FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1965071570> ?p ?o ?g. }

• W1965071570 endingPage "37612" @default.
• W1965071570 startingPage "37597" @default.
• W1965071570 abstract "A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, induces an inflammatory response in the host, and causes apoptosis of murine macrophages. In this study, we utilized five target cell types (a murine macrophage cell line (RAW 264.7), bone marrow-derived transformed macrophages, murine peritoneal macrophages, and two human intestinal epithelial cell lines (T84 and HT-29)) to investigate the effect of Act on mitogen-activated protein kinase (MAPK) pathways and mechanisms leading to apoptosis. As demonstrated by immunoprecipitation/kinase assays or Western blot analysis, Act activated stress-associated p38, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells. Act also induced phosphorylation of upstream MAPK factors (MAPK kinase 3/6 (MKK3/6), MKK4, and MAP/ERK kinase 1 (MEK1)) and downstream effectors (MAPK-activated protein kinase-2, activating transcription factor-2, and c-Jun). Act evoked cell membrane blebbing, caspase 3-cleavage, and activation of caspases 8 and 9 in these cells. In macrophages that do not express functional tumor necrosis factor receptors, apoptosis and caspase activities were significantly decreased. Immunoblotting of host whole cell lysates revealed Act-induced up-regulation of apoptosis-related proteins, including the mitochondrial proteins cytochrome c and apoptosis-inducing factor. However, mitochondrial membrane depolarization was not detected in response to Act. Taken together, the data demonstrated for the first time Act-induced activation of MAPK signaling and classical caspase-associated apoptosis in macrophages and intestinal epithelial cells. Given the importance of MAPK pathways and apoptosis in inflammation-associated diseases, this study provided new insights into the mechanism of action of Act on host cells. A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, induces an inflammatory response in the host, and causes apoptosis of murine macrophages. In this study, we utilized five target cell types (a murine macrophage cell line (RAW 264.7), bone marrow-derived transformed macrophages, murine peritoneal macrophages, and two human intestinal epithelial cell lines (T84 and HT-29)) to investigate the effect of Act on mitogen-activated protein kinase (MAPK) pathways and mechanisms leading to apoptosis. As demonstrated by immunoprecipitation/kinase assays or Western blot analysis, Act activated stress-associated p38, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells. Act also induced phosphorylation of upstream MAPK factors (MAPK kinase 3/6 (MKK3/6), MKK4, and MAP/ERK kinase 1 (MEK1)) and downstream effectors (MAPK-activated protein kinase-2, activating transcription factor-2, and c-Jun). Act evoked cell membrane blebbing, caspase 3-cleavage, and activation of caspases 8 and 9 in these cells. In macrophages that do not express functional tumor necrosis factor receptors, apoptosis and caspase activities were significantly decreased. Immunoblotting of host whole cell lysates revealed Act-induced up-regulation of apoptosis-related proteins, including the mitochondrial proteins cytochrome c and apoptosis-inducing factor. However, mitochondrial membrane depolarization was not detected in response to Act. Taken together, the data demonstrated for the first time Act-induced activation of MAPK signaling and classical caspase-associated apoptosis in macrophages and intestinal epithelial cells. Given the importance of MAPK pathways and apoptosis in inflammation-associated diseases, this study provided new insights into the mechanism of action of Act on host cells. Aeromonas species (spp.) are significant human pathogens that have been isolated from freshwater, salt water, and a variety of foods, and are frequently isolated from patients with diarrhea (1Albert M.J. Ansaruzzaman M. Talukder K.A. Chopra A.K. Kuhn I. Rahman M. Faruque A.S. Islam M.S. Sack R.B. Mollby R. J. Clin. Microbiol. 2000; 38: 3785-3790Crossref PubMed Google Scholar, 2Kirov S.M. Ardestani E.K. Hayward L.J. Int. J. Food Microbiol. 1993; 20: 159-168Crossref PubMed Scopus (45) Google Scholar, 3Majeed K.N. Egan A.F. Mac Rae I.C. J. Appl. Bacteriol. 1990; 69: 332-337Crossref PubMed Scopus (39) Google Scholar). Among Aeromonas spp., Aeromonas hydrophila is most commonly associated with human infections, leading to intestinal and non-intestinal diseases (4Janda J.M. Abbott S.L. Clin. Infect. Dis. 1998; 27: 332-344Crossref PubMed Scopus (553) Google Scholar). Furthermore, increased resistance of this organism to antibiotics and chlorination in water presents a significant threat to public health (2Kirov S.M. Ardestani E.K. Hayward L.J. Int. J. Food Microbiol. 1993; 20: 159-168Crossref PubMed Scopus (45) Google Scholar, 3Majeed K.N. Egan A.F. Mac Rae I.C. J. Appl. Bacteriol. 1990; 69: 332-337Crossref PubMed Scopus (39) Google Scholar, 5Alavandi S.V. Subashini M.S. Ananthan S. Indian J. Med. Res. 1999; 110: 50-55PubMed Google Scholar, 6Altwegg M. Martinetti Lucchini G. Luthy-Hottenstein J. Rohrbach M. Eur. J. Clin. Microbiol. Infect. Dis. 1991; 10: 44-45Crossref PubMed Scopus (76) Google Scholar, 7Brandi G. Sisti M. Giardini F. Schiavano G.F. Albano A. Lett. Appl. Microbiol. 1999; 29: 211-215Crossref PubMed Scopus (46) Google Scholar, 8Palumbo S.A. Bencivengo M.M. Del Corral F. Williams A.C. Buchanan R.L. J. Clin. Microbiol. 1989; 27: 854-859Crossref PubMed Google Scholar, 9Chopra A.K. Houston C.W. Microbes Infect. 1999; 1: 1129-1137Crossref PubMed Scopus (162) Google Scholar, 10Hanninen M.L. Oivanen P. Hirvela-Koski V. Int. J. Food Microbiol. 1997; 34: 17-26Crossref PubMed Scopus (94) Google Scholar, 11Hanninen M.L. Salmi S. Mattila L. Taipalinen R. Siitonen A. J. Med. Microbiol. 1995; 42: 26-31Crossref PubMed Scopus (56) Google Scholar, 12Yamada S. Matsushita S. Dejsirilert S. Kudoh Y. Epidemiol. Infect. 1997; 119: 121-126Crossref PubMed Scopus (57) Google Scholar). As a result, the Environmental Protection Agency has placed this organism on the “Candidate Contaminant List,” and the monitoring of United States water supplies for this organism began in 2002 (4Janda J.M. Abbott S.L. Clin. Infect. Dis. 1998; 27: 332-344Crossref PubMed Scopus (553) Google Scholar).A. hydrophila produces an array of virulence factors, one of the most significant of which is a 52-kDa cytotoxic enterotoxin (Act) 1The abbreviations used are: Act, cytotoxic enterotoxin; MAPK, mitogen-activated protein kinase; JNK, c-Jun, NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; MAP-KAPk-2, MAPK-activated protein kinase-2; ATF, activating transcription factor; AIF, apoptosis-inducing factor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; IL, interleukin; TNF-α, tumor necrosis factor-α; TNFR, tumor necrosis factor-α receptor; KO, knockout; WT, wild-type; PARP, poly(ADP-ribose) polymerase; PERP, p53 apoptosis effector related to PMP-22; CAS, cellular apoptosis susceptibility; LPS, lipopolysaccharide; C/EBP-β, CCAAAT/enhancer-binding protein-β; LEHD-pNA, N-acetyl-Leu-Glu-His-Asp-p-nitroaniline; IETD-pNA, N-acetyl-Ile-Glu-Thr-Asp-p-nitroaniline; FITC, fluorescein isothiocyanate; PI, propidium iodide; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolycarbocyanine iodide; CPM, trace quantity units; NF-κB, nuclear factor-κB; CCCP, carbonyl cyanide m-chlorophenylhydrazone; PBS, phosphate-buffered saline. 1The abbreviations used are: Act, cytotoxic enterotoxin; MAPK, mitogen-activated protein kinase; JNK, c-Jun, NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MKK, MAPK kinase; MAP-KAPk-2, MAPK-activated protein kinase-2; ATF, activating transcription factor; AIF, apoptosis-inducing factor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; IL, interleukin; TNF-α, tumor necrosis factor-α; TNFR, tumor necrosis factor-α receptor; KO, knockout; WT, wild-type; PARP, poly(ADP-ribose) polymerase; PERP, p53 apoptosis effector related to PMP-22; CAS, cellular apoptosis susceptibility; LPS, lipopolysaccharide; C/EBP-β, CCAAAT/enhancer-binding protein-β; LEHD-pNA, N-acetyl-Leu-Glu-His-Asp-p-nitroaniline; IETD-pNA, N-acetyl-Ile-Glu-Thr-Asp-p-nitroaniline; FITC, fluorescein isothiocyanate; PI, propidium iodide; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolycarbocyanine iodide; CPM, trace quantity units; NF-κB, nuclear factor-κB; CCCP, carbonyl cyanide m-chlorophenylhydrazone; PBS, phosphate-buffered saline. (13Ferguson M.R. Xu X.J. Houston C.W. Peterson J.W. Coppenhaver D.H. Popov V.L. Chopra A.K. Infect. Immunol. 1997; 65: 4299-4308Crossref PubMed Google Scholar). Our laboratory demonstrated that Act possessed several biological activities, including hemolysis, cytotoxicity, enterotoxicity, and lethality to mice, and induced acute inflammatory responses both in vitro and in vivo (9Chopra A.K. Houston C.W. Microbes Infect. 1999; 1: 1129-1137Crossref PubMed Scopus (162) Google Scholar). Additional studies by our laboratory revealed that Act significantly altered intracellular signaling in murine RAW 264.7 macrophages, which could lead to an inflammatory response in host cells (14Chopra A.K. Xu X. Ribardo D. Gonzalez M. Kuhl K. Peterson J.W. Houston C.W. Infect. Immunol. 2000; 68: 2808-2818Crossref PubMed Scopus (122) Google Scholar, 15Ribardo D.A. Kuhl K.R. Boldogh I. Peterson J.W. Houston C.W. Chopra A.K. Microb. Pathog. 2002; 32: 149-163Crossref PubMed Scopus (38) Google Scholar, 16Galindo C.L. Sha J. Ribardo D.A. Fadl A.A. Pillai L. Chopra A.K. J. Biol. Chem. 2003; 278: 40198-40212Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The pattern of Act-induced gene expression changes, based on our recent microarray studies, strongly suggested that Act might activate mitogen-activated protein kinase (MAPK) pathways (16Galindo C.L. Sha J. Ribardo D.A. Fadl A.A. Pillai L. Chopra A.K. J. Biol. Chem. 2003; 278: 40198-40212Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).MAPKs are activated by phosphorylation on both a threonine and tyrosine residue, which is accomplished by dual phosphorylation enzymes, called MAP/ERK kinases (MEKs) or MAPK kinases (MKKs). MEKs are themselves activated via phosphorylation by MEK kinases (MEKKs) or MAPK kinase kinases. There are four major MAPK cascades that lead to altered gene expression: ERK1/2, JNK, p38 kinase, and ERK5. ERK1 and ERK2 are activated via phosphorylation by MEK1 and MEK2, respectively (17Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3439) Google Scholar).The two MAPK pathways most frequently associated with immune responses are JNK and p38 kinases. The JNK signaling pathway is a stress-activated MAPK pathway involved in the regulation of cell proliferation and apoptosis (18Otto I.M. Raabe T. Rennefahrt U.E. Bork P. Rapp U.R. Kerkhoff E. Curr. Biol. 2000; 10: 345-348Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The p38 MAPK signaling pathway is associated with cell growth, differentiation, and death, and is activated by a variety of cytokines, including interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) (17Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3439) Google Scholar, 19Waetzig G.H. Seegert D. Rosenstiel P. Nikolaus S. Schreiber S. J. Immunol. 2002; 168: 5342-5351Crossref PubMed Scopus (346) Google Scholar). Three MAPKK proteins (MKK3, MKK4, and MKK6) can phosphorylate and activate p38 kinase (17Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3439) Google Scholar). The activation of p38 leads to the production of several cytokines, including IL-1β, TNF-α, IL-6, and IL-8, as well as cyclooxygenase-2 and inducible nitric-oxide synthase enzyme, and phosphorylation of phospholipase A2 (PLA2) (20Marie C. Roman-Roman S. Rawadi G. Infect. Immunol. 1999; 67: 688-693Crossref PubMed Google Scholar, 21Rawadi G. Ramez V. Lemercier B. Roman-Roman S. J. Immunol. 1998; 160: 1330-1339PubMed Google Scholar, 22Miyazawa K. Mori A. Miyata H. Akahane M. Ajisawa Y. Okudaira H. J. Biol. Chem. 1998; 273: 24832-24838Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 23Subbaramaiah K. Hart J.C. Norton L. Dannenberg A.J. J. Biol. Chem. 2000; 275: 14838-14845Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 24Shalom-Barak T. Quach J. Lotz M. J. Biol. Chem. 1998; 273: 27467-27473Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 25Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). The ERK5 pathway is the most recently described MAPK pathway and is primarily associated with response to stress (26Lee J.D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (285) Google Scholar, 27Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (491) Google Scholar). ERK5 is activated via MEK5, which is itself activated by MEKK3 (28Chao T.-H. Hayashi M. Tapping R.I. Kato Y. Lee J.-D. J. Biol. Chem. 1999; 274: 36035-36038Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar).In the current study, we investigated the effects of Act on MAPK signaling in RAW 264.7 cells, murine peritoneal macrophages, and human intestinal epithelial cells (T84 cells) to determine the role of MAPK signaling in host cells and ultimately dissect the definitive role of signaling molecules that lead to the disease state during Aeromonas infection.Because we previously demonstrated that Act caused apoptosis of RAW 264.7 murine macrophages (16Galindo C.L. Sha J. Ribardo D.A. Fadl A.A. Pillai L. Chopra A.K. J. Biol. Chem. 2003; 278: 40198-40212Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), we also investigated mechanisms associated with Act-induced apoptosis in murine peritoneal (primary) macrophages, TNF receptor (TNFR) knockout (KO) murine bone marrow-derived and transformed macrophages (29Mukhopadhyay A. Suttles J. Stout R.D. Aggarwal B.B. J. Biol. Chem. 2001; 276: 31906-31912Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and human intestinal epithelial cells. Taken together, the data clearly demonstrated for the first time that Act-induced host cell signaling involves activation of ERK1/2, p38, and JNK pathways. Furthermore, we delineated the mechanisms of apoptosis in Act-treated host cells.EXPERIMENTAL PROCEDURESCell Culture—The murine macrophage (RAW 264.7) and human intestinal epithelial (T84 and HT-29) cell lines were purchased from the American Type Culture Collection (Manassas, VA). WT, double KO murine bone marrow-derived and transformed macrophages (deficient in both TNFR-1 and TNFR-2), and TNFR1/TNFR2 single KO macrophages were graciously provided by Dr. Aggarwal (29Mukhopadhyay A. Suttles J. Stout R.D. Aggarwal B.B. J. Biol. Chem. 2001; 276: 31906-31912Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). RAW 264.7 and HT-29 cells were cultured at 37 °C and 5% CO2 in Dulbecco's minimal essential medium (Life Technologies, Inc., Gaithersburg, MD) containing 4.5 g of glucose/liter, 10% fetal bovine serum, 2 mm l-glutamine, and antibiotics, penicillin (100 units/ml) and streptomycin (0.1 mg/ml). T84 cells were cultured similarly, but in Dulbecco's minimal essential medium/F-12 (Invitrogen) and 5% fetal bovine serum. WT and TNFR KO macrophages were cultured in RPMI (Invitrogen) and 5% fetal bovine serum. For each experiment, 5 × 105 cells/ml were plated in 35-mm dishes and allowed to attach overnight. The medium was removed, and fresh medium containing the stimulant (6 ng/ml (unless otherwise stated) lipopolysaccharide (LPS)-free Act) was added (14Chopra A.K. Xu X. Ribardo D. Gonzalez M. Kuhl K. Peterson J.W. Houston C.W. Infect. Immunol. 2000; 68: 2808-2818Crossref PubMed Scopus (122) Google Scholar). After each time point, cells were lysed using 100 μl/well of 1× Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA), sonicated twice for 15 s, and centrifuged at 10,000 × g for 10 min.Peritoneal Macrophage Isolation—Adult 25–30 g Swiss Webster female mice (6–8 weeks old) were purchased from Taconic Farm (Germantown, CA). Mice were housed in the specific pathogen-free animal facility at the University of Texas Medical Branch, Galveston, and given free access to food and water prior to experiments. A 1-ml solution of 3% thioglycolate was injected into the peritoneal cavity of mice to elicit macrophage migration into the cavity. Mice were allowed to rest for 4 days and then sacrificed by an overdose of isoflurane followed by cervical dislocation. A partial midline incision was made to remove the dermis and expose the parietal peritoneum lining. Peritoneal exudate cells were harvested by flushing the peritoneal cavity with 7 ml of RPMI medium (Invitrogen) using a 10-ml syringe fitted with a 21-gauge, 1½-inch needle. Extracted cells were counted, centrifuged, and plated at 1 × 108 cells in 35-mm tissue culture dishes containing RPMI supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were allowed to adhere for 1 h, and then non-adherent cells were washed from the cultures. Complete medium was added to the cultures and Act (12 or 20 ng/ml) was added. Cells were incubated for the appropriate times before being lysed for subsequent Western blot analysis.Western Blot Analysis—The antibody for β-tubulin was purchased from Santa Cruz Biochemical Corp. (Santa Cruz, CA). All other antibodies and Jurkat T cell control extracts were purchased from Cell Signaling Technology, and Western blot analysis was performed by established procedures (30Rose J.M. Houston C.W. Kurosky A. Infect. Immun. 1989; 57: 1170-1176Crossref PubMed Google Scholar) with slight modifications according to specifications of the antibody manufacturer. Briefly, equal amounts of total protein were loaded and separated on SDS, 10% polyacrylamide gels and then transferred to nitrocellulose membranes. Membranes were blocked with 5% milk and washed in 1× Tween (0.1%), Tris-buffered saline three times for 5 min each. Primary antibodies diluted 1:1000 in 5% milk or bovine serum albumin (prepared in 1× Tween (0.1%), Tris-buffered saline) were allowed to incubate overnight at 4 °C. After washing, horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) was diluted 1:2000 in 5% milk and applied to membranes. Subsequently, membranes were washed and a chemiluminescence substrate (Pierce, Rockford, IL) was applied and allowed to incubate at room temperature for 5 min.Immunoprecipitation/Kinase Assay—Kinase assay kits for p38 and JNK were purchased from Cell Signaling Technology, and the procedure was performed as described by the manufacturer. Whole cell lysates were incubated with immobilized phospho-p38 antibody or c-Jun fusion protein overnight at 4 °C. Following microcentrifugation and washing, the pellet was suspended in kinase buffer supplemented with 200 μm ATP and 2 μg of ATF-2 fusion protein (for p38 kinase activity) or 100 μm ATP (for JNK activity) and incubated for 30 min at 30 °C. The reaction was terminated with SDS sample loading buffer and the samples subjected to Western blot analysis.Gel Shift Analysis—Gel shift assays were performed as previously described (14Chopra A.K. Xu X. Ribardo D. Gonzalez M. Kuhl K. Peterson J.W. Houston C.W. Infect. Immunol. 2000; 68: 2808-2818Crossref PubMed Scopus (122) Google Scholar, 16Galindo C.L. Sha J. Ribardo D.A. Fadl A.A. Pillai L. Chopra A.K. J. Biol. Chem. 2003; 278: 40198-40212Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Briefly, consensus oligonucleotide for CCAAAT/enhancer-binding protein-β (C/EBP-β) was labeled using T4 polynucleotide kinase (Promega, Madison, WI) according to the manufacturer. Next DNA binding reaction mixtures were assembled. We used unlabeled AP-1 consensus oligonucleotide as a specific competitor, unlabeled AP-2 or SP-1 consensus oligonucleotides as nonspecific competitors (Promega), and nuclear extracts from specific time points after Act treatment of RAW cells. Nuclear extracts were prepared using NE-PER kit (Pierce) as described by the manufacturer. The reaction mixtures were incubated at room temperature for 10 min, followed by addition of 20,000 cpm of 32P-labeled transcription factor consensus oligonucleotide, and incubation at room temperature for 20 min. Subsequently, 1 μl of gel loading 10× buffer (Promega) was added to each reaction mixture and samples (5 to 20 μg) were loaded on a nondenaturing 4% polyacrylamide gel. The gel was pre-run in 0.5× Tris borate-EDTA buffer for 30 min at 100 V before loading the samples. After completion of the run, the gel was transferred to Whatman 3MM paper, dried at 80 °C for 5 h, and exposed to x-ray film overnight to 48 h.Laser Scanning Confocal Microscopy—Cells (RAW 264.7 and T84) were grown in cover glass chamber slides (Nalge Nunc International, Rochester, NY) and observed on a Zeiss 510 UV meta confocal microscope (Carl Zeiss, Inc., Thornwood, NY). To view membrane blebbing, Act was added (20 ng/ml), and images were taken every 30 s for 6 h. To examine mitochondrial membrane potential, Act-treated RAW 264.7 cells were stained with 10 μm MitoTracker red CM-H2XRos dye (Molecular Probes, Eugene, OR) for 30 min at 37 °C, followed by washing with phosphate-buffered saline (PBS). All observations were made using a plan-neofluar ×40/0.85 objective lens with an electronic zoom of 2. The Nomarski differential interference contrast images were stored on an optical disk and analyzed with Zeiss LSM software.Electron Microscopy—Act-treated (20 ng/ml) RAW 264.7 cells were fixed with a mixture of 0.1% glutaraldehyde, 2.5% formaldehyde, 0.03% trinitrophenol, and 0.03% CaCl2 in 0.05 m cacodylate buffer (pH 7.3) (31Berryman M.A. Rodewald R.D. J. Histochem. Cytochem. 1990; 38: 159-170Crossref PubMed Scopus (173) Google Scholar), postfixed in 1% OsO4 in 0.1 m cacodylate buffer, stained en bloc with 1% aqueous uranyl acetate, and embedded in Poly/Bed 812. Ultrathin sections were cut on a Reichert-Leica Ultracut S ultramicrotome and examined in a Philips 201 electron microscope at 60 kV.Caspase Activity Assay—Colorimetric activity assay kits were purchased from BioVision Inc. (Mountain View, CA), and caspase activity assays were performed as described by the manufacturer. Briefly, reaction mixtures were assembled as follows: 50–200 μg of protein (whole cell lysates from Act-treated RAW 264.7 cells or from TNFR-1 and -2 single and double KO macrophages), 50 μl of 2× reaction buffer (containing 10 mm dithiothreitol), and 5 μl of peptide substrate (N-acetyl-Leu-Glu-His-Asp-p-nitroaniline (LEHD-pNA) for caspase 9 assays, N-acetyl-Ile-Glu-Thr-Asp-p-nitroaniline (IETD-pNA) for caspase 8 assays, or N-acetyl-Asp-Glu-Val-Asp-7-amino-4-p-nitroanilide (DEVD-pNA) for caspase 3 assays). Reactions were incubated at 37 °C for 1–2 h and then read at 405 nm in an enzyme-linked immunosorbent assay reader. Whole cell lysates from camptothecin-treated Jurkat T cells (BioVision Inc.) were used as positive controls.Flow Cytometry—For detection of apoptosis, variously stimulated HT-29 cells (1 × 106) were stained with annexin V-conjugated to fluorescein isothiocyanate (FITC) and propidium iodide (PI) by using the annexin-V-FITC staining kit (BioVision Inc.) according to the manufacturer's instruction. Briefly, cells were suspended in 500 μl of annexin V binding buffer and incubated with 5 μl of annexin V and 5 μl of PI for 5 min at room temperature in the dark. At least 20,000 cells were acquired in a FACSCalibur flow cytometer using CellQuest 3.0.1 software (BD Biosciences). Percentages of cells undergoing apoptosis were determined by dual-color analysis. This staining allowed us to distinguish 3 subsets of cells: viable cells (annexin V negative and PI negative), early apoptotic cells (annexin V positive and PI negative), and late apoptotic or necrotic cells (annexin V positive and PI positive). Immediately after staining, the cells were analyzed on a flow cytometer using 488-nm excitation and a 525-nm band pass filter for FITC and a 620-nm filter for PI detection.For detection of mitochondrial membrane potential, variously stimulated RAW 264.7 cells (1 × 106) were stained for 10 min at 37 °C with 10 μg/ml of the mitochondrial membrane potential sensor dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolycarbocyanine iodide (JC-1) (Molecular Probes). JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from 530 to 585 nm. At normal mitochondrial potential, fluorescent JC-1 aggregates predominate (585 nm). However, as mitochondrial potential drops, the aggregates separate, and the fluorescent monomeric form (530 nm) predominates. JC-1-stained cells were analyzed for relative 585 (FL2) to 530 nm (FL1) fluorescence, as described above.Apoptotic Protein Screens—Whole lysate protein samples (350 μg) from Act-treated (20 ng/ml) RAW 264.7 cells were analyzed by Kinetworks™ KAPS-1.0 Apoptosis Screens (Kinexus Bioinformatics Corp., Vancouver, British Columbia, Canada) as previously described (view the Kinexus website for details and publication links). The Kinetworks™ analysis involved resolution of a single lysate sample by SDS-PAGE and subsequent immunoblotting with panels of up to 3 primary antibodies per channel in a 20-lane Immunetics Multiblotter. The antibody mixtures were carefully selected to avoid overlapping cross-reactivity with target proteins. Normalized trace quantity units (CPM) were arbitrary based on the intensity of ECL fluorescence detection for target immunoreactive proteins recorded with a Fluor-S MultiImager and quantified using Quantity One Software (Bio-Rad).Isolation of Cytosolic and Mitochondrial Lysates—RAW 264.7 cells, treated with 20 ng/ml Act for 0, 2, 4, and 6 h, were washed with PBS and centrifuged at 1,000 × g for 5 min. Cell pellets were washed twice with PBS and suspended in ice-cold buffer containing 20 mm HEPES (pH 7.5), 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 μg/ml aprotinin, 10 μg/ml pepstatin A, and 250 mm sucrose. Cells were subsequently incubated on ice for 15 min and then homogenized by 15 passages though a 22-gauge needle. Homogenates were centrifuged at 1,000 × g for 5 min at 4 °C, and the resulting supernatants were centrifuged twice at 12,000 × g for 15 min at 4 °C. Supernatants (cytosolic fractions) were subsequently subjected to Western blot analysis. All of the experiments were performed at least in triplicate and representative data are presented.RESULTSAct Induces Phosphorylation/Activation of p38 Kinase in RAW 264.7 Cells, Murine Peritoneal Macrophages, and Human Intestinal Epithelial (T84) Cells—RAW 264.7 cells, thioglycolate-induced murine peritoneal macrophages, or T84 cells were treated with 6, 12, or 20 ng/ml of Act for various time points and whole cell lysates were subjected to electrophoresis and Western blot analysis. LPS-treated RAW 264.7 cells (100 ng/ml for 30 min) were used as a positive control in these experiments and other MAPK Western blot analyses. LPS has been shown to induce ERK, p38, and JNK pathways in RAW 264.7 cells (32Sanghera J.S. Weinstein S.L. Aluwalia M. Girn J. Pelech S.L. J. Immunol. 1996; 156: 4457-4465PubMed Google Scholar). Subsequent blots were probed with antibodies specific for the phosphorylated form of p38 (p-p38) or total p38α. As shown in Fig. 1A, Act (6 ng/ml) induced an increase in a 43-kDa sized band, consistent with the phosphorylated form of p38, by 1 h. This increase was observed up to 8 h but disappeared by 12 h. Western blot analysis on samples from RAW 264.7 cells treated with a higher dose (12 ng/ml) of Act (for up to 8 h) resulted in similar results, with the earliest induction of p38 phosphorylation observed at 30 min (data not shown). Increased phosphorylation of p38 observed upon Act treatment was not because of an increase in the protein concentration of total p38, as evidenced by similarly sized bands for each time point when blots were probed with an antibody that recognized both the phosphorylated and non-phosphorylated forms of p38α (Fig. 1B). Act-treated murine peritoneal macrophages exhibited a similar pattern of p38 phosphorylation for both concentrations of Act that were used (12 and 20 ng/ml): Act induced phosphorylation of p38 by 1 h (Fig. 1C), but no increase was observed for total p38α (Fig. 1D). Likewise, p38 phosphorylation was observed for Act-treated (12 ng/ml) T84 cells by 1 h (Fig. 1E), with no increase in total protein concentration (Fig. 1F). Because no bands were observed for total p38α in T84 cells (data not shown), we used an antibody specific for p38δ (Fig. 1F). Antibodies for p-p38 recognized both phosphorylated isoforms (p38α and p38δ), but it is likely that the phosphorylated form observed at 1 h in T84 cells was not the p38α isoform.To demonstrate that increased phosphorylation of p38 in Act-treated cells was biologically significant, we examined p38 kinase activity in Act-treated RAW cells. Whole cell lysates were immunoprecipitated using an antibody specific for the phosphorylated form of p38, and kinase assays were performed using ATF-2, a known substrate of p38. The resulting reactions were then subjected to electrophoresis and the blots were probed with an antibody specific for the phosphorylated form of ATF-2. As shown in Fig. 1G, a 70-kDa sized band consistent with phosphorylated ATF-2 was" @default.
• W1965071570 created "2016-06-24" @default.
• W1965071570 creator A5018398198 @default.
• W1965071570 creator A5022563446 @default.
• W1965071570 creator A5024831931 @default.
• W1965071570 creator A5033515303 @default.
• W1965071570 creator A5039282448 @default.
• W1965071570 creator A5041898039 @default.
• W1965071570 creator A5048675624 @default.
• W1965071570 creator A5077467093 @default.
• W1965071570 date "2004-09-01" @default.
• W1965071570 modified "2023-10-01" @default.
• W1965071570 title "Aeromonas hydrophila Cytotoxic Enterotoxin Activates Mitogen-activated Protein Kinases and Induces Apoptosis in Murine Macrophages and Human Intestinal Epithelial Cells" @default.
• W1965071570 cites W1494491432 @default.
• W1965071570 cites W1501833360 @default.
• W1965071570 cites W1507812324 @default.
• W1965071570 cites W1514719303 @default.
• W1965071570 cites W1524470153 @default.
• W1965071570 cites W1566737177 @default.
• W1965071570 cites W1572755918 @default.
• W1965071570 cites W1586365192 @default.
• W1965071570 cites W1637882022 @default.
• W1965071570 cites W1800165852 @default.
• W1965071570 cites W1826122008 @default.
• W1965071570 cites W1873493101 @default.
• W1965071570 cites W1963667478 @default.
• W1965071570 cites W1972085016 @default.
• W1965071570 cites W1975646272 @default.
• W1965071570 cites W1976226379 @default.
• W1965071570 cites W1978068134 @default.
• W1965071570 cites W1978365011 @default.
• W1965071570 cites W1979995768 @default.
• W1965071570 cites W1980773490 @default.
• W1965071570 cites W1981124481 @default.
• W1965071570 cites W1989981838 @default.
• W1965071570 cites W1991425488 @default.
• W1965071570 cites W1991972495 @default.
• W1965071570 cites W1996568613 @default.
• W1965071570 cites W1997739078 @default.
• W1965071570 cites W1999543220 @default.
• W1965071570 cites W2005706069 @default.
• W1965071570 cites W2009543520 @default.
• W1965071570 cites W2010095588 @default.
• W1965071570 cites W2012609273 @default.
• W1965071570 cites W2032615620 @default.
• W1965071570 cites W2035104435 @default.
• W1965071570 cites W2037783385 @default.
• W1965071570 cites W2039445898 @default.
• W1965071570 cites W2039570072 @default.
• W1965071570 cites W2041183642 @default.
• W1965071570 cites W2042196731 @default.
• W1965071570 cites W2051439699 @default.
• W1965071570 cites W2054248299 @default.
• W1965071570 cites W2054942069 @default.
• W1965071570 cites W2060630008 @default.
• W1965071570 cites W2063669247 @default.
• W1965071570 cites W2066671615 @default.
• W1965071570 cites W2068892595 @default.
• W1965071570 cites W2069104210 @default.
• W1965071570 cites W2070044720 @default.
• W1965071570 cites W2072858020 @default.
• W1965071570 cites W2073925624 @default.
• W1965071570 cites W2074329998 @default.
• W1965071570 cites W2074495535 @default.
• W1965071570 cites W2079081366 @default.
• W1965071570 cites W2080360077 @default.
• W1965071570 cites W2084037169 @default.
• W1965071570 cites W2084060660 @default.
• W1965071570 cites W2086614331 @default.
• W1965071570 cites W2087668188 @default.
• W1965071570 cites W2090707627 @default.
• W1965071570 cites W2094389804 @default.
• W1965071570 cites W2095424453 @default.
• W1965071570 cites W2100316337 @default.
• W1965071570 cites W2105699218 @default.
• W1965071570 cites W2109838780 @default.
• W1965071570 cites W2127551467 @default.
• W1965071570 cites W2130866079 @default.
• W1965071570 cites W2135612279 @default.
• W1965071570 cites W2140677435 @default.
• W1965071570 cites W2145722383 @default.
• W1965071570 cites W2149429457 @default.
• W1965071570 cites W2150573930 @default.
• W1965071570 cites W2152803022 @default.
• W1965071570 cites W2153734123 @default.
• W1965071570 cites W2154346380 @default.
• W1965071570 cites W2157026533 @default.
• W1965071570 cites W2158540015 @default.
• W1965071570 cites W2162226205 @default.
• W1965071570 cites W2165981458 @default.
• W1965071570 cites W2166221011 @default.
• W1965071570 cites W2168507682 @default.
• W1965071570 cites W2331647907 @default.
• W1965071570 doi "https://doi.org/10.1074/jbc.m404641200" @default.
• W1965071570 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15215244" @default.
• W1965071570 hasPublicationYear "2004" @default.
• W1965071570 type Work @default.
• W1965071570 sameAs 1965071570 @default.

• next