Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene
2019; Elsevier BV; Volume: 189; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2018.12.016
ISSN1525-2191
AutoresMeghali Nighot, Manmeet Rawat, Rana Al–Sadi, Eliseo F. Castillo, Prashant K. Nighot, Y. Thomas,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoLipopolysaccharides (LPSs) are a major component of Gram-negative bacterial cell wall and play an important role in promoting intestinal inflammatory responses. Recent studies have shown that physiologically relevant concentrations of LPS (0 to 2000 pg/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability without causing cell death. However, the intracellular pathways and the mechanisms that mediate LPS-induced increase in intestinal TJ permeability remain unclear. The aim was to delineate the intracellular pathways that mediate the LPS-induced increase in intestinal permeability using in vitro and in vivo intestinal epithelial models. LPS-induced increase in intestinal epithelial TJ permeability was preceded by an activation of transforming growth factor-β–activating kinase-1 (TAK-1) and canonical NF-κB (p50/p65) pathways. The siRNA silencing of TAK-1 inhibited the activation of NF-κB p50/p65. The siRNA silencing of TAK-1 and p65/p50 subunit inhibited the LPS-induced increase in intestinal TJ permeability and the increase in myosin light chain kinase (MLCK) expression, confirming the regulatory role of TAK-1 and NF-κB p65/p50 in up-regulating MLCK expression and the subsequent increase in TJ permeability. The data also showed that toll-like receptor (TLR)-4/myeloid differentiation primary response (MyD)88 pathway was crucial upstream regulator of TAK-1 and NF-κB p50/p65 activation. In conclusion, activation of TAK-1 by the TLR-4/MyD88 signal transduction pathway and MLCK by NF-κB p65/p50 regulates the LPS-induced increase in intestinal epithelial TJ permeability. Lipopolysaccharides (LPSs) are a major component of Gram-negative bacterial cell wall and play an important role in promoting intestinal inflammatory responses. Recent studies have shown that physiologically relevant concentrations of LPS (0 to 2000 pg/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability without causing cell death. However, the intracellular pathways and the mechanisms that mediate LPS-induced increase in intestinal TJ permeability remain unclear. The aim was to delineate the intracellular pathways that mediate the LPS-induced increase in intestinal permeability using in vitro and in vivo intestinal epithelial models. LPS-induced increase in intestinal epithelial TJ permeability was preceded by an activation of transforming growth factor-β–activating kinase-1 (TAK-1) and canonical NF-κB (p50/p65) pathways. The siRNA silencing of TAK-1 inhibited the activation of NF-κB p50/p65. The siRNA silencing of TAK-1 and p65/p50 subunit inhibited the LPS-induced increase in intestinal TJ permeability and the increase in myosin light chain kinase (MLCK) expression, confirming the regulatory role of TAK-1 and NF-κB p65/p50 in up-regulating MLCK expression and the subsequent increase in TJ permeability. The data also showed that toll-like receptor (TLR)-4/myeloid differentiation primary response (MyD)88 pathway was crucial upstream regulator of TAK-1 and NF-κB p50/p65 activation. In conclusion, activation of TAK-1 by the TLR-4/MyD88 signal transduction pathway and MLCK by NF-κB p65/p50 regulates the LPS-induced increase in intestinal epithelial TJ permeability. Intestinal epithelial tight junction (TJ) barrier dysfunction contributes to the development of inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC) by permitting increased paracellular permeation of luminal antigens that elicit and promote inflammatory response.1Turner J.R. Intestinal mucosal barrier function in health and disease.Nat Rev Immunol. 2009; 9: 799-809Crossref PubMed Scopus (2316) Google Scholar, 2Turner J.R. Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application.Am J Pathol. 2006; 169: 1901-1909Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 3Clark J.A. Doelle S.M. Halpern M.D. Saunders T.A. Holubec H. Dvorak K. Boitano S.A. Dvorak B. Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G938-G949Crossref PubMed Scopus (217) Google Scholar Several clinical and animal studies have highlighted the involvement of defective intestinal TJ barrier in the development and the prolongation of intestinal inflammation in IBD and NEC.1Turner J.R. Intestinal mucosal barrier function in health and disease.Nat Rev Immunol. 2009; 9: 799-809Crossref PubMed Scopus (2316) Google Scholar, 3Clark J.A. Doelle S.M. Halpern M.D. Saunders T.A. Holubec H. Dvorak K. Boitano S.A. Dvorak B. Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G938-G949Crossref PubMed Scopus (217) Google Scholar, 4Oriishi T. Sata M. Toyonaga A. Sasaki E. Tanikawa K. Evaluation of intestinal permeability in patients with inflammatory bowel disease using lactulose and measuring antibodies to lipid A.Gut. 1995; 36: 891-896Crossref PubMed Scopus (39) Google Scholar, 5Arrieta M.C. Madsen K. Doyle J. Meddings J. Reducing small intestinal permeability attenuates colitis in the IL10 gene-deficient mouse.Gut. 2009; 58: 41-48Crossref PubMed Scopus (222) Google Scholar Patients with IBD and NEC have a defective intestinal TJ barrier as shown by an increase in intestinal permeability,5Arrieta M.C. Madsen K. Doyle J. Meddings J. Reducing small intestinal permeability attenuates colitis in the IL10 gene-deficient mouse.Gut. 2009; 58: 41-48Crossref PubMed Scopus (222) Google Scholar, 6Benoit R. Rowe S. Watkins S.C. Boyle P. Garrett M. Alber S. Wiener J. Rowe M.I. Ford H.R. Pure endotoxin does not pass across the intestinal epithelium in vitro.Shock. 1998; 10: 43-48Crossref PubMed Scopus (40) Google Scholar and animal studies have shown that protecting the intestinal TJ barrier prevents the development of intestinal inflammation in animal models of IBD and NEC.7Mennigen R. Nolte K. Rijcken E. Utech M. Loeffler B. Senninger N. Bruewer M. Probiotic mixture VSL#3 protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in a murine model of colitis.Am J Physiol Gastrointest Liver Physiol. 2009; 296: G1140-G1149Crossref PubMed Scopus (375) Google Scholar, 8Ge Y. Ezzell R.M. Warren H.S. Localization of endotoxin in the rat intestinal epithelium.J Infect Dis. 2000; 182: 873-881Crossref PubMed Scopus (32) Google Scholar Lipopolysaccharides (LPSs) are complex amphiphilic molecules that have a hydrophobic (consisting of lipid A) and a hydrophilic (consisting of carbohydrate core and polysaccharide O-antigen) component and are released from the bacterial cell wall by shedding or through bacterial lysis. In pathologic conditions, the intestinal tissue and circulating LPS levels are markedly elevated and play an important role in mediating inflammatory response. Normally, LPS concentrations are maximum in the gut lumen where the gut bacteria reside and low or undetectable in the circulating plasma (<200 pg/mL) because the intestinal epithelial layer creates an effective barrier against LPS penetration across the healthy intestinal epithelium.6Benoit R. Rowe S. Watkins S.C. Boyle P. Garrett M. Alber S. Wiener J. Rowe M.I. Ford H.R. Pure endotoxin does not pass across the intestinal epithelium in vitro.Shock. 1998; 10: 43-48Crossref PubMed Scopus (40) Google Scholar, 9Hurley J.C. Endotoxemia: methods of detection and clinical correlates.Clin Microbiol Rev. 1995; 8: 268-292Crossref PubMed Google Scholar However, defective intestinal TJ barrier in IBD and NEC leads to paracellular permeation of LPS and other water-soluble luminal antigens, resulting in an increase in the intestinal tissue and plasma concentration of LPS.10Andreasen A.S. Krabbe K.S. Krogh-Madsen R. Taudorf S. Pedersen B.K. Moller K. Human endotoxemia as a model of systemic inflammation.Curr Med Chem. 2008; 15: 1697-1705Crossref PubMed Scopus (213) Google Scholar, 11Wellmann W. Fink P.C. Benner F. Schmidt F.W. Endotoxaemia in active Crohn's disease. Treatment with whole gut irrigation and 5-aminosalicylic acid.Gut. 1986; 27: 814-820Crossref PubMed Scopus (101) Google Scholar Studies have shown LPS to be an important contributing factor of intestinal inflammation, and removal of circulating LPS accelerates the clinical improvement of IBD and NEC. Clinically achievable concentrations of LPS during active and quiescent IBD and NEC ranges between 200 and 2000 pg/mL. Animal models of NEC and IBD also have increased levels of LPS in the intestinal tissue and in the serum.11Wellmann W. Fink P.C. Benner F. Schmidt F.W. Endotoxaemia in active Crohn's disease. Treatment with whole gut irrigation and 5-aminosalicylic acid.Gut. 1986; 27: 814-820Crossref PubMed Scopus (101) Google Scholar, 12Sharma R. Tepas III, J.J. Hudak M.L. Mollitt D.L. Wludyka P.S. Teng R.J. Premachandra B.R. Neonatal gut barrier and multiple organ failure: role of endotoxin and proinflammatory cytokines in sepsis and necrotizing enterocolitis.J Pediatr Surg. 2007; 42: 454-461Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 13Ma T.Y. Anderson J.M. Tight junctions and the intestinal barrier.in: Physiology of the Gastrointestinal Tract. ed 4. Elsevier Academic Press, Burlington, MA2006Crossref Scopus (48) Google Scholar The intestinal tissue expression of toll-like receptor-4 (TLR-4), the pattern recognition receptor that binds the LPS, is also markedly up-regulated in IBD and NEC.14Leal R.F. Milanski M. Ayrizono Mde L. Coope A. Rodrigues V.S. Portovedo M. Oliveira L.M. Fagundes J.J. Coy C.S. Velloso L.A. Toll-like receptor 4, F4/80 and pro-inflammatory cytokines in intestinal and mesenteric fat tissue of Crohn's disease.Int J Clin Exp Med. 2013; 6: 98-104PubMed Google Scholar, 15Le Mandat Schultz A. Bonnard A. Barreau F. Aigrain Y. Pierre-Louis C. Berrebi D. Peuchmaur M. Expression of TLR-2, TLR-4, NOD2 and pNF-kappaB in a neonatal rat model of necrotizing enterocolitis.PLoS One. 2007; 2: e1102Crossref PubMed Scopus (58) Google Scholar The TLR-4 polymorphism is associated with an increased risk of IBD and a more extensive colonic involvement in ulcerative colitis.16De Jager P.L. Franchimont D. Waliszewska A. Bitton A. Cohen A. Langelier D. Belaiche J. Vermeire S. Farwell L. Goris A. Libioulle C. Jani N. Dassopoulos T. Bromfield G.P. Dubois B. Cho J.H. Brant S.R. Duerr R.H. Yang H. Rotter J.I. Silverberg M.S. Steinhart A.H. Daly M.J. Podolsky D.K. Louis E. Hafler D.A. Rioux J.D. Quebec IBD Genetics ConsortiumNIDDK IBD Genetics ConsortiumThe role of the Toll receptor pathway in susceptibility to inflammatory bowel diseases.Genes Immun. 2007; 8: 387-397Crossref PubMed Scopus (121) Google Scholar Intestinal TJ barrier is primarily regulated by myosin light chain kinase (MLCK).13Ma T.Y. Anderson J.M. Tight junctions and the intestinal barrier.in: Physiology of the Gastrointestinal Tract. ed 4. Elsevier Academic Press, Burlington, MA2006Crossref Scopus (48) Google Scholar, 17Ma T.Y. Tran D. Hoa N. Nguyen D. Merryfield M. Tarnawski A. Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement.Microsc Res Tech. 2000; 51: 156-168Crossref PubMed Scopus (113) Google Scholar, 18Ma T.Y. Hoa N.T. Tran D.D. Bui V. Pedram A. Mills S. Merryfield M. Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G875-G885Crossref PubMed Google Scholar, 19Nighot M. Al-Sadi R. Guo S.H. Rawat M. Nighot P. Watterson M.D. Ma T.Y. Lipopolysaccharide-induced increase in intestinal epithelial tight permeability is mediated by Toll-like receptor 4/myeloid differentiation primary response 88 (MyD88) activation of myosin light chain kinase expression.Am J Pathol. 2017; 187: 2698-2710Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 20Ma 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 (370) Google Scholar, 21Yu L.C. Flynn A.N. Turner J.R. Buret A.G. SGLT-1-mediated glucose uptake protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a novel cellular rescue mechanism?.FASEB J. 2005; 19: 1822-1835Crossref PubMed Scopus (115) Google Scholar, 22Turner 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-C1385Crossref PubMed Google Scholar MLCK causes the contraction of the peri-junctional actomyosin filaments, leading to a mechanical tension-generated opening of the TJ barrier.17Ma T.Y. Tran D. Hoa N. Nguyen D. Merryfield M. Tarnawski A. Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement.Microsc Res Tech. 2000; 51: 156-168Crossref PubMed Scopus (113) Google Scholar, 18Ma T.Y. Hoa N.T. Tran D.D. Bui V. Pedram A. Mills S. Merryfield M. Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G875-G885Crossref PubMed Google Scholar, 22Turner 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-C1385Crossref PubMed Google Scholar, 23Turner J.R. Buschmann M.M. Romero-Calvo I. Sailer A. Shen L. The role of molecular remodeling in differential regulation of tight junction permeability.Semin Cell Dev Biol. 2014; 36: 204-212Crossref PubMed Scopus (137) Google Scholar The intestinal TJ permeability can be increased by physiological concentrations of LPS (0 to 1000 pg/mL) via activation of TLR-4 signal transduction pathway and an increase in MLCK expression and activity.19Nighot M. Al-Sadi R. Guo S.H. Rawat M. Nighot P. Watterson M.D. Ma T.Y. Lipopolysaccharide-induced increase in intestinal epithelial tight permeability is mediated by Toll-like receptor 4/myeloid differentiation primary response 88 (MyD88) activation of myosin light chain kinase expression.Am J Pathol. 2017; 187: 2698-2710Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24Guo S. Al-Sadi R. Said H.M. Ma T.Y. Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14.Am J Pathol. 2013; 182: 375-387Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 25Guo S. Nighot M. Al-Sadi R. Alhmoud T. Nighot P. Ma T.Y. Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR4 signal transduction pathway activation of FAK and MyD88.J Immunol. 2015; 195: 4999-5010Crossref PubMed Scopus (235) Google Scholar NF-κB is a nuclear transcription factor that plays an essential regulatory role in promoting intestinal inflammation. In quiescent cells, NF-κB heterodimers p50/p65 are sequestered in cytosol by a protein inhibitor inhibitory κ B (IκB). The IκB kinase (IKK) complex consists of two catalytic subunits, IKK-β and IKK-α, and a regulatory protein, IKK-γ (alias NF-κB essential modulator). Two distinct NF-κB pathways have been described, the canonical (or classic) and the noncanonical (or alternative) pathways that are involved in NF-κB activation.26Kim J.-Y. Morgan M. Kim D.-G. Lee J.-Y. Bai L. Lin Y. Liu Z.-g. Kim Y.-S. TNFα-induced noncanonical NF-κB activation is attenuated by RIP1 through stabilization of TRAF2.J Cell Sci. 2011; 124: 647-656Crossref PubMed Scopus (46) Google Scholar, 27Zarnegar B. Yamazaki S. He J.Q. Cheng G. Control of canonical NF-kappaB activation through the NIK–IKK complex pathway.Proc Natl Acad Sci U S A. 2008; 105: 3503-3508Crossref PubMed Scopus (151) Google Scholar, 28Ramakrishnan P. Wang W. Wallach D. Receptor-specific signaling for both the alternative and the canonical NF-kappaB activation pathways by NF-kappaB-inducing kinase.Immunity. 2004; 21: 477-489Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 29Baldwin Jr., A.S. The NF-kappaB and I kappa B proteins: new discoveries and insights.Annu Rev Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5572) Google Scholar, 30Ghosh S. May M.J. Kopp E.B. NF-kappa B and REL proteins: evolutionarily conserved mediators of immune responses.Annu Rev Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4605) Google Scholar, 31Pomerantz J.L. Baltimore D. Two pathways to NF-kappaB.Mol Cell. 2002; 10: 693-695Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar The activation of the canonical pathway leads to the phosphorylation and degradation of inhibitory IκB-α and activation of NF-κB dimer p50/p65,28Ramakrishnan P. Wang W. Wallach D. Receptor-specific signaling for both the alternative and the canonical NF-kappaB activation pathways by NF-kappaB-inducing kinase.Immunity. 2004; 21: 477-489Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 32Widera D. Mikenberg I. Elvers M. Kaltschmidt C. Kaltschmidt B. Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling.BMC Neurosci. 2006; 7: 64Crossref PubMed Scopus (183) Google Scholar whereas the activation of the noncanonical pathway results in the phosphorylation of the p100 subunit, leading to the generation and activation of RelB/p52 dimer.28Ramakrishnan P. Wang W. Wallach D. Receptor-specific signaling for both the alternative and the canonical NF-kappaB activation pathways by NF-kappaB-inducing kinase.Immunity. 2004; 21: 477-489Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 33Sun S.-C. The non-canonical NF-[kappa]B pathway in immunity and inflammation.Nat Rev Immunol. 2017; 17: 545-558Crossref PubMed Scopus (854) Google Scholar, 34Al-Sadi R. Guo S. Ye D. Rawat M. Ma T.Y. TNF-alpha modulation of intestinal tight junction permeability is mediated by NIK/IKK-alpha axis activation of the canonical NF-kappaB pathway.Am J Pathol. 2016; 186: 1151-1165Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar NF-κB activation can be triggered by a wide range of proinflammatory mediators, including tumor necrosis factor-α, IL-1β, interferon-γ, and LPS. LPS binds to TLR-4 complex at the cell surface, stimulating a cascade of intracellular signaling events that culminate in the phosphorylation of IκB-α. The LPS activation of both canonical and noncanonical NF-κB pathways has been shown in various models with LPS.35Guha M. Mackman N. LPS induction of gene expression in human monocytes.Cell Signal. 2001; 13: 85-94Crossref PubMed Scopus (1975) Google Scholar, 36Gay N.J. Gangloff M. Structure and function of Toll receptors and their ligands.Annu Rev Biochem. 2007; 76: 141-165Crossref PubMed Scopus (508) Google Scholar An important concern with the previous studies is that the LPS concentration used in those studies were at a pharmacologic concentration of 50 μg/mL, which is nonphysiologic and not achievable clinically.35Guha M. Mackman N. LPS induction of gene expression in human monocytes.Cell Signal. 2001; 13: 85-94Crossref PubMed Scopus (1975) Google Scholar, 36Gay N.J. Gangloff M. Structure and function of Toll receptors and their ligands.Annu Rev Biochem. 2007; 76: 141-165Crossref PubMed Scopus (508) Google Scholar At a concentration of 50 μg/mL, LPS causes rapid cell apoptosis and cell death.21Yu L.C. Flynn A.N. Turner J.R. Buret A.G. SGLT-1-mediated glucose uptake protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a novel cellular rescue mechanism?.FASEB J. 2005; 19: 1822-1835Crossref PubMed Scopus (115) Google Scholar, 37Forsythe R.M. Xu D.Z. Lu Q. Deitch E.A. Lipopolysaccharide-induced enterocyte-derived nitric oxide induces intestinal monolayer permeability in an autocrine fashion.Shock. 2002; 17: 180-184Crossref PubMed Scopus (76) Google Scholar, 38Liu C. Li A. Weng Y.B. Duan M.L. Wang B.E. Zhang S.W. Changes in intestinal mucosal immune barrier in rats with endotoxemia.World J Gastroenterol. 2009; 15: 5843-5850Crossref PubMed Scopus (31) Google Scholar In contrast, clinically achievable concentrations of LPS (0 to 2000 pg/mL) do not cause cell death.10Andreasen A.S. Krabbe K.S. Krogh-Madsen R. Taudorf S. Pedersen B.K. Moller K. Human endotoxemia as a model of systemic inflammation.Curr Med Chem. 2008; 15: 1697-1705Crossref PubMed Scopus (213) Google Scholar, 11Wellmann W. Fink P.C. Benner F. Schmidt F.W. Endotoxaemia in active Crohn's disease. Treatment with whole gut irrigation and 5-aminosalicylic acid.Gut. 1986; 27: 814-820Crossref PubMed Scopus (101) Google Scholar, 24Guo S. Al-Sadi R. Said H.M. Ma T.Y. Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14.Am J Pathol. 2013; 182: 375-387Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar Transforming growth factor-β–activated kinase-1 (TAK-1) has been shown to be a pivotal factor for NF-κB activation in response to the activation of various toll-like receptors, including TLR-2, TLR-4, and TLR-5.39Sato S. Sanjo H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Essential function for the kinase TAK1 in innate and adaptive immune responses.Nat Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (759) Google Scholar The phosphorylation of TAK-1 is a common signal for the activation of downstream targets, including IKK, Jun kinase, and p38 mitogen-activated protein kinase.40Cuevas B.D. Abell A.N. Johnson G.L. Role of mitogen-activated protein kinase kinase kinases in signal integration.Oncogene. 2007; 26: 3159-3171Crossref PubMed Scopus (215) Google Scholar, 41Chen Z.J. Bhoj V. Seth R.B. Ubiquitin, TAK1 and IKK: is there a connection?.Cell Death Differ. 2006; 13: 687-692Crossref PubMed Scopus (102) Google Scholar Previously, it was shown that the LPS-induced increase in intestinal TJ permeability depended on activation of a TLR-4 signal transduction pathway.24Guo S. Al-Sadi R. Said H.M. Ma T.Y. Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14.Am J Pathol. 2013; 182: 375-387Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar However, the intracellular signaling processes that mediate the TLR-4 signal transduction pathway modulation of MLCK expression and TJ permeability remain unclear. Here, the involvement of TAK-1 and NF-κB pathways was examined in the LPS-induced increase in intestinal TJ permeability with the use of intestinal epithelial model systems that consisted of filter-grown Caco-2 monolayers and live mice. Dulbecco's Modified Eagle Medium, trypsin, fetal bovine serum, penicillin, streptomycin, and phosphate-buffered saline (PBS) were purchased from Gibco BRL (Grand Island, NY). LPS (O111:B4) and TAK-1 inhibitor, 5Z-7-oxozeaenol, NF-κB inhibitors, ammonium pyrrolidinedithiocarbamate (PDTC) and Bay-11, 5Z-7-oxozeaenol were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies [IκB-α, p65, phospho- (p)65, p50, p52, p100, IKK-α, IKK-β, TAK-1, pTAK-1, IL-1 receptor-associated kinase 4, phospho–mitogen-activated kinase kinase (pMEKK)-1, MEKK-1, phospho NF-κB–inducing kinase (pNIK), NIK, MLCK, pMLC, TLR-4, myeloid differentiation primary response (MyD)88, and anti–β-actin] were obtained from Abcam (Cambridge, MA). Phospho–IKK-α/β antibodies were also obtained from Abcam and Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase–conjugated secondary antibodies for Western blot analysis were purchased from Invitrogen (San Francisco, CA). All other chemicals were purchased from Sigma-Aldrich, VWR (West Chester, PA), or Fisher Scientific (Pittsburgh, PA). Curcumin, PDTC, and Bay-11 were purchased from Sigma-Aldrich, VWR (Aurora, CO), or Fisher Scientific. siRNA for p65, p50, p100, p52, IKK-α, IKK-β TAK-1, NIK, MEKK-1, MLCK, TLR-4, and MyD88 were purchased from Dharmacon (Lafayette, CO). Caco-2 cells (passage 20) were purchased from the ATCC (Manassas, VA) and maintained at 37°C in Dulbecco's Modified Eagle Medium composed of 4.5 mg/mL glucose, 50 U/mL penicillin, 50 U/mL streptomycin, 4 mmol/L glutamine, 25 mmol/L HEPES, and 10% fetal bovine serum as previously described.19Nighot M. Al-Sadi R. Guo S.H. Rawat M. Nighot P. Watterson M.D. Ma T.Y. Lipopolysaccharide-induced increase in intestinal epithelial tight permeability is mediated by Toll-like receptor 4/myeloid differentiation primary response 88 (MyD88) activation of myosin light chain kinase expression.Am J Pathol. 2017; 187: 2698-2710Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Caco-2 cells were used between passages 22 and 30 in this study. The cells were kept at 37°C in a 5% CO2 environment. For filter-grown cells, high-density cells (1 × 105 cells) were plated on Transwell filters with 0.4-μm pore (Corning Incorporated, Corning, NY) and monitored regularly by visualization with an inverted microscope (Eclipse TS100/100-F; Nikon, Melville, NY) and by epithelial resistance measurements. Protein expression from Caco-2 cells and mouse tissue (intestinal scrapings that contained mostly enterocytes) was assessed by Western blot analysis as previously described.19Nighot M. Al-Sadi R. Guo S.H. Rawat M. Nighot P. Watterson M.D. Ma T.Y. Lipopolysaccharide-induced increase in intestinal epithelial tight permeability is mediated by Toll-like receptor 4/myeloid differentiation primary response 88 (MyD88) activation of myosin light chain kinase expression.Am J Pathol. 2017; 187: 2698-2710Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Cells and mouse intestinal scrapings were lyzed with lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 500 μmol/L NaF, 2 mmol/L EDTA, 100 μmol/L vanadate, 100 μmol/L phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, 1 μg/mL pepstatin A, 40 mmol/L paranitrophenyl phosphate, 1 μg/mL aprotinin, and 1% Triton X-100] on ice for 30 minutes. For cytosolic and nuclear fractionation the NE-PER kit from Thermo Scientific (Pittsburgh, PA) was used according to the manufacturer's instructions. The lysates were centrifuged at 10,000 × g for 10 minutes in an Eppendorf Centrifuge (5417R; Hauppauge, NY) to obtain a clear lysate. The supernatant was collected, and protein concentration was determined with the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Laemmli buffer (Bio-Rad Laboratories) was added to the lysate that contained 20 to 40 μg of protein and boiled at 100°C for 7 minutes, after which proteins were separated on an SDS-PAGE gel. Proteins from the gel were transferred to the membrane (Trans-Blot Transfer Medium, Nitrocellulose Membrane; Bio-Rad Laboratories) overnight. The membrane was incubated for 2 hours in blocking solution (5% dry milk or 5% bovine serum albumin in tris-buffered saline–Tween 20 buffer). The membrane was then incubated with antibody in blocking solution. After a wash in tris-buffered saline–1% Tween buffer, the membrane was incubated in secondary antibody and developed with the use of the Santa Cruz Western Blotting Luminal Reagents (Santa Cruz Biotechnology) on the Kodak Bio-Max MS film (Fisher Scientific, Pittsburgh, PA). ImageJ version 1.50d (NIH, Bethesda, MD; https://imagej.nih.gov/ij) was used for densitometry analysis for the quantification of the Western blot analyses. The filter-grown cells were fixed with absolute methanol and stored at −80°C until used. The cells were thawed, rinsed in PBS, permeabilized with 0.1% Triton X-100, blocked with normal serum, and incubated overnight at 4°C in primary antibody. The cells were washed thoroughly and incubated in secondary antibodies conjugated with fluorescent dyes Alexa Fluor 488 or cyanine 3. After washings in PBS, the cells were mounted in Prolong Gold antifade reagent (Invitrogen) that contained DAPI as a nuclear stain and examined with a Zeiss LSM 510 microscope (Zeiss, Jena, Germany) equipped with a digital camera (Fluorescence Microscopy Shared Resource, Health Sciences Center, University of New Mexico). For mice intestines, cryosections were fixed in methanol before immunofluorescence staining. Images were processed with ZEN LSM software version 1.0 en 04/2018 (Zeiss). Caco-2 transepithelial electrical resistance (TER) was measured by using an epithelial voltmeter (EVOM; World Precision Instruments, Sarasota, FL) as previously reported.19Nighot M. Al-Sadi R. Guo S.H. Rawat M. Nighot P. Watterson M.D. Ma T.Y. Lipopolysaccharide-induced increase in intestinal epithelial tight permeability is mediated by Toll-like receptor 4/myeloid differentiation primary response 88 (MyD88) activation of myosin light chain kinase expression.Am J Pathol. 2017; 187: 2698-2710Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Both apical and basolateral sides of the epithelium were bathed with buffer solution. Electrical resistance was measured until similar values were recorded on three consecutive measurements. Caco-2 paracellular permeability was determined by using an established paracellular marker inulin.24Guo S. Al-Sadi R. Said H.M. Ma T.Y. Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14.Am J Pathol. 2013; 182: 375-387Abstract Full Text Full Text PDF PubMed Scopus (405) Google Sc
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