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• W2022364566 abstract "Background & Aims: LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a tumor necrosis factor core family member that regulates T-cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT is capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling. Methods: The effects of LIGHT and interferon-γ on barrier function, cytoskeletal regulation, and tight junction structure were assessed in mice and intestinal epithelial monolayers. Results: LIGHT induced barrier loss in cultured epithelia via myosin II regulatory light chain (MLC) phosphorylation; both barrier loss and MLC phosphorylation were reversed by MLC kinase (MLCK) inhibition. Pretreatment with interferon-γ, which induced lymphotoxin β receptor (LTβR) expression, was required for these effects, and neither barrier dysfunction nor intestinal epithelial MLC phosphorylation occurred in LTβR knockout mice. In cultured monolayers, endocytosis of the tight junction protein occludin correlated with barrier loss. Internalized occludin colocalized with caveolin-1. LIGHT-induced occludin endocytosis and barrier loss were both prevented by inhibition of caveolar endocytosis. Conclusions: T cell–derived LIGHT activates intestinal epithelial LTβR to disrupt barrier function. This requires MLCK activation and caveolar endocytosis. These data suggest a novel role for LIGHT in disease pathogenesis and suggest that inhibition of MLCK-dependent caveolar endocytosis may represent an approach to restoring barrier function in inflammatory bowel disease. Background & Aims: LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a tumor necrosis factor core family member that regulates T-cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT is capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling. Methods: The effects of LIGHT and interferon-γ on barrier function, cytoskeletal regulation, and tight junction structure were assessed in mice and intestinal epithelial monolayers. Results: LIGHT induced barrier loss in cultured epithelia via myosin II regulatory light chain (MLC) phosphorylation; both barrier loss and MLC phosphorylation were reversed by MLC kinase (MLCK) inhibition. Pretreatment with interferon-γ, which induced lymphotoxin β receptor (LTβR) expression, was required for these effects, and neither barrier dysfunction nor intestinal epithelial MLC phosphorylation occurred in LTβR knockout mice. In cultured monolayers, endocytosis of the tight junction protein occludin correlated with barrier loss. Internalized occludin colocalized with caveolin-1. LIGHT-induced occludin endocytosis and barrier loss were both prevented by inhibition of caveolar endocytosis. Conclusions: T cell–derived LIGHT activates intestinal epithelial LTβR to disrupt barrier function. This requires MLCK activation and caveolar endocytosis. These data suggest a novel role for LIGHT in disease pathogenesis and suggest that inhibition of MLCK-dependent caveolar endocytosis may represent an approach to restoring barrier function in inflammatory bowel disease. LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a member of the tumor necrosis factor (TNF) core family involved in T-cell regulation during innate and adaptive immune responses. Although related, it is notable that LIGHT and TNF activate distinct, nonoverlapping surface receptors. Transgenic overexpression of LIGHT in murine T cells leads to development of experimental inflammatory bowel disease (IBD).1Wang J. Anders R.A. Wu Q. Peng D. Cho J.H. Sun Y. Karaliukas R. Kang H.S. Turner J.R. Fu Y.X. Dysregulated LIGHT expression on T cells mediates intestinal inflammation and contributes to IgA nephropathy.J Clin Invest. 2004; 113: 826-835Crossref PubMed Scopus (99) Google Scholar, 2Shaikh R.B. Santee S. Granger S.W. Butrovich K. Cheung T. Kronenberg M. Cheroutre H. Ware C.F. Constitutive expression of LIGHT on T cells leads to lymphocyte activation, inflammation, and tissue destruction.J Immunol. 2001; 167: 6330-6337PubMed Google Scholar The pattern of disease in these transgenic mice is similar to Crohn’s disease and, like Crohn’s disease, is ameliorated by neutralization of TNF.1Wang J. Anders R.A. Wu Q. Peng D. Cho J.H. Sun Y. Karaliukas R. Kang H.S. Turner J.R. Fu Y.X. Dysregulated LIGHT expression on T cells mediates intestinal inflammation and contributes to IgA nephropathy.J Clin Invest. 2004; 113: 826-835Crossref PubMed Scopus (99) Google Scholar, 2Shaikh R.B. Santee S. Granger S.W. Butrovich K. Cheung T. Kronenberg M. Cheroutre H. Ware C.F. Constitutive expression of LIGHT on T cells leads to lymphocyte activation, inflammation, and tissue destruction.J Immunol. 2001; 167: 6330-6337PubMed Google Scholar While a specific role for LIGHT in human IBD has not yet been shown, it is notable that LIGHT expression is markedly increased in mucosal biopsy specimens from patients with active IBD3Wang J. Anders R.A. Wang Y. Turner J.R. Abraham C. Pfeffer K. Fu Y.X. The critical role of LIGHT in promoting intestinal inflammation and Crohn’s disease.J Immunol. 2005; 174: 8173-8182PubMed Google Scholar and that LIGHT maps to 19p13.3,4Granger S.W. Butrovich K.D. Houshmand P. Edwards W.R. Ware C.F. Genomic characterization of LIGHT reveals linkage to an immune response locus on chromosome 19p13.3 and distinct isoforms generated by alternate splicing or proteolysis.J Immunol. 2001; 167: 5122-5128PubMed Google Scholar a known IBD susceptibility locus.5Rioux J.D. Silverberg M.S. Daly M.J. Steinhart A.H. McLeod R.S. Griffiths A.M. Green T. Brettin T.S. Stone V. Bull S.B. Bitton A. Williams C.N. Greenberg G.R. Cohen Z. Lander E.S. Hudson T.J. Siminovitch K.A. Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci.Am J Hum Genet. 2000; 66: 1863-1870Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar Moreover, LIGHT enhances TNF and interferon (IFN)-γ release from T cells,3Wang J. Anders R.A. Wang Y. Turner J.R. Abraham C. Pfeffer K. Fu Y.X. The critical role of LIGHT in promoting intestinal inflammation and Crohn’s disease.J Immunol. 2005; 174: 8173-8182PubMed Google Scholar, 6Cohavy O. Zhou J. Granger S.W. Ware C.F. Targan S.R. LIGHT expression by mucosal T cells may regulate IFN-gamma expression in the intestine.J Immunol. 2004; 173: 251-258PubMed Google Scholar consistent with the known contributions of these cytokines to human disease. Thus, available data suggest that LIGHT, which is released by T cells, participates in IBD pathogenesis primarily via T-cell regulation.6Cohavy O. Zhou J. Granger S.W. Ware C.F. Targan S.R. LIGHT expression by mucosal T cells may regulate IFN-gamma expression in the intestine.J Immunol. 2004; 173: 251-258PubMed Google Scholar, 7Wang J. Fu Y.X. Tumor necrosis factor family members and inflammatory bowel disease.Immunol Rev. 2005; 204: 144-155Crossref PubMed Scopus (61) Google Scholar, 8Mackay F. Browning J.L. Lawton P. Shah S.A. Comiskey M. Bhan A.K. Mizoguchi E. Terhorst C. Simpson S.J. Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis.Gastroenterology. 1998; 115: 1464-1475Abstract Full Text Full Text PDF PubMed Scopus (133) Google ScholarIntestinal epithelial barrier defects are well recognized in IBD.9Hollander D. The intestinal permeability barrier A hypothesis as to its regulation and involvement in Crohn’s disease.Scand J Gastroenterol. 1992; 27: 721-726Crossref PubMed Scopus (253) Google Scholar, 10Yacyshyn B.R. Meddings J.B. CD45RO expression on circulating CD19+ B cells in Crohn’s disease correlates with intestinal permeability.Gastroenterology. 1995; 108: 132-137Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 11Wyatt J. Vogelsang H. Hubl W. Waldhoer T. Lochs H. Intestinal permeability and the prediction of relapse in Crohn’s disease.Lancet. 1993; 341: 1437-1439Abstract PubMed Scopus (528) Google Scholar While a primary barrier defect may be present in IBD kindreds,12Hollander D. Permeability in Crohn’s disease: altered barrier functions in healthy relatives?.Gastroenterology. 1993; 104: 1848-1851Abstract PubMed Google Scholar, 13Buhner S. Buning C. Genschel J. Kling K. Herrmann D. Dignass A. Kuechler I. Krueger S. Schmidt H.H. Lochs H. Genetic basis for increased intestinal permeability in families with Crohn’s disease: role of CARD15 3020insC mutation?.Gut. 2006; 55: 342-347Crossref PubMed Scopus (264) Google Scholar, 14Suenaert P. Bulteel V. Vermeire S. Noman M. Van Assche G. Rutgeerts P. Hyperresponsiveness of the mucosal barrier in Crohn’s disease is not tumor necrosis factor-dependent.Inflamm Bowel Dis. 2005; 11: 667-673Crossref PubMed Scopus (33) Google Scholar it is also clear that epithelial barrier defects can be induced by inflammatory cytokines.15Hollander D. Crohn’s disease, TNF-alpha, and the leaky gut The chicken or the egg?.Am J Gastroenterol. 2002; 97: 1867-1868Crossref PubMed Scopus (33) Google Scholar, 16Suenaert P. Bulteel V. Lemmens L. Noman M. Geypens B. Van Assche G. Geboes K. Ceuppens J.L. Rutgeerts P. Anti-tumor necrosis factor treatment restores the gut barrier in Crohn’s disease.Am J Gastroenterol. 2002; 97: 2000-2004Crossref PubMed Google Scholar, 17Heller F. Florian P. Bojarski C. Richter J. Christ M. Hillenbrand B. Mankertz J. Gitter A.H. Burgel N. Fromm M. Zeitz M. Fuss I. Strober W. Schulzke J.D. Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution.Gastroenterology. 2005; 129: 550-564PubMed Google Scholar, 18Prasad S. Mingrino R. Kaukinen K. Hayes K.L. Powell R.M. MacDonald T.T. Collins J.E. Inflammatory processes have differential effects on Claudins 2, 3 and 4 in colonic epithelial cells.Lab Invest. 2005; 85: 1139-1162Crossref PubMed Scopus (345) Google Scholar, 19Bruewer M. Luegering A. Kucharzik T. Parkos C.A. Madara J.L. Hopkins A.M. Nusrat A. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms.J Immunol. 2003; 171: 6164-6172PubMed Google Scholar, 20Clayburgh D.R. Musch M.W. Leitges M. Fu Y.X. Turner J.R. Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo.J Clin Invest. 2006; 116: 2682-2694Crossref PubMed Scopus (172) Google Scholar, 21Nusrat A. Turner J.R. Madara J.L. Molecular physiology and pathophysiology of tight junctions IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G851-G857PubMed Google Scholar, 22Utech M. Ivanov A.I. Samarin S.N. Bruewer M. Turner J.R. Mrsny R.J. Parkos C.A. Nusrat A. Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: Myosin II-dependent vacuolarization of the apical plasma membrane.Mol Biol Cell. 2005; 16: 5040-5052Crossref PubMed Scopus (272) Google Scholar, 23Mullin J.M. Snock K.V. Effect of tumor necrosis factor on epithelial tight junctions and transepithelial permeability.Cancer Res. 1990; 50: 2172-2176PubMed Google Scholar, 24Taylor C.T. Dzus A.L. Colgan S.P. Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha.Gastroenterology. 1998; 114: 657-668Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar Recent work has shown that barrier dysfunction induced by inflammatory processes can be due to epithelial damage as well as nonapoptotic regulation of tight junction permeability.19Bruewer M. Luegering A. Kucharzik T. Parkos C.A. Madara J.L. Hopkins A.M. Nusrat A. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms.J Immunol. 2003; 171: 6164-6172PubMed Google Scholar, 22Utech M. Ivanov A.I. Samarin S.N. Bruewer M. Turner J.R. Mrsny R.J. Parkos C.A. Nusrat A. Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: Myosin II-dependent vacuolarization of the apical plasma membrane.Mol Biol Cell. 2005; 16: 5040-5052Crossref PubMed Scopus (272) Google Scholar, 25Abreu M.T. Palladino A.A. Arnold E.T. Kwon R.S. McRoberts J.A. Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.Gastroenterology. 2000; 119: 1524-1536Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 26Gitter A.H. Bendfeldt K. Schulzke J.D. Fromm M. Leaks in the epithelial barrier caused by spontaneous and TNF-alpha-induced single-cell apoptosis.FASEB J. 2000; 14: 1749-1753Crossref PubMed Scopus (226) Google Scholar, 27Gitter A.H. Wullstein F. Fromm M. Schulzke J.D. Epithelial barrier defects in ulcerative colitis: characterization and quantification by electrophysiological imaging.Gastroenterology. 2001; 121: 1320-1328Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 28Blair S.A. Kane S.V. Clayburgh D.R. Turner J.R. Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease.Lab Invest. 2006; 86: 191-201Crossref PubMed Scopus (218) Google Scholar In vitro and in vivo studies have shown that TNF signals directly to intestinal epithelia to regulate barrier function via myosin light chain kinase (MLCK) activation.20Clayburgh D.R. Musch M.W. Leitges M. Fu Y.X. Turner J.R. Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo.J Clin Invest. 2006; 116: 2682-2694Crossref PubMed Scopus (172) Google Scholar, 29Ma T.Y. Boivin M.A. Ye D. Pedram A. Said H.M. Mechanism of TNF-{alpha} modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G422-G430Crossref PubMed Scopus (362) Google Scholar, 30Wang F. Graham W.V. Wang Y. Witkowski E.D. Schwarz B.T. Turner J.R. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression.Am J Pathol. 2005; 166: 409-419Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 31Wang F. Schwarz B.T. Graham W.V. Wang Y. Su L. Clayburgh D.R. Abraham C. Turner J.R. IFN-gamma-induced TNFR2 upregulation is required for TNF-dependent intestinal epithelial barrier dysfunction.Gastroenterology. 2006; 131: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 32Zolotarevsky Y. Hecht G. Koutsouris A. Gonzalez D.E. Quan C. Tom J. Mrsny R.J. Turner J.R. A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.Gastroenterology. 2002; 123: 163-172Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 33Clayburgh D.R. Barrett T.A. Tang Y. Meddings J.B. Van Eldik L.J. Watterson D.M. Clarke L.L. Mrsny R.J. Turner J.R. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo.J Clin Invest. 2005; 115: 2702-2715Crossref PubMed Scopus (318) Google Scholar We recently reported that acute LIGHT administration also causes MLCK-dependent intestinal epithelial barrier dysfunction in vivo.20Clayburgh D.R. Musch M.W. Leitges M. Fu Y.X. Turner J.R. Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo.J Clin Invest. 2006; 116: 2682-2694Crossref PubMed Scopus (172) Google Scholar However, due to the complexities of the in vivo system used, these data could not discriminate between direct effects of LIGHT on intestinal epithelia and those mediated by intermediates, such as TNF or immune cells. Thus, although some reports suggest that LIGHT may be capable of signaling to epithelial-derived cancer cells,34Zhang M. Guo R. Zhai Y. Yang D. LIGHT sensitizes IFNgamma-mediated apoptosis of MDA-MB-231 breast cancer cells leading to down-regulation of anti-apoptosis Bcl-2 family members.Cancer Lett. 2003; 195: 201-210Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar direct LIGHT signaling to epithelia has not been explored or considered in intestinal disease.The aim of this study was to determine if LIGHT is capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling. The data show that LIGHT signals directly to intestinal epithelia via the lymphotoxin β receptor (LTβR). This induces both transcriptional and enzymatic MLCK activation and results in caveolar endocytosis of tight junction components, including occludin. In addition to demonstrating LIGHT-mediated barrier regulation, these data are the first to show a functional requirement for endocytosis during cytokine-induced barrier dysfunction.Materials and MethodsMonolayer Preparation and Transepithelial Electrical Resistance MeasurementCaco-2BBE cell35Turner J.R. Rill B.K. Carlson S.L. Carnes D. Kerner R. Mrsny R.J. Madara J.L. Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.Am J Physiol. 1997; 273: C1378-C1385PubMed Google Scholar, 36Peterson M.D. Mooseker M.S. Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2.J Cell Sci. 1992; 102: 581-600Crossref PubMed Google Scholar cultures were grown as monolayers on collagen-coated polycarbonate membrane Transwell supports (Corning, Cambridge, MA) with 0.4-μm pores for 17–20 days after confluence, as described previously.30Wang F. Graham W.V. Wang Y. Witkowski E.D. Schwarz B.T. Turner J.R. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression.Am J Pathol. 2005; 166: 409-419Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar Transwell supports with 0.33- and 5-cm2 surface areas were used for electrophysiologic and biochemical studies, respectively. Cytokines (R&D Systems, Minneapolis, MN) were added to the basal chamber without manipulating the apical media unless otherwise specified. Sulfasalazine (MP Biochemicals, Aurora, OH), curcumin (Calbiochem, San Diego, CA), BAY 11-7085 (Calbiochem), MG132 (Calbiochem), chlorpromazine (Sigma Chemical Co, St Louis, MO), amiloride (Sigma Chemical Co), methyl-β-cyclodextrin (Sigma Chemical Co), and monodansylcadaverine (Sigma Chemical Co) were added to apical and basal chambers. Transepithelial resistance (TER) was measured with an epithelial voltohmmeter under open-circuit conditions (World Precision Instruments, Sarasota, FL) as described previously.30Wang F. Graham W.V. Wang Y. Witkowski E.D. Schwarz B.T. Turner J.R. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression.Am J Pathol. 2005; 166: 409-419Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar TER averaged 250 Ω · cm2, after subtraction of a blank that includes filter and fluid resistances, before cytokine treatment. To facilitate comparisons between experiments, the TER of all monolayers was normalized to that of control monolayers in the same experiment.Sodium Dodecyl Sulfate/Polyacrylamide Gel Electrophoresis and ImmunoblotMonolayers were scraped directly into 0.5 mL sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, sonicated, separated on SDS-PAGE gels (Cambrex, Rockland, ME), and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). Lysates of isolated colonocytes were processed similarly.33Clayburgh D.R. Barrett T.A. Tang Y. Meddings J.B. Van Eldik L.J. Watterson D.M. Clarke L.L. Mrsny R.J. Turner J.R. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo.J Clin Invest. 2005; 115: 2702-2715Crossref PubMed Scopus (318) Google Scholar Immunoblots were performed using antibodies specific for MLCK (clone K36; Sigma Chemical Co), total myosin II regulatory light chain (MLC),33Clayburgh D.R. Barrett T.A. Tang Y. Meddings J.B. Van Eldik L.J. Watterson D.M. Clarke L.L. Mrsny R.J. Turner J.R. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo.J Clin Invest. 2005; 115: 2702-2715Crossref PubMed Scopus (318) Google Scholar phosphorylated MLC,37Berglund J.J. Riegler M. Zolotarevsky Y. Wenzl E. Turner J.R. Regulation of human jejunal transmucosal resistance and MLC phosphorylation by Na-glucose cotransport.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G1487-G1493PubMed Google Scholar ZO-1 (Invitrogen, Carlsbad, CA), occludin (Invitrogen), claudin-1 (Invitrogen), caspase-3 (Cell Signaling Technology, Beverly, MA), caspase-8 (Cell Signaling Technology), herpes virus entry mediator (HVEM; R&D Systems), and LTβR (R&D Systems). After incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology), blots were visualized by enhanced chemiluminescence using Super Signal West Pico Reagents (Pierce Biotechnology Inc, Rockford, IL). Quantitative analysis was performed using Metamorph 6.2 (Molecular Devices Corp, Downingtown, PA).Real-Time Reverse-Transcription Polymerase Chain ReactionMonolayers were scraped directly into TRIzol and sonicated. RNA was extracted and further purified as described previously.38Graham W.V. Wang F. Clayburgh D.R. Cheng J.X. Yoon B. Wang Y. Lin A. Turner J.R. Tumor necrosis factor-induced long myosin light chain kinase transcription is regulated by differentiation-dependent signaling events Characterization of the human long myosin light chain kinase promoter.J Biol Chem. 2006; 281: 26205-26215Crossref PubMed Scopus (111) Google Scholar Long (epithelial) MLCK messenger RNA expression was determined by SYBR green real-time polymerase chain reaction using the MyiQ Real-Time PCR Detection System (Bio-Rad Laboratories), as described previously.38Graham W.V. Wang F. Clayburgh D.R. Cheng J.X. Yoon B. Wang Y. Lin A. Turner J.R. Tumor necrosis factor-induced long myosin light chain kinase transcription is regulated by differentiation-dependent signaling events Characterization of the human long myosin light chain kinase promoter.J Biol Chem. 2006; 281: 26205-26215Crossref PubMed Scopus (111) Google Scholar Glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard for normalization.In Vivo StudiesSeven- to 10-week-old wild-type, HVEM−/−,39Wang Y. Subudhi S.K. Anders R.A. Lo J. Sun Y. Blink S. Wang J. Liu X. Mink K. Degrandi D. Pfeffer K. Fu Y.X. The role of herpesvirus entry mediator as a negative regulator of T cell-mediated responses.J Clin Invest. 2005; 115: 711-717Crossref PubMed Scopus (150) Google Scholar and LTβR−/−40 mice on C57BL/6 genetic background, as described previously,39Wang Y. Subudhi S.K. Anders R.A. Lo J. Sun Y. Blink S. Wang J. Liu X. Mink K. Degrandi D. Pfeffer K. Fu Y.X. The role of herpesvirus entry mediator as a negative regulator of T cell-mediated responses.J Clin Invest. 2005; 115: 711-717Crossref PubMed Scopus (150) Google Scholar, 40Futterer A. Mink K. Luz A. Kosco-Vilbois M.H. Pfeffer K. The lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues.Immunity. 1998; 9: 59-70Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar were used for all studies. Knockout mice were generously provided by Klaus Pfeffer (Technical University of Munich, Munich, Germany). Genotypes were confirmed by polymerase chain reaction, as described.39Wang Y. Subudhi S.K. Anders R.A. Lo J. Sun Y. Blink S. Wang J. Liu X. Mink K. Degrandi D. Pfeffer K. Fu Y.X. The role of herpesvirus entry mediator as a negative regulator of T cell-mediated responses.J Clin Invest. 2005; 115: 711-717Crossref PubMed Scopus (150) Google Scholar, 40Futterer A. Mink K. Luz A. Kosco-Vilbois M.H. Pfeffer K. The lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues.Immunity. 1998; 9: 59-70Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar Animal experiments were performed in accordance with National Institutes of Health guidelines under protocols approved by the Institutional Animal Care and Use Committee at the University of Chicago.Mice were injected intraperitoneally with either 5 μg TNF or 5 μg LIGHT in 250 μL phosphate-buffered saline (PBS) or with PBS alone, as described previously.20Clayburgh D.R. Musch M.W. Leitges M. Fu Y.X. Turner J.R. Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo.J Clin Invest. 2006; 116: 2682-2694Crossref PubMed Scopus (172) Google Scholar To determine paracellular permeability, 250 μL of 1 mg/mL Alexa 488 conjugated bovine serum albumin (BSA; Invitrogen) was injected intravenously.33Clayburgh D.R. Barrett T.A. Tang Y. Meddings J.B. Van Eldik L.J. Watterson D.M. Clarke L.L. Mrsny R.J. Turner J.R. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo.J Clin Invest. 2005; 115: 2702-2715Crossref PubMed Scopus (318) Google Scholar A ∼5-cm loop of jejunum was cannulated and perfused from 1.5 to 3.5 hours after cytokine injection. BSA clearance was calculated as described previously.20Clayburgh D.R. Musch M.W. Leitges M. Fu Y.X. Turner J.R. Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo.J Clin Invest. 2006; 116: 2682-2694Crossref PubMed Scopus (172) Google ScholarTo isolate intestinal epithelial cells, a fresh section of jejunum was opened lengthwise, washed in 4°C Ca2+- and Mg+-free Hanks’ buffered saline solution, transferred to Ca2+- and Mg+-free Hanks’ buffered saline solution containing 10 mmol/L dithiothreitol and 50 nmol/L calyculin A (Calbiochem), and incubated for 30 minutes at 4°C. After incubation, the tube was shaken briefly and the tissue transferred to a fresh tube containing Ca2+- and Mg+-free Hanks’ buffered saline solution with 1 mmol/L EDTA and 50 nmol/L calyculin A. After 1 hour, epithelial cells were dislodged by vigorous shaking and large pieces of tissue were removed from the tube and discarded. Epithelial cells were harvested by centrifugation at 500g for 10 minutes, and pellets were resuspended in SDS-PAGE sample buffer.ImmunofluorescenceCultured monolayers were fixed with 1% paraformaldehyde in PBS, pH 7.4, with 1 mmol/L CaCl2 for 30 minutes at room temperature. After 3 washes in PBS and a 10-minute incubation in PBS with 50 mmol/L NH4Cl, cells were permeabilized in PBS with 3% BSA and 0.05% saponin (wash buffer) in five 5-minute incubations. Monolayers were then incubated with anti–claudin-1, anti–ZO-1, or anti-occludin antibodies in wash buffer for 2 hours, washed 5 times, and incubated for 1 hour with appropriate Alexa Fluor 488– or 594–conjugated secondary antibodies (Invitrogen) and Hoechst 33342 (Invitrogen). After 5 washes, monolayers were rinsed in water and mounted in Prolong Gold (Invitrogen).For colocalization studies, monolayers were fixed in methanol overnight at −20°C, air dried, rehydrated with 100 μmol/L bis(sulfosuccinimidyl)suberate in PBS with 0.1% n-octyl-glutaraldehyde (PBS+) for 30 minutes, washed in PBS+, quenched in 100 mmol/L ethylenediamine, pH 7.5, and washed once more in PBS+. Monolayers were then blocked in 1% nonfat dry milk, 1% fish gelatin, and 1% normal donkey serum in PBS+ for 1 hour, incubated with mouse anti-occludin and either rabbit anti–caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-clathrin heavy chain antibodies (Santa Cruz Biotechnology) for 2 hours, washed, and incubated with Alexa Fluor–conjugated secondary antibodies for 1 hour. After 5 washes, monolayers were rinsed in water and mounted in Prolong Gold.For immunofluorescence of mouse jejunum, tissues were snap frozen in OCT and stored at −80°C. Frozen sections (5 μm) were collected on coated slides, fixed in 1% PFA, washed 3 times with PBS, and blocked and quenched for 30 minutes in PBS with 10% normal goat serum and 50 mmol/L NH4Cl. After incubation for 2 hours with rabbit anti-occludin (Invitrogen), sections were washed and incubated for 1 hour with Alexa 594–conjugated goat anti-rabbit immunoglobulin and Alexa 488–conjugated phalloidin (Invitrogen). Stained sections were mounted using Prolong Gold antifade reagent (Invitrogen, Eugene, OR).Samples were imaged using a Leica DMLB epifluorescence microscope equipped with 63× and 100× PL-APO objectives, an 88000 filter set (Chroma Technology, Brattleboro, VT), and a Retiga EXi camera (Q Imaging, Burnaby, British Columbia, Canada) controlled by MetaMorph. Monolayers were imaged as z-stacks at 0.2-μm intervals and deconvolved using Autodeblur 9.3 (Media Cybernetics, Silver Spring, MD) for 10 iterations.MorphometryDeconvolved z-stacks were merged, after pseudocolor assignment, using MetaMorph. Vesicles were defined as round or oval structures present in 3 or more z-planes. The number of vesicles in a single cell was counted over the full height of the cell. Signals were considered to colocalize if there was ≥80% overlap between channels. For each measurement, 15 randomly chosen average-shaped and -sized cells were counted.Statistical AnalysisAll data are pre" @default.
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• W2022364566 date "2007-06-01" @default.
• W2022364566 modified "2023-10-12" @default.
• W2022364566 title "LIGHT Signals Directly to Intestinal Epithelia to Cause Barrier Dysfunction via Cytoskeletal and Endocytic Mechanisms" @default.
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