by indomethacin.Gastroenterology. 1989; 96: 393-402PubMed Scopus (172) Google Scholar, 2Tarnawski A Stachura J Douglass TG Krause WJ Gergely H Sarfeh IJ Indomethacin impairs quality of experimental gastric ulcer healing: a quality histologic and ultrastructural analysis.in: Garner A O'Brien PE Mechanism of Injury, Protection and Repair of the Upper Gastrointestinal Tract. J. Willey & Sons, Chichester1991: 521-531Google Scholar, 3Schmassmann A Tarnawski A Peskar BM Varga L Flogerzi B Halter F Influence of acid and angiogenesis on kinetics of gastric ulcer healing in rats: interaction with indomethacin.Am J Physiol. 1995; 268: G276-G285PubMed Google Scholar, 4Levi S Goodlad RA Lee CY Stamp G Walport MJ Wright NA Hodgson HJ Inhibitory effect of non-steroidal anti-inflammatory drugs on mucosal cell proliferation associated with gastric ulcer healing.Lancet. 1990; 336: 840-843Abstract PubMed Scopus (198) Google Scholar Although cellular and molecular mechanisms of gastric ulcer healing have been extensively studied and well characterized,5Tarnawski A Cellular mechanisms of gastric ulcer healing.in: Domschke W Konturek SJ The Stomach. Springer-Verlag, Berlin, New York1993: 177-192Crossref Google Scholar, 6Pai R Ohta M Itani RM Sarfeh IJ Tarnawski AS Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats.Gastroenterology. 1998; 114: 706-713Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7Tarnawski A Pai R Wang H Tomikawa M Translocation of MAP (ERK-1 and -2) kinases to cell nuclei and activation of c-fos gene during healing of experimental gastric ulcers.J Physiol Pharmacol. 1998; 49: 479-487PubMed Google Scholar, 8Pai R Jones MK Tomikawa M Tarnawski AS Activation of Raf-1 during gastric ulcer healing in rats is ras mediated and protein kinase C-independent.Am J Pathol. 1999; 155: 1759-1766Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 9Schmassmann A Stettler C Poulsom R Tarasova N Hirschi C Flogerzi B Matsumoto K Nakamura T Halter F Roles of hepatocyte growth factor and its receptor met during gastric ulcer healing in rats.Gastroenterology. 1997; 113: 1858-1872Abstract Full Text PDF PubMed Scopus (82) Google Scholar the mechanisms involved in the healing of esophageal ulcers remain virtually unexplored. Our preliminary study demonstrated that, similar to gastric ulcers, epithelial cell proliferation is increased at the esophageal ulcer margin.10Baatar D Jones MK Pai R Kawanaka H Szabo IL Kitano S Tarnawski AS Esophageal ulceration triggers activation of genes encoding keratinocyte growth factor and its receptor: a key to esophageal ulcer healing?.Gastroenterology. 2001; 120: A816PubMed Google Scholar Whether NSAIDs affect esophageal ulcer healing, epithelial cell proliferation, and the molecular mechanisms involved in the healing process remain unknown. NSAIDs inhibit cyclooxygenase (COX), a rate-limiting enzyme in prostaglandin synthesis.11Vane JR Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs.Nat New Biol. 1971; 231: 232-235Crossref PubMed Scopus (7443) Google Scholar Two isoforms of this enzyme are known: COX-1, constitutively expressed in most tissues, is responsible for the physiological production of prostaglandins; and COX-2, induced by cytokines, mitogens, and endotoxins in inflammatory cells, is responsible for the increased production of prostaglandins during inflammation.12Smith WL Garavito RM DeWitt DL Prostaglandin endoperoxidase H synthases (cyclooxygenases)-1 and -2.J Biol Chem. 1996; 271: 33157-33160Crossref PubMed Scopus (1868) Google Scholar Early studies examining the effect of NSAIDs on experimental gastric ulcer healing1Wang JY Yamasaki S Takeuchi K Okabe S Delayed healing of acetic acid-induced gastric ulcers in rats by indomethacin.Gastroenterology. 1989; 96: 393-402PubMed Scopus (172) Google Scholar, 2Tarnawski A Stachura J Douglass TG Krause WJ Gergely H Sarfeh IJ Indomethacin impairs quality of experimental gastric ulcer healing: a quality histologic and ultrastructural analysis.in: Garner A O'Brien PE Mechanism of Injury, Protection and Repair of the Upper Gastrointestinal Tract. J. Willey & Sons, Chichester1991: 521-531Google Scholar, 3Schmassmann A Tarnawski A Peskar BM Varga L Flogerzi B Halter F Influence of acid and angiogenesis on kinetics of gastric ulcer healing in rats: interaction with indomethacin.Am J Physiol. 1995; 268: G276-G285PubMed Google Scholar have been conducted with the use of nonselective NSAIDs (eg, indomethacin) that inhibit both COX-1 and COX-2 enzymes.13Meade EA Smith WL DeWitt DL Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs.J Biol Chem. 1993; 268: 6610-6614Abstract Full Text PDF PubMed Google Scholar However, more recent studies indicate that experimental gastric ulceration induces COX-2, but not COX-1, expression14Mizuno H Sakamoto C Matsuda K Wada K Uchida T Noguchi H Akamatsu T Kasuga M Induction of cyclooxygenase 2 in gastric mucosal lesions and its inhibition by the specific antagonist delays healing in mice.Gastroenterology. 1997; 112: 387-397Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, 15Takahashi S Shigeta J Inoue H Tanabe T Okabe S Localization of cyclooxygenase-2 and regulation of its mRNA expression in gastric ulcers in rats.Am J Physiol. 1998; 275: G1137-G1145PubMed Google Scholar and that COX-2 selective NSAIDs delay gastric ulcer healing similarly to nonselective NSAIDs.16Schmassmann A Peskar BM Stettler C Netzer P Stroff T Flogerzi B Halter F Effects of inhibition of prostaglandin endoperoxide synthase-2 in chronic gastrointestinal ulcer models in rats.Br J Pharmacol. 1998; 123: 795-804Crossref PubMed Scopus (294) Google Scholar The expression of COX in ulcerated esophageal mucosa has not been studied and it is not known whether the COX-2 selective inhibitors affect esophageal ulcer healing. Various growth factors, including epidermal growth factor and hepatocyte growth factor (HGF), have been implicated in the stimulation of epithelial proliferation during gastric ulcer healing.9Schmassmann A Stettler C Poulsom R Tarasova N Hirschi C Flogerzi B Matsumoto K Nakamura T Halter F Roles of hepatocyte growth factor and its receptor met during gastric ulcer healing in rats.Gastroenterology. 1997; 113: 1858-1872Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 17Tarnawski A Stachura J Durbin T Sarfeh IJ Gergely H Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats.Gastroenterology. 1992; 102: 695-698PubMed Google Scholar, 18Wright NA Pike C Elia G Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells.Nature. 1990; 343: 82-85Crossref PubMed Scopus (434) Google Scholar Suppression of HGF production has been suggested as a key factor involved in the inhibitory action of NSAIDs on gastric ulcer healing.19Takahashi M Ota S Hata Y Mikami Y Azuma N Nakamura T Terano A Omata M Hepatocyte growth factor as a key to modulate anti-ulcer action of prostaglandins in stomach.J Clin Invest. 1996; 98: 2604-2611Crossref PubMed Scopus (93) Google Scholar, 20Bamba H Ota S Kato A Matsuzaki F Nonsteroidal anti-inflammatory drugs may delay the repair of gastric mucosa by suppressing prostaglandin-mediated increase of hepatocyte growth factor production.Biochem Biophys Res Commun. 1998; 245: 567-571Crossref PubMed Scopus (59) Google Scholar However, the role of endogenous HGF in the healing of esophageal ulcers remains unexplored. In regard to the esophagus, previous studies demonstrated that exogenous HGF is the most potent stimulator of proliferation and restitution of esophageal epithelial cells in vitro, suggesting that it might be involved in the repair process of esophageal mucosal damage.21Jimenez P Lanas A Piazuelo E Esteva F Effect of growth factors and prostaglandin E2 on restitution and proliferation of rabbit esophageal epithelial cells.Dig Dis Sci. 1998; 43: 2309-2316Crossref PubMed Scopus (25) Google Scholar, 22Takahashi M Ota S Ogura K Nakamura T Omata M Hepatocyte growth factor stimulates wound repair of the rabbit esophageal epithelial cells in primary culture.Biochem Biophys Res Commun. 1995; 216: 298-305Crossref PubMed Scopus (34) Google Scholar In previous studies we have demonstrated that the extracellular signal-regulated kinases (ERKs) and their upstream kinase, Raf-1, which mediate the mitogenic effects of growth factors, are activated during gastric ulcer healing6Pai R Ohta M Itani RM Sarfeh IJ Tarnawski AS Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats.Gastroenterology. 1998; 114: 706-713Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7Tarnawski A Pai R Wang H Tomikawa M Translocation of MAP (ERK-1 and -2) kinases to cell nuclei and activation of c-fos gene during healing of experimental gastric ulcers.J Physiol Pharmacol. 1998; 49: 479-487PubMed Google Scholar, 8Pai R Jones MK Tomikawa M Tarnawski AS Activation of Raf-1 during gastric ulcer healing in rats is ras mediated and protein kinase C-independent.Am J Pathol. 1999; 155: 1759-1766Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar and that interruption of this signaling pathway dramatically delays the healing process.6Pai R Ohta M Itani RM Sarfeh IJ Tarnawski AS Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats.Gastroenterology. 1998; 114: 706-713Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar However, the signaling pathways involved in esophageal ulcer healing, and stimulation of esophageal epithelial cell proliferation essential for the healing, remain unknown. The present study was aimed to: 1) explore molecular events associated with esophageal ulcer healing: expression of COX-1, COX-2, HGF and its receptor c-Met, and ERK2 phosphorylation levels and activity; and, 2) determine whether the selective COX-2 inhibitor, celecoxib, affects esophageal ulcer healing, ulceration-triggered cell proliferation and the above stated molecular events. The RNeasy Mini Kit was purchased from Qiagen (Valencia, CA), the bicinchoninic acid protein assay kit was purchased from Pierce Chemical (Rockford, IL), and the enhanced chemiluminescence Western blotting detection reagents were purchased from Amersham Life Science (Arlington Heights, IL). Monoclonal mouse anti-COX-1 and polyclonal rabbit anti-COX-2 antibodies were purchased from Cayman Chemical Co. (Ann Arbor, MI), polyclonal rabbit anti-HGF-α, anti-c-Met p140, anti-ERK2 antibodies and monoclonal mouse anti-pERK antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal mouse anti-proliferating cell nuclear antigen (PCNA) antibody and 3,3′-diaminobenzidine tetrahydrochloride were purchased from DAKO (Carpinteria, CA). [γ-32P]ATP was purchased from Dupont NEN Research Products (Boston, MA). Keratinocyte basal media was purchased from Clonetics (San Diego, CA). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). This study was approved by the Subcommittee for Animal Studies of the Long Beach Department of Veterans Affairs Medical Center. Male Sprague-Dawley rats (weighing 225 to 250 g) were used in the experiments. Rats were kept individually in wire-bottom cages with free access to a standard rat chow (Rodent Diet No.8504; Harlan Teklad, Madison, WI) and water. The animal room was illuminated on 12-hour light-dark cycles. Room temperature was kept at 18 to 22°C and humidity at 60 to 70%. Rats fasted for 12 hours underwent laparotomy under ketamine-xylazine anesthesia (40 mg/kg body wt of ketamine and 5 mg/kg body wt of xylazine, intraperitoneally). Esophageal ulcers were induced by modification of the method described by Tsuji and colleagues.23Tsuji H Fuse Y Kawamoto K Fujino H Kodama T Healing process of experimental esophageal ulcers induced by acetic acid in rats.Scand J Gastroenterol. 1989; 24: 6-10Crossref Scopus (18) Google Scholar In brief, the stomach was pulled down and the intra-abdominal esophagus was exposed. The length of esophagus, brought below the diaphragm by this procedure, was ∼1 cm providing sufficient space for application of acetic acid. After careful separation of vessels and nerves, 100% acetic acid (30 μl) was applied through a polyethylene tube (3-mm inner diameter) to the anterior wall of intra-abdominal esophagus for 3 minutes. The area was then washed with isotonic saline and the abdomen was closed. The rats undergoing the operation as described above but without application of acetic acid (sham operation) served as controls. All rats survived the scheduled study design. To evaluate the effects of ulceration on the studied parameters, rats with esophageal ulcers and sham-operated (SO) rats were euthanized 3 or 7 days after ulcer induction. To evaluate the effects of celecoxib on ulcer healing, rats with esophageal ulcers were treated once daily with either 10 mg/kg of celecoxib (4-[5-(4-methylphenyl)-3-(trifluoro-methyl)-1H-pyrazol-1-yl]benzenesulfonamide; Pfizer Inc., New York, NY) or its vehicle (1 ml of 1% carboxymethyl cellulose sodium salt) starting 3 days after ulcer induction. Celecoxib was given at 10 mg/kg because this dose has been shown to significantly inhibit the COX-2-induced generation of prostaglandin E2 in the rat air pouch without affecting whole-blood thromboxane synthesis, an index of COX-1 activity.24Wallace JL Chapman K McKnight W Limited anti-inflammatory efficacy of cyclo-oxygenase-2 inhibition in carrageenan-airpouch inflammation.Br J Pharmacol. 1999; 126: 1200-1204Crossref PubMed Scopus (120) Google Scholar Treatments were given intragastrically for 2 or 4 days, and rats were euthanized 5 and 7 days after ulcer induction. The starting of treatment (day 3) and ending (day 7) time points were chosen because our previous preliminary studies demonstrated that esophageal ulcers are fully developed by day 3 after application of acetic acid and that ∼60% of ulcers are completely re-epithelialized by day 9.10Baatar D Jones MK Pai R Kawanaka H Szabo IL Kitano S Tarnawski AS Esophageal ulceration triggers activation of genes encoding keratinocyte growth factor and its receptor: a key to esophageal ulcer healing?.Gastroenterology. 2001; 120: A816PubMed Google Scholar A one-cm long segment of the lower esophagus (including ulcer if present) was excised, opened longitudinally, and photographed. The ulcer area was measured using a computerized video-image analysis system (Image 1/FL; Universal Imaging Corp., Westchester, PA). Esophageal tissue samples including ulcer and immediately adjacent tissue (ulcerated tissue) or esophageal tissue from SO rats (normal tissue) were snap-frozen in liquid nitrogen, and stored at −80°C for RNA isolation and protein extraction or fixed in 10% formalin. In addition, a 1-cm long segment of the esophagus distal to the resected segment was excised and fixed in formalin for immunohistochemical staining. RNA was isolated using the RNeasy Mini Kit according to the manufacturer's instructions. Reverse transcription (RT) and polymerase chain reaction (PCR) were performed to determine mRNA levels. RT was performed using a GeneAmp RNA PCR kit and a DNA thermal cycler (Perkin Elmer, Norwalk, CT), which were also used for PCR. Total RNA (0.3 μg) was used as the template to synthesize complementary DNA (cDNA) with 2.5 U of Moloney murine leukemia virus reverse transcriptase in 10 μl of buffer containing 10 mmol of Tris-HCl, pH 8.3, 50 mmol KCl, 5 mmol random hexamer, 1.4 U of ribonuclease inhibitor. RT was performed at 42°C for 15 minutes. The resulting cDNA was used as a template for subsequent PCR. The specific primer set for rat COX-1 was 5′-ACGCCCTCATTCACCCATTT-3′ (sense) and 5′-CACGGACGCCTGTTCTACGG-3′ (anti-sense) and for COX-2 was 5′-TGGTGCCGGGTCTGATGATG-3′ (sense) and 5′-GCAATGCGGTTCTGATACTG-3′ (anti-sense), and the sizes of amplified fragments were 561 bp for COX-1 and 253 bp for COX-2, respectively.25Jones MK Sasaki E Halter F Pai R Nakamura T Arakawa T Kuroki T Tarnawski AS HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.FASEB J. 1999; 13: 2186-2194Crossref PubMed Scopus (88) Google Scholar The primers for rat c-Met were 5′-GCGAACTAATTCACTGCCCA-3′ (sense) and 5′-GCATCTGTGTTGTGTACGGT-3′ (antisense), and the size of the amplified fragment was 242 bp.26Masson S Scotte M Francois A Coeffier M Provot F Hiron M Teniere P Fallu J Salier JP Daveau M Changes in growth factor and cytokine mRNA levels after hepatectomy in rat with CCl4-induced cirrhosis.Am J Physiol. 1999; 277: G838-G846PubMed Google Scholar The PCR for β-actin was used as a positive control and an internal standard. The specific primer set for rat β-actin was purchased from Clontech Laboratories, Inc., Palo Alto, CA. The PCR was performed in 50 μl of buffer containing 10 mmol Tris-HCl, pH 8.3, 2 mmol MgCl2, 50 mmol KCl, 0.2 mmol each of deoxyribonucleoside triphosphates, 0.4 μmol of each primer, 2 U of Taq DNA polymerase. For the amplification of COX-1, COX-2, and rat c-Met cDNAs, 35 cycles of 1 minute at 94°C for denaturing, 1 minute at 55°C for annealing, and 2 minutes at 72°C for extension were performed. Nine-μl aliquots of the PCR products were subjected to electrophoresis on a 1.25% agarose gel, and the DNA was visualized by ethidium bromide staining. Location of the products and their sizes were determined using a 100-bp ladder (Life Technologies, Inc., Gaithersburg, MD). The gel was then photographed under UV transillumination. For the quantitative assessment of the PCR products, a computerized video analysis system (Image-1/FL, Universal Imaging Corp.) was used. The results are expressed as target cDNA/β-actin ratio. Esophageal tissues were homogenized with a Polytron homogenizer (Kinematica, Littau, Switzerland) in a lysis buffer containing 62.5 mmol ethylenediaminetetraacetic acid, 50 mmol Tris, pH 8.0, 0.4% deoxycholic acid, 1% Nonidet P-40, 0.5 mg/ml leupeptin, 0.5 mg/ml pepstatin, 0.5 mg/ml aprotinin, 0.2 mmol phenylmethylsulfonyl fluoride, and 0.05 mmol aminoethyl benzene sulfonyl fluoride. The homogenates were then centrifuged at 14,000 rpm for 10 minutes at 4°C. The protein concentration of the supernatant was determined by the bicinchoninic acid protein assay kit. Equal amounts of protein (0.15 mg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with specific antibodies at room temperature for 1 hour. The membranes were washed and incubated with corresponding IgG peroxidase conjugates at room temperature for 1 hour. The signal of the bound antibody was visualized using enhanced chemiluminescence Western blotting detection reagents. Protein expression was measured using a computerized video analysis system (Image-1/FL, Universal Imaging Corp.). COX-1 and COX-2 protein levels were determined using monoclonal mouse anti-COX-1 antibody and polyclonal rabbit anti-COX-2 antibody (Cayman Chemical Co.) diluted 1:1000. For the determination of HGF and c-Met protein expression, polyclonal rabbit anti-HGF-α antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and polyclonal rabbit anti-c-Met p140 antibody (Santa Cruz Biotechnology) were used at 1:250 dilution. ERK2 activity was determined as described previously.6Pai R Ohta M Itani RM Sarfeh IJ Tarnawski AS Induction of mitogen-activated protein kinase signal transduction pathway during gastric ulcer healing in rats.Gastroenterology. 1998; 114: 706-713Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Briefly, 50 μg of protein from tissue lysates was added to a conjugate of protein A Sepharose and 1 μg of polyclonal rabbit anti-ERK2 antibody and mixed at 4°C for 2 hours. The conjugates were then pelleted by centrifugation and washed four times. After the final wash, buffer was removed completely and 40 μl of protein kinase assay mixture (10 mmol/L HEPES, pH 7.5, 10 mmol/L MgCl2, 50 μmol/L ATP, 30 μg myelin basic protein, and 4 μCi [γ-32P]ATP) were added to each sample. The samples were incubated at 30°C for 20 minutes and the reaction was terminated by the addition of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer. The samples were then electrophoresed on 15% acrylamide gels. After electrophoresis, the gels were stained with Coomassie brilliant blue and dried. The gels were autoradiographed; the myelin basic protein bands were cut out and the radioactivity was counted in a scintillation counter. The activity was expressed as pmol of [γ-32P]ATP incorporated into 1 mg of MBP protein (pmol/mg). ERK2 and phosphorylated ERK (pERK) protein levels were determined by Western blot analysis using polyclonal rabbit anti-ERK2 antibody and monoclonal mouse anti-pERK antibody. The ERK2 phosphorylation levels were expressed as a percentage of total ERK2 protein levels. Immunohistochemical staining with specific antibodies was performed to determine localization of COX-2 and c-Met expression in normal and ulcerated esophageal sections. Deparaffinized sections were incubated overnight at 4°C with polyclonal rabbit anti-COX-2 antibody diluted 1:300 in phosphate-buffered saline (PBS) or polyclonal rabbit anti-c-Met antibody diluted 1:50 in PBS. After washing, sections were then incubated for 30 minutes with rabbit fluorescein-conjugated IgG diluted 1:100 in PBS. Immunofluorescence signal was evaluated under a Nikon Optiphot epifluorescence microscope with B-filter composition (Nikon, Garden City, NY). Expression of PCNA in formalin-fixed paraffin-embedded esophageal tissue sections was determined by the enhanced polymer one-step staining method.27Sano K Sekine J Inokuchi T Pe MB Ma G Enhanced polymer one-step staining for proliferating cell nuclear antigen in squamous cell carcinoma of the tongue.Biotech Histochem. 1996; 71: 273-287Crossref PubMed Scopus (11) Google Scholar Deparaffinized sections were incubated with monoclonal mouse anti-PCNA antibody for 1 hour at room temperature. The color was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAKO) and the sections were counterstained with Mayer's hematoxylin. Coded specimens were evaluated quantitatively under ×400 microscopic magnification by two investigators unaware of the code. Cell nuclei that stained dark brown were considered as labeled. Labeled cells were counted in the epithelium above the edge of interrupted muscularis mucosa at each ulcer margin corresponding to 700 μm of mucosal section length, in the epithelium distant from the ulcer, and in normal esophageal epithelium of SO rats. The length of the basement membrane was measured on the photographed images of corresponding hematoxylin and eosin (H&E) sections using a computerized video analysis system (Image-1/FL, Universal Imaging Corp.) and the number of PCNA-labeled cells per 100-μm length of the basement membrane was calculated. The results are expressed as percentage of increase in the number of labeled cells in the epithelium of the ulcer margin over the number of labeled cells in the epithelium distant from the ulcer. Sections from six rats per group were evaluated and the mean ± SD was calculated. To assess angiogenesis, enhanced polymer one-step immunostaining with monoclonal mouse anti-factor VIII-related antigen antibody (DAKO) that visualizes endothelial cells of vessels was used. Five days after ulcer induction, only a few endothelial cells forming microvessels were present in granulation tissue at the ulcer bed. Therefore, microvessels were counted only in sections obtained 7 days after ulcer induction. Coded specimens were evaluated quantitatively under ×200 microscopic magnification by two investigators unaware of the code. Microvessels with distinct lumen were counted in granulation tissue below the regenerating epithelium of the ulcer margin at each side. The results are expressed as a number of microvessels (mean ± SD) per ×200 microscopic field. Sections from six rats per group were evaluated. Rats (n = 18) were euthanized and a 1-cm long segment of the lower esophagus was excised and opened longitudinally. The esophageal mucosa was stripped from its muscle layers by sharp dissection as described previously.28Tobey NA Reddy SP Keku TO Cragoe Jr, EJ Orlando RC Studies of pHi in rabbit esophageal basal and squamous epithelial cells in culture.Gastroenterology. 1992; 103: 830-839Abstract PubMed Scopus (42) Google Scholar Explants consisted of squamous epithelium, lamina propria, and muscularis mucosa as determined by H&E staining. Esophageal mucosal explants were incubated in serum-free keratinocyte basal medium at 37°C with 5% CO2 and 95% air in a humidified incubator in the presence of 10 μmol/L or 100 μmol/L of SC-236 (Searle, Skokie, IL), a structural analog of celecoxib,29Penning TD Talley JJ Bertenshaw SR Carter JS Collins PW Docter S Graneto MJ Lee LF Malecha JW Miyashiro JM Rogers RS Rogier DJ Yu SS Anderson GD Burton EG Cogburn JN Gregory SA Koboldt CM Perkins WE Seibert K Veenhuizen AW Zhang YY Isakson PC Synthesis and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-58635, celecoxib).J Med Chem. 1997; 40: 1347-1365Crossref PubMed Scopus (2039) Google Scholar or its vehicle (dimethyl sulfoxide) for 6 hours. SC-236 was used because of its better solubility compared to celecoxib. Then explants were treated with either human recombinant HGF (100 ng/ml) or its vehicle (PBS) and were incubated for an additional 30 minutes. Protein extraction and Western blotting for pERK2 and ERK2 were performed as described above. Values are expressed as the mean ± SD. Student's t-test was used to determine the statistical significance of the differences. One-way analysis of variance followed by Bonferroni correction was used for multiple comparisons. A P value of <0.05 was considered statistically significant. Three days after ulcer induction, COX-2 mRNA and protein levels in ulcerated esophageal tissue were increased ≈2.5-fold and ≈threefold, respectively, versus normal esophageal tissue (Figure 1, Figure 2). COX-2 protein levels in ulcerated esophageal tissue were decreased at 7 days versus 3
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