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• W2015356544 abstract "Clostridium difficile toxin A induces acute colitis with neutrophil infiltration and up-regulation of numerous pro-inflammatory mediators, but the contribution of cyclooxygenase-2 (COX-2) induction in this infection is unknown. We report here that toxin A induces expression of COX-2 and secretion of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cultured NCM460 human colonocytes and in human intestinal xenografts. This induction was blocked by SB203580, a p38 MAPK inhibitor, which also decreased the phosphorylation of MSK-1, CREB/ATF-1, and COX-2 promoter activity following toxin A stimulation. Gel shift assays indicated that CREB/ATF-1 was the major proteins binding to the COX-2-CRE. Moreover, colonocytes exposed to toxin A produced reactive oxygen species (ROS), which activated p38 MAPK, MSK-1, and CREB/ATF-1, leading to subsequent COX-2 induction and PGE2 secretion. In intact mice, blockage of p38 MAPK inhibited toxin A-mediated induction of COX-2 in enterocytes as well as lamina propria cells, and significantly blocked the toxin A-induced ileal secretion of fluid and PGE2. Furthermore, a selective COX-2 inhibitor also diminished toxin A-associated ileal fluid and PGE2 secretion. The main signaling pathway for toxin A induction of human COX-2 involves ROS-mediated activation of p38 MAPK, MSK-1, CREB, and ATF-1. Toxin A triggers ileal inflammation and secretion of fluid via COX-2 induction and release of PGE2. Clostridium difficile toxin A induces acute colitis with neutrophil infiltration and up-regulation of numerous pro-inflammatory mediators, but the contribution of cyclooxygenase-2 (COX-2) induction in this infection is unknown. We report here that toxin A induces expression of COX-2 and secretion of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cultured NCM460 human colonocytes and in human intestinal xenografts. This induction was blocked by SB203580, a p38 MAPK inhibitor, which also decreased the phosphorylation of MSK-1, CREB/ATF-1, and COX-2 promoter activity following toxin A stimulation. Gel shift assays indicated that CREB/ATF-1 was the major proteins binding to the COX-2-CRE. Moreover, colonocytes exposed to toxin A produced reactive oxygen species (ROS), which activated p38 MAPK, MSK-1, and CREB/ATF-1, leading to subsequent COX-2 induction and PGE2 secretion. In intact mice, blockage of p38 MAPK inhibited toxin A-mediated induction of COX-2 in enterocytes as well as lamina propria cells, and significantly blocked the toxin A-induced ileal secretion of fluid and PGE2. Furthermore, a selective COX-2 inhibitor also diminished toxin A-associated ileal fluid and PGE2 secretion. The main signaling pathway for toxin A induction of human COX-2 involves ROS-mediated activation of p38 MAPK, MSK-1, CREB, and ATF-1. Toxin A triggers ileal inflammation and secretion of fluid via COX-2 induction and release of PGE2. Clostridium difficile is the major cause of antibiotic-associated colitis, a disease with significant morbidity and mortality (1Kelly C.P. Pothoulakis C. LaMont J.T. N. Engl. J. Med. 1994; 330: 257-262Crossref PubMed Scopus (1044) Google Scholar), and a major economic burden for hospitalized patients (2Kyne L. Hamel M.B. Polavaram R. Kelly C.P. Clin. Infect. Dis. 2002; 34: 346-353Crossref PubMed Scopus (613) Google Scholar). C. difficile produces intestinal damage and diarrhea by releasing two exotoxins, A and B, into the intestinal lumen (3Chaves-Olarte E. Weidmann M. Eichel-Streiber C. Thelestam M. J. Clin. Invest. 1997; 100: 1734-1741Crossref PubMed Scopus (117) Google Scholar). Toxin A, a 308-kDa heat-labile protein, elicits acute enteritis and secretion of fluid from ileum and colon of several animal species (4Pothoulakis C. Lamont J.T. Am. J. Physiol. 2001; 280: G178-G183Crossref PubMed Google Scholar). The toxin elicits an inflammatory exudate containing lymphocytes, neutrophils, and serum proteins and pro-inflammatory cytokines that mediate a profound and rapid inflammatory response (5Triadafilopoulos G. Pothoulakis C. Weiss R. Giampaolo C. Lamont J.T. Gastroenterology. 1989; 97: 1186-1192Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 6Pothoulakis C. Karmeli F. Kelly C.P. Eliakim R. Joshi M.A. O'Keane C.J. Castagliuolo I. LaMont J.T. Rachmilewitz D. Gastroenterology. 1993; 105: 701-707Abstract Full Text PDF PubMed Google Scholar, 7Ishida Y. Maegawa T. Kondo T. Kimura A. Iwakura Y. Nakamura S. Mukaida N. J. Immunol. 2004; 172: 3018-3025Crossref PubMed Scopus (84) Google Scholar). The induction of fluid secretion and inflammation by toxin A involves extensive signaling cross-talk between epithelial cells, mast cells, sensory neurons, and inflammatory cells of the intestinal lamina propria (4Pothoulakis C. Lamont J.T. Am. J. Physiol. 2001; 280: G178-G183Crossref PubMed Google Scholar). The cellular mechanism of toxin A involves glucosylation of a threonine residue at position 37 on Rho, Rac, and cdc42 (8Just I. Wilm M. Selzer J. Rex G. von Eichel-Streiber C. Mann M. Aktories K. J. Biol. Chem. 1995; 270: 13932-13936Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar), small GTP-binding proteins that regulate cell shape through modulation of the actin cytoskeleton. Monoglucosylation and inactivation of Rho proteins by the toxin causes severe cytoskeletal abnormalities in cultured and intact human colonocytes (9Hecht G. Pothoulakis C. LaMont J.T. Madara J.L. J. Clin. Invest. 1988; 82: 1516-1524Crossref PubMed Scopus (298) Google Scholar, 10Riegler M. Sedivy R. Pothoulakis C. Hamilton G. Zacherl J. Bischof G. Cosentini E. Feil W. Schiessel R. LaMont J.T. Wenzl E. J. Clin. Invest. 1995; 95: 2004-2011Crossref PubMed Scopus (265) Google Scholar). However, the signal transduction pathways by which toxin A induces intestinal inflammation are not entirely known. Toxin A binds to a G protein-coupled receptor (11Pothoulakis C. LaMont J.T. Eglow R. Gao N. Rubins J.B. Theoharides T.C. Dickey B.F. J. Clin. Invest. 1991; 88: 1119-1125Crossref Scopus (69) Google Scholar) on the luminal aspect of the apical intestinal epithelial cell membrane (12He D. Hagen S.J. Pothoulakis C. Chen M. Medina N.D. Warny M. LaMont J.T. Gastroenterology. 2000; 119: 139-150Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and is then internalized where it activates MAPKs 1The abbreviations used are: MAPK, mitogen-activated protein kinase; COX-2, cyclooxygenase-2; COX-1, cyclooxygenase-1; CRE, cAMP-responsive element; CREB, CRE-binding protein; ERK, extracellular signal-regulated protein kinase; p38, p38 MAPK; ATF-1, -2, activating transcription factors 1 and 3; MSK-1, mitogen- and stress-activated protein kinase-1; NAC, N-acetyl-l-cysteine; ROS, reactive oxygen species; NFκB, nuclear factor kappa B; IκB, inhibitory kappa B; PGE2, prostaglandin E2; JAK, Janus tyrosine kinase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; CMV, cytomegalovirus; DCFH-DA, 2,7-dichlorofluorescin-diacetate; RT, reverse transcription; STAT5, signal transducers and activators of transcription 5.1The abbreviations used are: MAPK, mitogen-activated protein kinase; COX-2, cyclooxygenase-2; COX-1, cyclooxygenase-1; CRE, cAMP-responsive element; CREB, CRE-binding protein; ERK, extracellular signal-regulated protein kinase; p38, p38 MAPK; ATF-1, -2, activating transcription factors 1 and 3; MSK-1, mitogen- and stress-activated protein kinase-1; NAC, N-acetyl-l-cysteine; ROS, reactive oxygen species; NFκB, nuclear factor kappa B; IκB, inhibitory kappa B; PGE2, prostaglandin E2; JAK, Janus tyrosine kinase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; CMV, cytomegalovirus; DCFH-DA, 2,7-dichlorofluorescin-diacetate; RT, reverse transcription; STAT5, signal transducers and activators of transcription 5. (13Warny M. Keates A.C. Keates S. Castagliuolo I. Zacks J.K. Aboudola S. Qamar A. Pothoulakis C. LaMont J.T. Kelly C.P. J. Clin. Invest. 2000; 105: 1147-1156Crossref PubMed Scopus (178) Google Scholar), intracellular calcium release (14Pothoulakis C. Gilbert R.J. Cladaras C. Castagliuolo I. Semenza G. Hitti Y. Montcrief J.S. Linevsky J. Kelly C.P. Nikulasson S. Desai H.P. Wilkins T.D. LaMont J.T. J. Clin. Invest. 1996; 98: 641-649Crossref PubMed Scopus (85) Google Scholar, 15Jefferson K.K. Smith M.F. Bobak Jr, D.A. J. Immunol. 1999; 163: 5183-5191PubMed Google Scholar), release of reactive oxygen species (ROS) (12He D. Hagen S.J. Pothoulakis C. Chen M. Medina N.D. Warny M. LaMont J.T. Gastroenterology. 2000; 119: 139-150Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 16He D. Sougioultzis S. Hagen S. Liu J. Keates S. Keates A.C. Pothoulakis C. Lamont J.T. Gastroenterology. 2002; 122: 1048-1057Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and secretion of pro-inflammatory mediators (16He D. Sougioultzis S. Hagen S. Liu J. Keates S. Keates A.C. Pothoulakis C. Lamont J.T. Gastroenterology. 2002; 122: 1048-1057Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 17Mahida Y.R. Makh S. Hyde S. Gray T. Borriello S.P. Gut. 1996; 38: 337-347Crossref PubMed Scopus (165) Google Scholar). We previously reported that toxin A releases prostaglandin E2 (PGE2) into the ileal lumen of intact rats (5Triadafilopoulos G. Pothoulakis C. Weiss R. Giampaolo C. Lamont J.T. Gastroenterology. 1989; 97: 1186-1192Abstract Full Text PDF PubMed Scopus (78) Google Scholar) and Alcantara et al. (18Alcantara C. Stenson W.F. Steiner T.S. Guerrant R.L. J. Infect. Dis. 2001; 184: 648-652Crossref PubMed Scopus (45) Google Scholar) reported that toxin A-induced water and electrolyte secretion in vivo was significantly blocked by a COX-2 inhibitor. COX-2 is induced by pro-inflammatory cytokines, lipopolysaccharide, growth factors, and infectious agents in a variety of cell types (19Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2212) Google Scholar, 20Smith W.L. Dewitt D.L. Adv. Immunol. 1996; 62: 167-215Crossref PubMed Google Scholar, 21Saukkonen K. Tomasetto C. Narko K. Rio M.C. Ristimaki A. Cancer Res. 2003; 63: 3032-3036PubMed Google Scholar, 22Nie M. Pang L. Inoue H. Knox A.J. Mol. Cell. Biol. 2003; 23: 9233-9244Crossref PubMed Scopus (106) Google Scholar, 23Devaux Y. Seguin C. Grosjean S. de Talance N. Camaeti V. Burlet A. Zannad F. Meistelman C. Mertes P.M. Longrois D. J. Immunol. 2001; 167: 3962-3971Crossref PubMed Scopus (49) Google Scholar). PGE2 is a potent stimulator of intestinal chloride and water secretion in mammalian gut (24Powell D.W. Am. Physiol. Soc. 1991; 4: 591-641Google Scholar), and PGE2 is released during various forms of intestinal inflammation and infection. For example, infection of cultured enterocytes with Salmonella organisms induces COX-2 expression, followed by PGE2-induced apical chloride secretion (25Eckmann L. Stenson W.F. Savidge T.C. Lowe D.C. Barrett K.E. Fierer J. Smith J.R. Kagnoff M.F. J. Clin. Invest. 1997; 100: 296-309Crossref PubMed Scopus (173) Google Scholar). In view of the potential importance of COX-2 in toxin A-induced enteritis, we studied the signaling pathway of toxin A induction of COX-2 in colonocytes, the natural target of toxin A, using the non-transformed human colonic epithelial line, NCM460. Because toxin B also elicits inflammatory and cytotoxic responses in human colon in vitro (10Riegler M. Sedivy R. Pothoulakis C. Hamilton G. Zacherl J. Bischof G. Cosentini E. Feil W. Schiessel R. LaMont J.T. Wenzl E. J. Clin. Invest. 1995; 95: 2004-2011Crossref PubMed Scopus (265) Google Scholar) and in vivo (26Savidge T.C. Pan W.H. Newman P. O'Brien M. Anton P.M. Pothoulakis C. Gastroenterology. 2003; 125: 413-420Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), we also examined whether it is also able to stimulate COX-2 expression in these cells. We found that toxin A, but not toxin B, induced COX-2 expression at both mRNA and protein levels through ROS-mediated activation of p38 MAPK, mitogen- and stress-activated protein kinase-1 (MSK-1), ATF-1, and CREB. We also demonstrate that a p38 inhibitor and a selective COX-2 inhibitor reduced toxin A-induced fluid and PGE2 secretion in vivo. C. difficile Toxin A—Toxin A and toxin B were purified from culture supernatants of C. difficile strain VPI 10463 (American Type Culture Collection, Rockville, MD) using anion exchange chromatography and fast protein liquid chromatography as previously described (13Warny M. Keates A.C. Keates S. Castagliuolo I. Zacks J.K. Aboudola S. Qamar A. Pothoulakis C. LaMont J.T. Kelly C.P. J. Clin. Invest. 2000; 105: 1147-1156Crossref PubMed Scopus (178) Google Scholar, 26Savidge T.C. Pan W.H. Newman P. O'Brien M. Anton P.M. Pothoulakis C. Gastroenterology. 2003; 125: 413-420Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Reagents—The transfection reagent TransIT-LT1 was from Mirus (Madison, WI). The luciferase reporter constructs for human COX-2 were previously described (27Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). All plasmids were prepared using the Endo Free plasmid kit as recommended by the manufacturer (Qiagen, Valencia, CA). The polyclonal antibodies for IκB, COX-1, and COX-2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-ATF-2, ATF-2, phospho-CREB, CREB, and phospho-MSK1 were from Cell Signaling Technology (Beverly, MA). The MAPK/ERK kinase specific inhibitor, PD98059, the JAK inhibitor, AG490, the NF-κB inhibitor, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline, the p38 MAPK inhibitor, SB203580 and the selective COX-2 inhibitor, NS-398, were from Calbiochem. N-Acetyl-l-cysteine (NAC), sodium formate, and 2,7-dichlorofluorescin-diacetate (DCFH-DA) were from Sigma. The human non-transformed colonocytes NCM460 and the culture medium M3D were obtained from INCELL Corp. (San Antonio, TX) (28Zhao D. Keates A.C. Kuhnt-Moore S. Moyer M.P. Kelly C.P. Pothoulakis C. J. Biol. Chem. 2001; 276: 44464-44471Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). NCM460 cells were cultivated in M3D media supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 1% l-glutamine, 10 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37 °C in air supplemented with 5% CO2. PGE2 and cAMP Measurements in Cultured Colonocytes—Enzyme-linked immunosorbent assay (ELISA) was used to measure the level of human PGE2 using the appropriate kits from R&D Systems (Minneapolis, MN). Colonocytes were plated 1 day prior to the experiment and then incubated with toxin A (3 nm) and/or the selective COX-2 inhibitor NS-398 (100 μm), or toxin B (20 nm). Conditioned medium was used for ELISA following the manufacturer's protocol. To measure intracellular cAMP, cells were initially treated with toxin A and lysed in 500 μl of HCl (0.1 n, 10 min). The suspension was centrifuged (4 min, 3,000 × g), and the supernatant was assayed for cAMP by ELISA (R&D Systems, Minneapolis, MN) following the manufacturer's protocol. Immunoblot Analysis—Cells were harvested and washed once with ice-cold 1× PBS (pH 7.5), and then lysed for 60 min on ice in lysis buffer (150 mm NaCl, 50 mm Tris-Cl (pH 8.0), 5 mm EDTA, 1% Nonidet P-40) with a protease inhibitor mixture (Roche Applied Science) and a phosphatase inhibitor mixture (Sigma). Protein concentrations of the lysates were measured by the Bradford method (Bio-Rad, Hercules, CA), and equal amounts of total protein were fractionated on 10% SDS-polyacrylamide gels. Membranes were first incubated overnight at 4 °C with the primary antibodies (1:1000 in 5% bovine serum albumin/in Tris-buffered saline with Tween 20 (150 mm NaCl, 10 nm Tris at pH 8.0, 0.005% Tween 20 [TBST]), then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (in 5% nonfat dry milk/TBST) at room temperature for 1 h and then washed three times with TBST. Antigen-antibody complexes were detected with LumiGlo reagent (New England Biolabs). Transfection and Luciferase Reporter Assays—NCM460 cells were plated in 6-well plates (0.4 × 106 cells/well) and transfected with 1 μg of the human COX-2 promoter (27Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Cells were cotransfected with 0.1 μg of the pRL-CMV plasmid containing the Renilla gene (Promega, Madison WI). One day after transfection, cells were incubated with toxin A (3 nm) and toxin B (20 nm) for 12 h, and firefly luciferase values were standardized to the Renilla values. The total amount of plasmid DNA was kept constant by adding the empty vector for each transfection. All assays were performed in triplicate, and data are expressed as mean values ± S.E. Measurement of ROS Generation—DCFH-DA is a non-polar compound, which enters the cell and is cleaved to form DCFH. Trapped DCFH is oxidized by oxygen free radicals to produce fluorescent DCF. NCM460 cells were preincubated on a 96-well microplate (2 × 103 cells per well) in M3D medium lacking serum for 1 h at 37 °Cinthe presence of 10 μm DCFH-DA. Cells were washed three times in pre-warmed PBS and then exposed to toxin A (3 nm). Fluorescence intensity was analyzed by fluorospectroscan (Fluoroscan Ascent FL, Labsystems) using 485 nm excitation and 538 nm emission filters. DCF fluorescence was also measured by fluorescence microscopy. To avoid photo-oxidation of DCFH, fluorescent images were collected by a rapid scan (total scan time, 3 s). Five groups of 10–20 subconfluent cells were randomly selected for analysis from each sample. Electrophoretic Mobility Shift Assays—The following sequences derived from the human COX-2 promoter containing the CRE was used: CRE-sense, 5′-AAACAGTCATTTCGTCACATGGGCTTG-3′; CRE-antisense, 5′-CAAGCCCATGTGACGAAATGACTGTTT-3′. Probes were annealed and 5′-overhangs were labeled by incorporation of [32P]dATP (PerkinElmer Life Sciences) with T4 polynucleotide kinase. For gel shift assays, 2 μg of nuclear protein extracts were incubated at room temperature for 5 min with a mixture containing 6 mm HEPES (pH 7.9), 0.4 mm EDTA, 125 mm KCl, 10% glycerol, 0.05 mg/ml poly(dIdC), 1 mm dithiothreitol, 2.5 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 10 mm NaF, 50 mg/ml aprotinin, 50 μg/ml leupeptin. Approximately 1 ng of labeled probe was added, and the reactions were incubated at room temperature for another 20 min. In antibody supershift experiments, mixtures were preincubated with various amounts of antibodies on ice for 1 h before the addition of probe. DNA·protein complexes were separated on 5% polyacrylamide gel in low ionic strength Tris borate buffer, dried under vacuum, and visualized with a Phosphor-Imager (Amersham Biosciences). RNA Isolation and Semi-quantitative RT-PCR—Total cellular RNA was isolated and RT-PCR was conducted according to Nakayama et al. (29Nakayama H. Yokoi H. Fujita J. Nucleic Acids Res. 1992; 20: 4939Crossref PubMed Scopus (158) Google Scholar). Two micrograms of total RNA was reverse transcribed at 42 °C for 1hin20 μl of the reaction mixture containing mouse Moloney leukemia virus reverse transcriptase with oligo(dT) primers. Thereafter, the resultant cDNA was amplified together with Taq polymerase (PerkinElmer Life Sciences) using the specific sets of primers. The primers used were: human COX-2 (300 bp), 5′-ATGAGATTGTGGGAAAATTGCT-3′ (sense), 5′-GATCATCTCTGCCTGAGTATC-3′ (antisense), and mouse COX-2 (333 bp), 5′-GCAAATCCTTGCTGTTCCAATC-3′ (sense) 5′-GGAGAAGGCTTCCCAGCTTTTG-3′ (antisense). β-Actin amplification was used as the control, respectively. PCR of each molecule was conducted with the optimal numbers of cycles consisting of 94 °C for 1 min, optimal annealing temperature for 1 min, and 72 °C for 1 min, followed by incubation at 72 °C for 3 min. The generated PCR products did not reach a saturable level with the determined optimal cycle numbers. The amplified PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. Effects of SB203580 and NS-398 on Toxin A-induced Secretion of PGE2 and Fluid and Immunohistochemistry for COX-2 in Mouse Intestine in Vivo—This study was approved by the Beth Israel Deaconess Medical Center Animal Care and Use Committee (Boston, MA). CD1 male mice (Charles River Laboratories, Wilmington, MA) weighing 30–35 g had free access to food and water in a 12-h light/dark cycle. Mice were acclimated to these conditions at least 7 days before the experiment. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and ileal loops (3–4 cm) were prepared and injected with buffer alone or with the specific p38 inhibitor SB203580 hydrochloride (100 μg in water) and the selective COX-2 inhibitor, NS-398 (1 mg/kg), in a volume of 200 μl. After 30 min, toxin A (10 μg in PBS) or PBS alone was injected intraluminally, and animals were sacrificed 2 h later by CO2. Ileal loop fluid was collected and centrifuged at 50,000 × g for 15 min. Secretion of PGE2 was assayed in the supernatant with specific ELISA kits according to the manufacturer's instructions. Ileal loops were excised and weighed, and length was measured. Fluid secretion was expressed as the loop weight-to-length ratio (mg/cm). Ileal tissue samples were quick frozen for immunohistochemical analysis and for protein determination. Frozen intestinal sections were placed on Fisher Superfrost slides, fixed in 80% acetone at room temperature for 60 s, and air-dried. Before labeling, sections were rehydrated in PBS and incubated with 10% normal goat serum for 20 min. Sections were incubated with a fluorescein isothiocyanate conjugated goat anti-COX-2 antibody (Santa Cruz, CA, sc-1745-fluorescein isothiocyanate) (1:100) for 16 h. The slide was then counter-stained for 30 min in propidium iodide staining solution at a concentration of 5 μg/ml in PBS. After final washing, slides were coverslipped with Vectashield (Vector Laboratories) and viewed on a fluorescence microscopy (MRC 1024, Bio-Rad). To confirm expression of COX-2 mRNA, tissue samples (100 mg) were homogenized in 1 ml of TRIzol reagent (Invitrogen) using a power homogenizer, total RNA was isolated and RT-PCR performed using a specific mouse COX-2 primer. Human Intestinal Xenografts—Human fetal intestine was obtained from Brigham and Woman's Hospital (Boston, MA) (mean age, 14.2 ± 1.6 weeks) after therapeutic abortion as previously described (30Savidge T.C. Morey A.L. Ferguson D.J.P. Fleming K.A. Shmakov A.N. Phillips A.D. Differentiation. 1995; 58: 361-371Crossref PubMed Scopus (73) Google Scholar). Procurement and procedures involving xenografting of human fetal tissues into C.B.-17 SCID/SCID mice were performed with full approval from the Institutional Review Board. Fetal small intestine and colon were washed in Dulbecco's modified Eagle's medium and xeno-transplanted subcutaneously into SCID mice. Twelve weeks after xenografting, the graft lumen was injected subcutaneously with 400 μl of serum-free Dulbecco's modified Eagle's medium containing 40 nm purified toxin A (n = 4) or medium control (n = 2). Mice were killed after 6 h, and the xenografts were excised. We isolated total proteins from these tissue and performed immunoassays with specific COX-2 antibody and loading control, β-actin antibody (Sigma). Induction of COX-2 by C. difficile Toxin A in Colonocytes— Because COX-2 has been associated with intestinal inflammation, monocyte infiltration, and prostaglandin production, we examined COX-2 expression levels and PGE2 synthesis in colonocytes exposed to purified toxin A (3 nm). Toxin A induced the expression of COX-2, but had no effect on the expression of COX-1 (Fig. 1A). This response was evident at 1–3 h and peaked at 6 h. Under basal conditions, we observed weak expression of COX-2 mRNA in cultured colonocytes. Toxin A also increased COX-2 mRNA in a time pattern that paralleled the COX-2 protein expression (Fig. 1B). The effect of toxin A on COX-2 protein levels was concentration-dependent between doses of 0.8 and 3 nm (Fig. 1C). Despite the close structural relationship between toxins A and B and their common enzymatic activity against rho proteins, toxin B at 20 nm showed no effect on COX-2 expression (Fig. 1D). Colonocytes were transiently transfected with a construct containing the nucleotide sequence from –1432 to +59 of the human COX-2 promoter and then treated with 1.5 and 3 nm of toxin A or with 10 and 20 nm of toxin B. Toxin A, but not toxin B, increased COX-2 promoter activity in a dose-dependent manner (Fig. 1E). Next, we examined whether toxin A induces prostaglandin E2 secretion in colonocytes and determined the COX-2 dependence of this response using the selective COX-2 inhibitor NS-398. The basal concentration of secreted prostaglandin E2 was ∼17 pg/ml/2 × 106 cells (Fig. 1F). Toxin A increased PGE2 secretion by 2- to 3-fold at 6–12 h (Fig. 1F), which was completely blocked by the selective COX-2 inhibitor NS-398. As expected, toxin B did not induce PGE2 secretion up to 12 h (Fig. 1F). These results indicate that toxin A-mediated secretion of PGE2 in colonocytes required COX-2 induction. Toxin A Induction of COX-2 Expression in Human Intestinal Xenografts—To confirm the induction of COX-2 by toxin A in human intestine, we injected toxin A into the lumen of human intestinal xenografts growing subcutaneously in SCID mice. We previously reported (26Savidge T.C. Pan W.H. Newman P. O'Brien M. Anton P.M. Pothoulakis C. Gastroenterology. 2003; 125: 413-420Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) that epithelial cells in the grafted intestine are exclusively of human origin and that injection of toxin A induces an acute inflammatory response, similar to that seen in animal models and in the colon of patients with C. difficile-associated colitis. We found no expression of COX-2 in control xenografts, but robust increases of COX-2 expression 6 h after toxin A exposure (Fig. 2), similar to the results observed in cultured colonocytes (Fig. 1, A and B). Phosphorylation of CREB, ATF-1, ATF-2, MSK-1, and p38 by Toxin A in Colonocytes—We next investigated toxin A-associated phosphorylation of p38 MAPK and its downstream transcription factors ATF-1, ATF-2, and CREB. Toxin A strongly increased the phosphorylation of p38, CREB, ATF-1, and ATF-2 after 1 and 3 h of exposure with return to basal levels at 6 h (Fig. 3A). Mitogen- and stress-activated protein kinase-1 (MSK-1) is directly activated by p38 MAPK and may mediate activation of CREB, as shown in embryonal human kidney cells activated by growth factors and oxidative stress (31Deak M. Clifton A.D. Lucocq L.M. Alessi D.R. EMBO J. 1998; 17: 4426-4441Crossref PubMed Scopus (842) Google Scholar). We therefore determined phosphorylation of threonine 581 of MSK-1 following toxin A stimulation. We observed phosphorylation of MSK-1 at 1 and 3 h after toxin A exposure, similar to the time course of CREB/ATF-1 phosphorylation. Because NF-κB, and ERK1/2 are known regulators of COX-2 expression (32Wu D. Marko M. Claycombe K. Paulson K.E. Meydani S.N. J. Biol. Chem. 2003; 278: 10983-10992Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 33Jones M.K. Sasaki E. Halter F. Pai R. Nakamura T. Arakawa T. Kuroki T. Tarnawski A.S. FASEB J. 1999; 13: 2186-2194Crossref PubMed Scopus (88) Google Scholar), we examined phosphorylation of IκB, and ERK1/2 in colonocytes exposed to toxin A. As shown in Fig. 3B, we did not observe phosphorylation of IκB and only slight phosphorylation of ERK1/2. Activation Pathways for COX-2 in Colonocytes Exposed to Toxin A—To define specific induction pathways of COX-2 by toxin A, we treated colonocytes with the p38 MAPK inhibitor SB203580, the JAK1 (Janus tyrosine kinase 1) inhibitor AG490, the NF-κB inhibitor 6-amino-4-quinazoline, the ERK1/2 kinase inhibitor PD98059, and toxin A for 6 h. We found that inhibition of p38 by SB203580 completely prevented the expression of COX-2 by toxin A (Fig. 4A). None of the other inhibitors had any significant effect on toxin A-induced COX-2 expression, apart from the JAK1 inhibitor AG490, which slightly inhibited COX-2 protein expression (Fig. 4A). Similarly, only the p38 MAPK inhibitor, SB203580 reduced the secretion of PGE2 in supernatants (from 49 pg/ml/2 × 106 cells to 21 pg/ml/2 × 106 cells, p < 0.001) (Fig. 4B), whereas, none of the other inhibitors exerted any significant effect at the doses used. Blockage of JAK1 by AG490 weekly inhibited toxin A-induced COX-2 protein (Fig. 4A), but failed to inhibit toxin A-mediated PGE2 secretion (Fig. 4B). In view of the lack of inhibition of COX-2 expression by the ERK1/2 kinase inhibitor PD98059 (Fig. 4A) as well as by the weak phosphorylation of ERK1/2 induced by toxin A (Fig. 3B), we conclude that the ERK1/2 and JAK1 pathways are not significantly involved in the regulation of COX-2 expression by toxin A. We next examined the involvement of p38 MAPK on toxin A-induction of COX-2 mRNA. The p38 MAPK inhibitor, SB203580, at 10 μm completely blocked the toxin A effect on COX-2 mRNA in colonocytes (Fig. 4C). Because weak expression of COX-2 mRNA was detected in basal condition of cultured colonocytes and previous studies report that p38 activation stabilizes COX-2 messenger RNA (34Mifflin R.C. Saada J.I. Di Mari J.F. Valentich J.D. Adegboyega P.A. Powell D.W. Mol. Pharmacol. 2004; 65: 470-478Crossref PubMed Scopus (36) Google Scholar, 35Faour W.H. Mancini A. He Q.W. Di Battista J.A. J. Biol. Chem. 2003; 278: 26897-26907Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 36Dixon D.A. Tolley N.D. King P.H. Nabors L.B. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Clin. Invest. 2001; 108: 1657-1" @default.
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• W2015356544 title "Clostridium difficile Toxin A Regulates Inducible Cyclooxygenase-2 and Prostaglandin E2 Synthesis in Colonocytes via Reactive Oxygen Species and Activation of p38 MAPK" @default.
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