FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2970492078> ?p ?o ?g. }

• W2970492078 endingPage "2232" @default.
• W2970492078 startingPage "2221" @default.
• W2970492078 abstract "Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTP–binding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cell–signaling events functioning in intestinal repair. Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTP–binding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cell–signaling events functioning in intestinal repair. Injury to the intestinal epithelium can occur because of multiple clinical conditions that include infectious or idiopathic inflammatory diseases, ischemia, or irradiation. Generally, the epithelium has a remarkable capacity to repair itself, thereby preventing fluid/electrolyte imbalance and systemic exposure to luminal antigens or pathogens. However, many clinical conditions, such as inflammatory bowel disease (ulcerative colitis or Crohn disease), lead to intestinal injury that does not heal. Therefore, the molecular mechanisms and functional elements that mediate epithelial barrier wound repair are of intense investigative interest. The intestinal epithelium is a dynamic barrier separating the luminal stream from the underlying subepithelial compartments. This barrier is actively renewed by proliferation of progenitor stem cells within crypts, migration of epithelial cells along the crypt-villous axis, and programmed shedding at the luminal surface. This process occurs in 5 to 7 days in humans, while concomitantly maintaining barrier function.1Peterson L.W. Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis.Nat Rev Immunol. 2014; 14: 141-153Crossref PubMed Scopus (1391) Google Scholar, 2Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration.Nat Rev Mol Cell Biol. 2014; 15: 19-33Crossref PubMed Scopus (677) Google Scholar Many cell-signaling pathways have been shown to function in wound repair, including networks that are activated in response to the controlled generation of reactive oxygen within cells.3Alam A. Leoni G. Wentworth C.C. Kwal J.M. Wu H. Ardita C.S. Swanson P.A. Lambeth J.D. Jones R.M. Nusrat A. Neish A.S. Redox signaling regulates commensal-mediated mucosal homeostasis and restitution and requires formyl peptide receptor 1.Mucosal Immunol. 2014; 7: 645-655Crossref PubMed Scopus (101) Google Scholar, 4Campbell E.L. Colgan S.P. Control and dysregulation of redox signalling in the gastrointestinal tract.Nat Rev Gastroenterol Hepatol. 2019; 16: 106-120Crossref PubMed Scopus (43) Google Scholar, 5Jones R.M. Neish A.S. Redox signaling mediated by the gut microbiota.Free Radic Biol Med. 2017; 105: 41-47Crossref PubMed Scopus (83) Google Scholar, 6Leoni G. Neumann P.A. Kamaly N. Quiros M. Nishio H. Jones H.R. Sumagin R. Hilgarth R.S. Alam A. Fredman G. Argyris I. Rijcken E. Kusters D. Reutelingsperger C. Perretti M. Parkos C.A. Farokhzad O.C. Neish A.S. Nusrat A. Annexin A1-containing extracellular vesicles and polymeric nanoparticles promote epithelial wound repair.J Clin Invest. 2015; 125: 1215-1227Crossref PubMed Scopus (182) Google Scholar The controlled and deliberate generation of reactive oxygen can occur as a result of the catalytic activity harbored within cells, either intrinsically at low levels or at elevated levels in response to sensing exogenous stimuli. For example, sensing of molecular elements specific to bacteria and subsequent generation of reactive oxygen by neutrophils are a long-established host microbicidal response to bacteria, termed the respiratory burst.7Uhlinger D.J. Tyagi S.R. Inge K.L. Lambeth J.D. The respiratory burst oxidase of human neutrophils: guanine nucleotides and arachidonate regulate the assembly of a multicomponent complex in a semirecombinant cell-free system.J Biol Chem. 1993; 268: 8624-8631PubMed Google Scholar This process is catalyzed by a specific NADPH oxidase 2 (Nox2), of which gp91phox is a membrane-bound component found primarily in macrophages and neutrophils and required for their bactericidal action.8Lambeth J.D. Neish A.S. Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited.Annu Rev Pathol. 2014; 9: 119-145Crossref PubMed Scopus (302) Google Scholar Circulating immune cells recruited to the site of a wound exert indiscriminate microbicidal activity by the generation of reactive oxygen.9Babior B.M. Oxygen-dependent microbial killing by phagocytes: 1.N Engl J Med. 1978; 298: 659-668Crossref PubMed Scopus (1645) Google Scholar In addition to microbicidal activity, deliberate generation of reactive oxygen within epithelial cells via the NADPH oxidase Nox1 has an established role in modulating cell signaling, including regulatory events that initiate and promote restitution and healing of a damaged epithelium.3Alam A. Leoni G. Wentworth C.C. Kwal J.M. Wu H. Ardita C.S. Swanson P.A. Lambeth J.D. Jones R.M. Nusrat A. Neish A.S. Redox signaling regulates commensal-mediated mucosal homeostasis and restitution and requires formyl peptide receptor 1.Mucosal Immunol. 2014; 7: 645-655Crossref PubMed Scopus (101) Google Scholar, 10Hall C.H.T. Campbell E.L. Colgan S.P. Neutrophils as components of mucosal homeostasis.Cell Mol Gastroenterol Hepatol. 2017; 4: 329-337Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 11Alam A. Leoni G. Quiros M. Wu H. Desai C. Nishio H. Jones R.M. Nusrat A. Neish A.S. The microenvironment of injured murine gut elicits a local pro-restitutive microbiota.Nat Microbiol. 2016; 1: 15021Crossref PubMed Scopus (114) Google Scholar Reactive oxygen–mediated signaling occurs through the rapid and reversible oxidation of cysteine residues within specific target proteins, usually regulatory enzymes, thus allowing for graded perception of intracellular reactive oxygen concentrations and control of critical steps in signal transduction pathways.12Rhee S.G. Kang S.W. Jeong W. Chang T.S. Yang K.S. Woo H.A. Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins.Curr Opin Cell Biol. 2005; 17: 183-189Crossref PubMed Scopus (596) Google Scholar, 13Barford D. The role of cysteine residues as redox-sensitive regulatory switches.Curr Opin Struct Biol. 2004; 14: 679-686Crossref PubMed Scopus (263) Google Scholar The specificity of biological responses to the altered levels of reactive oxygen is dependent on the specific reactive oxygen species molecule type generated, the intensity of the signal, the subcellular sites of production, or the developmental stage of the cell.14Terada L.S. Specificity in reactive oxidant signaling: think globally, act locally.J Cell Biol. 2006; 174: 615-623Crossref PubMed Scopus (172) Google Scholar, 15Ushio-Fukai M. Compartmentalization of redox signaling through NADPH oxidase-derived ROS.Antioxid Redox Signal. 2009; 11: 1289-1299Crossref PubMed Scopus (273) Google Scholar Our research group recently reported that oxidation-reduction (redox) signaling could influence the phosphorylation of Crk-associated substrate (Cas) via a mechanism that requires the activity of the Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase.16Matthews J.D. Sumagin R. Hinrichs B. Nusrat A. Parkos C.A. Neish A.S. Redox control of Cas phosphorylation requires Abl kinase in regulation of intestinal epithelial cell spreading and migration.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G458-G465Crossref PubMed Scopus (5) Google Scholar Specifically, Cas, which acts as a mechanosensor in focal adhesions and is essential for cell movement is phosphorylated at residue Y410 in response to elevated levels of exposure to hydrogen peroxide. In addition, within a restituting, mechanically inflicted wound, phosphorylated CAS delocalizes from focal adhesions to cell junctions, which is a phenotype observed in actively restituting intestine tissue.16Matthews J.D. Sumagin R. Hinrichs B. Nusrat A. Parkos C.A. Neish A.S. Redox control of Cas phosphorylation requires Abl kinase in regulation of intestinal epithelial cell spreading and migration.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G458-G465Crossref PubMed Scopus (5) Google Scholar Interestingly, focal adhesion kinase (FAK) has also been shown to localize to cell junctions and control permeability in both the endothelium17Arnold K.M. Goeckeler Z.M. Wysolmerski R.B. Loss of focal adhesion kinase enhances endothelial barrier function and increases focal adhesions.Microcirculation. 2013; 20: 637-649Crossref PubMed Scopus (21) Google Scholar, 18Quadri S.K. Cross talk between focal adhesion kinase and cadherins: role in regulating endothelial barrier function.Microvasc Res. 2012; 83: 3-11Crossref PubMed Scopus (39) Google Scholar, 19Mehta D. Tiruppathi C. Sandoval R. Minshall R.D. Holinstat M. Malik A.B. Modulatory role of focal adhesion kinase in regulating human pulmonary arterial endothelial barrier function.J Physiol. 2002; 539: 779-789Crossref PubMed Scopus (80) Google Scholar, 20Yuan S.Y. Shen Q. Rigor R.R. Wu M.H. Neutrophil transmigration, focal adhesion kinase and endothelial barrier function.Microvasc Res. 2012; 83: 82-88Crossref PubMed Scopus (58) Google Scholar, 21Lee J. Borboa A.K. Chun H.B. Baird A. Eliceiri B.P. Conditional deletion of the focal adhesion kinase FAK alters remodeling of the blood-brain barrier in glioma.Cancer Res. 2010; 70: 10131-10140Crossref PubMed Scopus (38) Google Scholar, 22Schmidt T.T. Tauseef M. Yue L. Bonini M.G. Gothert J. Shen T.L. Guan J.L. Predescu S. Sadikot R. Mehta D. Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rac1 activities.Am J Physiol Lung Cell Mol Physiol. 2013; 305: L291-L300Crossref PubMed Scopus (38) Google Scholar and epithelium.23Cheng C.Y. Mruk D.D. Regulation of blood-testis barrier dynamics by focal adhesion kinase (FAK): an unexpected turn of events.Cell Cycle. 2009; 8: 3493-3499Crossref PubMed Scopus (22) Google Scholar, 24Ilic D. Mao-Qiang M. Crumrine D. Dolganov G. Larocque N. Xu P. Demerjian M. Brown B.E. Lim S.T. Ossovskaya V. Schlaepfer D.D. Fisher S.J. Feingold K.R. Elias P.M. Mauro T.M. Focal adhesion kinase controls pH-dependent epidermal barrier homeostasis by regulating actin-directed Na+/H+ exchanger 1 plasma membrane localization.Am J Pathol. 2007; 170: 2055-2067Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar These findings point to the possibility of cross talk between focal adhesion proteins and cell junction proteins.25Usatyuk P.V. Parinandi N.L. Natarajan V. Redox regulation of 4-hydroxy-2-nonenal-mediated endothelial barrier dysfunction by focal adhesion, adherens, and tight junction proteins.J Biol Chem. 2006; 281: 35554-35566Crossref PubMed Scopus (139) Google Scholar As stated above, immune cells also secrete high levels of extracellular reactive oxygen into the tissue wound environment.26Dupre-Crochet S. Erard M. Nuss O. ROS production in phagocytes: why, when, and where?.J Leukoc Biol. 2013; 94: 657-670Crossref PubMed Scopus (242) Google Scholar This event has long been assumed to represent deleterious collateral damage necessary for killing of phagocytosed bacteria. However, this reactive oxygen can form a concentration gradient with signaling function. Therefore, at a certain distance, the gradient of immune cell–generated reactive oxygen is inevitably sensed by cells within the adjacent epithelium and possibly results in the physiological reversible oxidation of cysteine residues within specific target proteins. To date, the extent to which immune cell–derived reactive oxygen orchestrates cell-signaling events during intestinal epithelium wound repair has not been fully described. The aim of this study was to investigate the cell signaling pathways regulated by reactive oxygen that function to induce wound restitution. A variety of genetic, imaging, biochemical, and proteomic techniques were used, which showed that neutrophil-derived reactive oxygen produced at the site of intestinal injury function in activating signaling events in the epithelium that regulate cytoskeleton dynamics, focal adhesion, and cell junction activity. Wild-type C57BL/6 (stock number 000664) and gp91phox-null (stock number 002365) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in the Emory University (Atlanta, GA) Whitehead Animal facility for 2 weeks before experimentation. To account for host-microbiome reciprocal interactions, the microbiome of each group of mice was normalized by routinely exchanging bedding between cages for 2 weeks before experimentation. Biopsy wounding was performed, as previously described.16Matthews J.D. Sumagin R. Hinrichs B. Nusrat A. Parkos C.A. Neish A.S. Redox control of Cas phosphorylation requires Abl kinase in regulation of intestinal epithelial cell spreading and migration.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G458-G465Crossref PubMed Scopus (5) Google Scholar Briefly, to generate discrete mucosal injuries in the mouse colon and to monitor their regeneration, a high-resolution miniaturized colonoscope system (Coloview Veterinary Endoscope; Karl Storz, Goleta, CA) was used. This system consisted of a miniature rigid endoscope (1.9-mm outer diameter), a xenon light source, a triple-chip, high-resolution charge-coupled device camera, and an operating sheath with 3F instrument channel and air/water injection bulb to regulate inflation of the mouse colon (all from Karl Storz). Endoscopic procedures were viewed with high-resolution (1024 × 768 pixel) images on a flat-panel color monitor. The night before the initial biopsy injury, food was removed from the mouse cages. The following morning, mice were anesthetized by using ketamine and xylazine, and the endoscope with outer operating sheath was inserted to the mid–descending colon and the mucosa was surveyed to the anorectal junction. Then, a flexible biopsy forceps with a diameter of 3F was used to remove single full-thickness areas of the entire mucosa and submucosa. Particular attention was taken to avoid penetration of the muscularis propria. Each mouse received biopsy injuries at three to five sites along the dorsal side of the colon (spacing was >5 mm). After the defined experimental period, colons were dissected and tissue was embedded for histopathologic and immunofluorescence analysis. C57BL/6 and gp91phox-null mice were given drinking water with dextran sodium sulfate (DSS) at 3%. This procedure causes erosions of the intestinal epithelium and consequently results in acute inflammation within 5 days. At 5 days, DSS is then discontinued and healing is monitored by assessing the disease activity index up until that extended to day 14 after initial addition of DSS in the water. The disease activity index is measured and calculated on the basis of stool consistency, weight loss, and presence or absence of detectable fecal blood. Colonic inflammation and damage were assessed by histologic scoring of immune cell infiltration, ulcerations, adhesions, and bowel wall thickness. Polymorphonuclear neutrophil depletion was achieved by two administrations of an anti-Ly6G antibody (100 μg, intraperitoneally; Thermo Fisher Scientific, Waltham, MA): once, 12 hours before wounding; and second, 24 hours (day 1) after infliction of colonic wounds. The control group received a similar dose of the relevant IgG antibody. Polymorphonuclear neutrophil depletion (>90%) was confirmed by flow cytometry. To measure transepithelial resistance, SK-CO-15 (a gift from Charles Parkos, University of Michigan, Ann Arbor, MI) cultured human epithelial cells were plated onto Corning transwells (24-well format; catalog number 3470; Corning Life Sciences, Tewksbury, MA) and monitored with an epithelial voltohmmeter. Transwells generate a cell culture that more closely mimics an in vivo environment, where chemical inhibitors or stimulants can be added to either the upper compartment (apical) or the lower compartment (basolateral) of the transwell. SK-CO-15 cells were also used for plasmid transfection assays and biochemical experiments to analyze protein signaling or protein-protein interactions. Transfections were performed in a 24-well plate, with each well receiving 0.5 μg plasmid DNA/0.75 μL of Lipofectamine 2000 overnight treatment in Optimem cell culture media (both from Thermo Fisher Scientific). Unless otherwise noted, transfected or chemically treated cells were harvested and analyzed after 24 hours of incubation. FAK inhibitor PF-562271 was used at 500 nmol/L final concentration, and the p21-activated kinase 2 (PAK2) inhibitor FRAX597 (Selleckchem, Houston, TX) was used at 2.5 μmol/L final concentration. Cells were fixed in 4% formaldehyde, ice cold, for 10 minutes and then permeabilized with 0.5% Triton X-100 for 7 minutes. For mouse sections, tissues were fixed in ice-cold formaldehyde for 15 minutes and permeabilized in Triton X-100 for 15 minutes. Samples were blocked in phosphate-buffered saline/bovine serum albumin and then probed consecutively with primary and fluorescent conjugate secondary antibodies (goat anti-mouse, anti-rabbit, or anti-rat) with appropriate species reactivity (Molecular Probes, Eugene, OR). For proximity ligation, the samples were processed according to the Duolink proximity ligation assay technology (Sigma-Aldrich, St. Louis, MO) instructions, with slight modifications. Briefly, after incubation with primary antibody, samples were exposed to the Duolink secondary antibodies containing conjugated nucleic acid probes for 45 minutes, and then ligated for 30 minutes at 37°C, after which DNA polymerase was added with a master mix of fluorescent nucleotides and allowed to react for 1 hour. Images were obtained on an Olympus F1000 confocal microscope (Olympus America Inc., Center Valley, PA). The biotin-iodoacetamide (BIAM) pull-down assay was performed, as described previously,27Matthews J.D. Reedy A.R. Wu H. Hinrichs B.H. Darby T.M. Addis C. Robinson B.S. Go Y.M. Jones D.P. Jones R.M. Neish A.S. Proteomic analysis of microbial induced redox-dependent intestinal signaling.Redox Biol. 2019; 20: 526-532Crossref PubMed Scopus (14) Google Scholar without modification. Briefly, epithelial cell monolayers were lysed, the insoluble material was removed by centrifugation, and the lysates were adjusted to 25 μmol/L (biotinylated iodoacetamide) BIAM and incubated on ice in the dark for 45 minutes. After removal of unbound biotin label with a desalting column, biotinylated proteins were immobilized onto streptavidin agarose for 1 hour. After extensive washing and heat denaturing, SDS-PAGE and Western blot analysis were performed. The Cleavable ICAT Reagent for Protein Labeling (stock keeping unit number 4339036) was obtained from SCIEX (Framingham, MA). Isotope-coded affinity tag (ICAT) and tandem mass spectrometry analysis was performed essentially, as previously described,27Matthews J.D. Reedy A.R. Wu H. Hinrichs B.H. Darby T.M. Addis C. Robinson B.S. Go Y.M. Jones D.P. Jones R.M. Neish A.S. Proteomic analysis of microbial induced redox-dependent intestinal signaling.Redox Biol. 2019; 20: 526-532Crossref PubMed Scopus (14) Google Scholar with minor modification. Briefly, trichloroacetic acid precipitated proteins from cell lysates were incubated with a heavy ICAT reagent to label reduced cysteines, followed by a second trichloroacetic acid precipitation, disulfide reduction with tris(2-carboxyethyl)phosphine (TCEP), and then labeling of newly reduced cysteines with a light ICAT reagent. After overnight trypsin digestion and an on-column purification (first-round anion exchange, second-round streptavidin) of biotinylated peptides with heavy and light labels, samples were vacuum dried before processing. Peptides were resuspended in trifluoroacetic acid/acetonitrile and loaded onto a C18-fused silica column before elution and injection into a mass spectrometer, where they were then scanned, resolved, and trapped before being dynamically excluded; and the raw values were used to search a human database for corresponding peptide matches, with a false discovery rate set to 1%. Protein lysates (approximately 1 mg protein) were mixed with preloaded protein-G agarose beads (Sigma-Aldrich) containing the appropriate immunoprecipitating antibody and incubated with end-over-end mixing for 2 hours at 4°C. Beads with immune complexes were isolated by centrifugation and washed several times with lysis buffer (1% Triton X-100/tris-buffered saline) before being heat denatured and analyzed by Western blot analysis. To this end, blotting was performed using standard SDS-PAGE and transfer conditions (100 V for gel, 350 mA for transfer). Blots were blocked in milk or, in the case for phosphoblotting, 5% bovine serum albumin was used. Primary antibodies were incubated with blots overnight at 4°C, and the secondary electrochemiluminescence conjugate (donkey anti-mouse or donkey anti-rabbit; Jackson Immunoresearch Inc., West Grove, PA) was incubated for 2 hours after multiple washes with tris-buffered saline/Tween-20. The electrochemiluminescence signal was captured by a digital camera system (KwikQuant; Kindle Biosciences, Greenwich, CT) and adjusted for brightness and contrast. Experiments were repeated multiple times to ensure accuracy. Other investigations have reported on the negative effects of the loss of neutrophil activity on recovery using the 2,4,6-trinitrobenzene sulfonic acid–colitis model that includes the development of a transmural inflammation that resembles the histopathologic lesions that develop in human Crohn disease.28Campbell E.L. Bruyninckx W.J. Kelly C.J. Glover L.E. McNamee E.N. Bowers B.E. Bayless A.J. Scully M. Saeedi B.J. Golden-Mason L. Ehrentraut S.F. Curtis V.F. Burgess A. Garvey J.F. Sorensen A. Nemenoff R. Jedlicka P. Taylor C.T. Kominsky D.J. Colgan S.P. Transmigrating neutrophils shape the mucosal microenvironment through localized oxygen depletion to influence resolution of inflammation.Immunity. 2014; 40: 66-77Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar The effect of the loss of reactive oxygen generated by infiltrating immune cells was assessed on disease intensity and recovery rates using a DSS model of colitis and gut injury. Mice that are null for Nox2 activity by genetic ablation of gp91phox exhibited significantly increased disease activity index during DSS treatment and recovered at a significantly slower rate after the removal of DSS from the drinking water, compared with wild-type controls (Figure 1A–C ). These data point to the contributing influence of immune cell–generated reactive oxygen in disease intensity and, in particular, in the restitution of injured gut tissue. Within biopsy wounds inflicted on the colonic mucosa, the localization of phosphorylated Cas (p-Cas) deviates from being restricted to focal adhesions at the base of columnar enterocytes in uninjured tissue, to also emerge on the lateral edges of enterocytes in wounded tissue. The emergence of p-Cas on the lateral edges of enterocytes also occurs after the addition of hydrogen peroxide to polarized colonic epithelial monolayers.16Matthews J.D. Sumagin R. Hinrichs B. Nusrat A. Parkos C.A. Neish A.S. Redox control of Cas phosphorylation requires Abl kinase in regulation of intestinal epithelial cell spreading and migration.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G458-G465Crossref PubMed Scopus (5) Google Scholar Because hydrogen peroxide alone could recapitulate the phenotype of the emergence of p-Cas on the lateral edges of enterocytes, and because high levels of hydrogen peroxide are generated by immune cells recruited to the site of the wound, we hypothesized that immune cell–generated hydrogen peroxide potentiates the emergence of p-Cas on the lateral edges of enterocytes within wounds. To test this hypothesis, biopsy wounds were inflicted in the colons of mice that lack Nox2 activity (null for gp91phox, the primary driver of superoxide generation), with Nox2 being the holoenzyme that is essential for catalyzing the generation of reactive oxygen in immune cells. Although the emergence of p-Cas occurred on the lateral edges of enterocytes of wild-type C57BL/6 control mice at 48 hours after wound infliction, no p-Cas was detected on the lateral edges of enterocytes within wounds lacking Nox2 activity (Figure 1D), demonstrating the requirement of immune cell–generated reactive oxygen for this phenotype. In addition, depletion of circulating neutrophils from mice by i.p. administration of anti-Ly6G elicited the same results as observed in Nox2-null mice, in which no p-Cas was detected within wounds after neutrophil depletion. These data indicate that a major source of exogenous reactive oxygen within wounds emanates from recruited neutrophils at the site of intestinal injury (Figure 1D). Next, the epithelial signaling pathways affected by exposure to high levels of exogenous reactive oxygen species were studied. A proteome-wide screen was performed to detect proteins oxidized after exposure to 1 mmol/L H2O2 using the ICAT method.29Go Y.M. Roede J.R. Orr M. Liang Y. Jones D.P. Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of Cd toxicity.Toxicol Sci. 2014; 139: 59-73Crossref PubMed Scopus (74) Google Scholar This concentration of hydrogen peroxide was selected because it was the lowest concentration found to elicit detectable phosphorylation of Cas in vitro.16Matthews J.D. Sumagin R. Hinrichs B. Nusrat A. Parkos C.A. Neish A.S. Redox control of Cas phosphorylation requires Abl kinase in regulation of intestinal epithelial cell spreading and migration.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G458-G465Crossref PubMed Scopus (5) Google Scholar The ICAT approach involves a two-step labeling of reduced and reversibly oxidized cysteine residues within proteins using iodoacetamide. First, reduced cysteines are labeled with a heavy isotope. Then, in a second reaction, oxidized cysteines are reduced with TCEP and reacted with a light isotope. Therefore, by contrasting the ratio of cysteine residues labeled in untreated cells to ratios of cysteine residues labeled in cells exposed to hydrogen peroxide, it is possible to identify residues that are sensitive to oxidation by exposure to reactive oxygen (Figure 2A). Cultured SK-CO-1 cells were exposed to 1 mmol/L H2O2 for 15 minutes, whereupon cells were harvested and analyzed by ICAT. Approximately 2000 peptides corresponding to 115 different proteins that are significantly oxidized on hydrogen peroxide exposure were identified (Supplemental Table S1). The ICAT protocol relies on column purification of biotinylated peptides and, therefore, the whole proteome is not fully captured; thus, the data set only represents a fraction of the possible oxidized proteome. Graphical representation of the percentage oxidation for each of the captured peptides shows an overall shift from left to right, indicating that a large portion of the assayed proteome was oxidized when exposed to exogenous hydrogen peroxide (Figure 2B)." @default.
• W2970492078 created "2019-09-05" @default.
• W2970492078 creator A5007051084 @default.
• W2970492078 creator A5021969411 @default.
• W2970492078 creator A5032480830 @default.
• W2970492078 creator A5046682255 @default.
• W2970492078 creator A5052166300 @default.
• W2970492078 creator A5056153030 @default.
• W2970492078 creator A5056514518 @default.
• W2970492078 creator A5057779997 @default.
• W2970492078 creator A5068450657 @default.
• W2970492078 creator A5078257803 @default.
• W2970492078 date "2019-11-01" @default.
• W2970492078 modified "2023-10-15" @default.
• W2970492078 title "Neutrophil-Derived Reactive Oxygen Orchestrates Epithelial Cell Signaling Events during Intestinal Repair" @default.
• W2970492078 cites W1592451467 @default.
• W2970492078 cites W1969606022 @default.
• W2970492078 cites W1971443244 @default.
• W2970492078 cites W1971867535 @default.
• W2970492078 cites W1981068608 @default.
• W2970492078 cites W1982159359 @default.
• W2970492078 cites W1985776151 @default.
• W2970492078 cites W1986454658 @default.
• W2970492078 cites W1990479344 @default.
• W2970492078 cites W1994949724 @default.
• W2970492078 cites W1999763927 @default.
• W2970492078 cites W2010081021 @default.
• W2970492078 cites W2014280822 @default.
• W2970492078 cites W2015232280 @default.
• W2970492078 cites W2020285880 @default.
• W2970492078 cites W2021819728 @default.
• W2970492078 cites W2023122744 @default.
• W2970492078 cites W2045549203 @default.
• W2970492078 cites W2054697465 @default.
• W2970492078 cites W2059716736 @default.
• W2970492078 cites W2067659428 @default.
• W2970492078 cites W2068307494 @default.
• W2970492078 cites W2068752808 @default.
• W2970492078 cites W2091355057 @default.
• W2970492078 cites W2094555042 @default.
• W2970492078 cites W2100807457 @default.
• W2970492078 cites W2103156288 @default.
• W2970492078 cites W2105924489 @default.
• W2970492078 cites W2106453911 @default.
• W2970492078 cites W2122292170 @default.
• W2970492078 cites W2129191533 @default.
• W2970492078 cites W2131917465 @default.
• W2970492078 cites W2134466886 @default.
• W2970492078 cites W2142258398 @default.
• W2970492078 cites W2148042107 @default.
• W2970492078 cites W2153972277 @default.
• W2970492078 cites W2160826303 @default.
• W2970492078 cites W2162752530 @default.
• W2970492078 cites W2169818209 @default.
• W2970492078 cites W2463255563 @default.
• W2970492078 cites W2529915577 @default.
• W2970492078 cites W2539598072 @default.
• W2970492078 cites W2737081341 @default.
• W2970492078 cites W2901399106 @default.
• W2970492078 cites W2901694258 @default.
• W2970492078 doi "https://doi.org/10.1016/j.ajpath.2019.07.017" @default.
• W2970492078 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6892184" @default.
• W2970492078 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31472109" @default.
• W2970492078 hasPublicationYear "2019" @default.
• W2970492078 type Work @default.
• W2970492078 sameAs 2970492078 @default.
• W2970492078 citedByCount "12" @default.
• W2970492078 countsByYear W29704920782020 @default.
• W2970492078 countsByYear W29704920782021 @default.
• W2970492078 countsByYear W29704920782022 @default.
• W2970492078 countsByYear W29704920782023 @default.
• W2970492078 crossrefType "journal-article" @default.
• W2970492078 hasAuthorship W2970492078A5007051084 @default.
• W2970492078 hasAuthorship W2970492078A5021969411 @default.
• W2970492078 hasAuthorship W2970492078A5032480830 @default.
• W2970492078 hasAuthorship W2970492078A5046682255 @default.
• W2970492078 hasAuthorship W2970492078A5052166300 @default.
• W2970492078 hasAuthorship W2970492078A5056153030 @default.
• W2970492078 hasAuthorship W2970492078A5056514518 @default.
• W2970492078 hasAuthorship W2970492078A5057779997 @default.
• W2970492078 hasAuthorship W2970492078A5068450657 @default.
• W2970492078 hasAuthorship W2970492078A5078257803 @default.
• W2970492078 hasBestOaLocation W29704920781 @default.
• W2970492078 hasConcept C126322002 @default.
• W2970492078 hasConcept C1491633281 @default.
• W2970492078 hasConcept C203014093 @default.
• W2970492078 hasConcept C2776914184 @default.
• W2970492078 hasConcept C3019368612 @default.
• W2970492078 hasConcept C48349386 @default.
• W2970492078 hasConcept C529295009 @default.
• W2970492078 hasConcept C54355233 @default.
• W2970492078 hasConcept C62478195 @default.
• W2970492078 hasConcept C71924100 @default.
• W2970492078 hasConcept C86803240 @default.
• W2970492078 hasConcept C95444343 @default.
• W2970492078 hasConceptScore W2970492078C126322002 @default.
• W2970492078 hasConceptScore W2970492078C1491633281 @default.
• W2970492078 hasConceptScore W2970492078C203014093 @default.

• next