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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

2017; Elsevier BV; Volume: 187; Issue: 12 Linguagem: Inglês

10.1016/j.ajpath.2017.08.005

ISSN

1525-2191

Autores

Meghali Nighot, Rana Al–Sadi, Shuhong Guo, Manmeet Rawat, Prashant K. Nighot, Martin D. Watterson, Y. Thomas,

Tópico(s)

Immune Response and Inflammation

Resumo

Lipopolysaccharides (LPSs) are a major component of the Gram-negative bacterial cell wall and play an important role in mediating intestinal inflammatory responses in inflammatory bowel disease. Although recent studies suggested that physiologically relevant concentrations of LPS (0 to 1 ng/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability, the mechanisms that mediate an LPS-induced increase in intestinal TJ permeability remain unclear. Herein, we show that myosin light chain kinase (MLCK) plays a central role in the LPS-induced increase in TJ permeability. Filter-grown Caco-2 intestinal epithelial monolayers and C57BL/6 mice were used as an in vitro and in vivo intestinal epithelial model system, respectively. LPS caused a dose- and time-dependent increase in MLCK expression and kinase activity in Caco-2 monolayers. The pharmacologic MLCK inhibition and siRNA-induced knock-down of MLCK inhibited the LPS-induced increase in Caco-2 TJ permeability. The LPS increase in TJ permeability was mediated by toll-like receptor 4 (TLR-4)/MyD88 signal-transduction pathway up-regulation of MLCK expression. The LPS-induced increase in mouse intestinal permeability also required an increase in MLCK expression. The LPS-induced increase in intestinal permeability was inhibited in MLCK−/− and TLR-4−/− mice. These data show, for the first time, that the LPS-induced increase in intestinal permeability was mediated by TLR-4/MyD88 signal-transduction pathway up-regulation of MLCK. Therapeutic targeting of these pathways can prevent an LPS-induced increase in intestinal permeability. Lipopolysaccharides (LPSs) are a major component of the Gram-negative bacterial cell wall and play an important role in mediating intestinal inflammatory responses in inflammatory bowel disease. Although recent studies suggested that physiologically relevant concentrations of LPS (0 to 1 ng/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability, the mechanisms that mediate an LPS-induced increase in intestinal TJ permeability remain unclear. Herein, we show that myosin light chain kinase (MLCK) plays a central role in the LPS-induced increase in TJ permeability. Filter-grown Caco-2 intestinal epithelial monolayers and C57BL/6 mice were used as an in vitro and in vivo intestinal epithelial model system, respectively. LPS caused a dose- and time-dependent increase in MLCK expression and kinase activity in Caco-2 monolayers. The pharmacologic MLCK inhibition and siRNA-induced knock-down of MLCK inhibited the LPS-induced increase in Caco-2 TJ permeability. The LPS increase in TJ permeability was mediated by toll-like receptor 4 (TLR-4)/MyD88 signal-transduction pathway up-regulation of MLCK expression. The LPS-induced increase in mouse intestinal permeability also required an increase in MLCK expression. The LPS-induced increase in intestinal permeability was inhibited in MLCK−/− and TLR-4−/− mice. These data show, for the first time, that the LPS-induced increase in intestinal permeability was mediated by TLR-4/MyD88 signal-transduction pathway up-regulation of MLCK. Therapeutic targeting of these pathways can prevent an LPS-induced increase in intestinal permeability. Lipopolysaccharide (LPS) plays an important pathogenic role in intestinal inflammation of Crohn disease and other inflammatory conditions.1Guo 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 (233) Google Scholar, 2Guo S. Al-Sadi R. Said H.M. Ma T.Y. 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Most of the published studies used extreme pharmacologic concentrations of LPS, ranging between 50 and 1000 μg/mL, which exceed the physiologically achievable concentrations by 104- to 107-fold, to assess various biological responses.23Forsythe 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, 24Hirotani Y. Ikeda K. Kato R. Myotoku M. Umeda T. Ijiri Y. Tanaka K. Protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal Caco-2 cells.Yakugaku Zasshi. 2008; 128: 1363-1368Crossref PubMed Scopus (54) Google Scholar, 25Kim I.D. Ha B.J. Paeoniflorin protects RAW 264.7 macrophages from LPS-induced cytotoxicity and genotoxicity.Toxicol In Vitro. 2009; 23: 1014-1019Crossref PubMed Scopus (53) Google Scholar, 26Liu C. Li A. Weng Y.B. Duan M.L. Wang B.E. Zhang S.W. 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In contrast, we previously showed, for the first time, that LPS at physiologically achievable concentrations (0 to 1000 pg/mL) and clinically achievable concentrations (0 to 10 ng/mL) does not cause acute cell death.1Guo 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 (233) Google Scholar, 2Guo 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 (400) Google Scholar Moreover, LPS at concentrations up to 10 ng/mL did not affect intestinal TJ permeability up to 3 days of LPS exposure. LPS caused a selective increase in intestinal epithelial TJ permeability between days 3.5 and 4 that was associated with an increase in TLR-4 and CD14 basolateral membrane expression. Myosin light chain kinase (MLCK) is a Ca2+/calmodulin-activated enzyme that catalyzes the myosin light chain phosphorylation, triggering actin/myosin contraction and subsequent muscle contraction.29Ohlmann P. Tesse A. Loichot C. Ralay Ranaivo H. Roul G. Philippe C. Watterson D.M. Haiech J. Andriantsitohaina R. Deletion of MLCK210 induces subtle changes in vascular reactivity but does not affect cardiac function.Am J Physiol Heart Circ Physiol. 2005; 289: H2342-H2349Crossref PubMed Scopus (25) Google Scholar MLCK exists in two isoforms: the short isoform (MLCK108) is ubiquitous in adult tissues, with smooth muscle cells containing the highest amounts; and the long isoform (MLCK210) is prominently expressed in embryonic smooth muscle cells and in adult cells of nonmuscular lineage, including enterocytes.30Verin A.D. Lazar V. Torry R.J. Labarrere C.A. Patterson C.E. Garcia J.G. Expression of a novel high molecular-weight myosin light chain kinase in endothelium.Am J Respir Cell Mol Biol. 1998; 19: 758-766Crossref PubMed Scopus (67) Google Scholar The long isoform MLCK210 has been shown to play a crucial role in both physiological and pathologic regulation of intestinal TJ permeability.30Verin A.D. Lazar V. Torry R.J. Labarrere C.A. Patterson C.E. Garcia J.G. Expression of a novel high molecular-weight myosin light chain kinase in endothelium.Am J Respir Cell Mol Biol. 1998; 19: 758-766Crossref PubMed Scopus (67) Google Scholar, 31Lazar V. Garcia J.G. A single human myosin light chain kinase gene (MLCK; MYLK).Genomics. 1999; 57: 256-267Crossref PubMed Scopus (102) Google Scholar, 32Garcia J.G. Lazar V. Gilbert-McClain L.I. Gallagher P.J. Verin A.D. Myosin light chain kinase in endothelium: molecular cloning and regulation.Am J Respir Cell Mol Biol. 1997; 16: 489-494Crossref PubMed Scopus (173) Google Scholar The overexpression of MLCK was sufficient to cause an increase in TJ permeability,33Clayburgh D.R. Rosen S. Witkowski E.D. Wang F. Blair S. Dudek S. Garcia J.G. Alverdy J.C. Turner J.R. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability.J Biol Chem. 2004; 279: 55506-55513Crossref PubMed Scopus (124) Google Scholar and pharmacologic activation of MLCK produced an increase in epithelial TJ permeability. Previous studies have shown that a cytokine-induced increase in intestinal TJ permeability was also mediated by an increase in MLCK expression.34Al-Sadi R. Boivin M. Ma T. Mechanism of cytokine modulation of epithelial tight junction barrier.Front Biosci (Landmark Ed). 2009; 14: 2765-2778Crossref PubMed Scopus (411) Google Scholar, 35Al-Sadi R. Ye D. Dokladny K. Ma T.Y. Mechanism of IL-1beta-induced increase in intestinal epithelial tight junction permeability.J Immunol. 2008; 180: 5653-5661Crossref PubMed Scopus (297) Google Scholar, 36Ma T.Y. Iwamoto G.K. Hoa N.T. Akotia V. Pedram A. Boivin M.A. Said H.M. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G367-G376Crossref PubMed Scopus (688) Google Scholar As mentioned above, high pharmacologic doses of LPS cause rapid and diffuse epithelial cell death, leading to a rapid loss in intestinal epithelial barrier.37Johnston D.G. Corr S.C. Toll-like receptor signalling and the control of intestinal barrier function.Methods Mol Biol. 2016; 1390: 287-300Crossref PubMed Scopus (23) Google Scholar, 38Chang J.X. Chen S. Ma L.P. Jiang L.Y. Chen J.W. Chang R.M. Wen L.Q. Wu W. Jiang Z.P. Huang Z.T. Functional and morphological changes of the gut barrier during the restitution process after hemorrhagic shock.World J Gastroenterol. 2005; 11: 5485-5491Crossref PubMed Scopus (99) Google Scholar In contrast, low physiological levels (0.3 to 1.0 ng/mL) of LPS do not cause cell death, but cause a functional opening of the TJ barrier with an increase in TJ permeability.1Guo 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 (233) Google Scholar, 2Guo 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 (400) Google Scholar However, the specific intracellular mechanisms or the effector proteins responsible for the physiological or clinically achievable concentrations of LPS on intestinal TJ permeability remain unknown. The purpose of these studies was to determine the protein kinase and the intracellular processes responsible for the LPS-induced functional opening of the intestinal TJ barrier. Our results suggested that the TJ proteins, including zonula occludens protein 1, claudins 1, 2, 3, and 5, and occludin, were not affected by LPS. Instead, the LPS-induced increase in intestinal TJ permeability mediated the TLR-4/Myd88 signaling pathway–regulated increase in MLCK transcript and protein expression and subsequent MLCK-induced opening of the TJ barrier. Dulbecco’s modified Eagle’s medium, trypsin, fetal bovine serum, glutamine, penicillin, streptomycin, phosphate-buffered saline (PBS), and horseradish peroxidase–conjugated secondary antibodies for Western blot analysis were purchased from Invitrogen Life Technologies (Carlsbad, CA). siRNA of MLCK, TLR-4, and transfection reagents were from Dharmacon (Lafayette, CO). LPS (O111:B4) and ML-7 were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were of reagent grade and were purchased from Sigma-Aldrich, VWR (Aurora, CO), or Fisher Scientific (Pittsburgh, PA). Caco-2 cells (passage 20) were purchased from ATCC (Manassas, VA) and maintained at 37°C in a culture medium composed of Dulbecco’s modified Eagle’s medium with 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. The cells were kept at 37°C in a 5% CO2 environment. Culture medium was changed every 2 days. The cells were subcultured by partial digestion with 0.25% trypsin and 0.9 mmol/L EDTA in Ca2+- and Mg2+-free PBS. For growth on filters, high-density Caco-2 cells (1 × 105 cells) were plated on Transwell filters, with a 0.4-μm pore (Corning Inc., Corning, NY), and monitored regularly by visualization with an inverted microscope (Eclipse TS100/100-F; Nikon, Melville, NY) and by epithelial resistance measurements. The human intestinal Caco-2 cell line has been extensively used over the past 20 years as an in vitro model system of functional epithelial barriers. The transepithelial electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured using an epithelial voltmeter (EVOM; World Precision Instruments, Sarasota, FL), as previously reported.35Al-Sadi R. Ye D. Dokladny K. Ma T.Y. Mechanism of IL-1beta-induced increase in intestinal epithelial tight junction permeability.J Immunol. 2008; 180: 5653-5661Crossref PubMed Scopus (297) Google Scholar, 39Ye D. Ma I. Ma T.Y. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G496-G504Crossref PubMed Scopus (313) Google Scholar After treatment, including LPS (LPS was refreshed every 24 hours), ML-7, and siRNA, both apical and basolateral sides of the epithelium were bathed with Dulbecco’s modified Eagle’s medium. Electrical resistance was measured using 5% difference on three consecutive measurements. The Caco-2 monolayer paracellular permeability was determined using inulin, an established paracellular marker.35Al-Sadi R. Ye D. Dokladny K. Ma T.Y. Mechanism of IL-1beta-induced increase in intestinal epithelial tight junction permeability.J Immunol. 2008; 180: 5653-5661Crossref PubMed Scopus (297) Google Scholar, 39Ye D. Ma I. Ma T.Y. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G496-G504Crossref PubMed Scopus (313) Google Scholar, 40Al-Sadi R.M. Ma T.Y. IL-1beta causes an increase in intestinal epithelial tight junction permeability.J Immunol. 2007; 178: 4641-4649Crossref PubMed Scopus (432) Google Scholar Unless specified otherwise, Dulbecco’s modified Eagle’s medium (pH 7.4) was used as the incubation solution during the experiments. Buffered solution (0.5 mL) was added to the apical compartment, and 1.5 mL was added to the basolateral compartment to ensure equal hydrostatic pressure, as recommended by the manufacturer. Known concentrations of permeability marker inulin (10 μmol/L) and its radioactive tracer were added to the apical solution. Low concentrations of permeability marker were used to ensure that a negligible osmotic or concentration gradient was introduced. All flux studies were performed at 37°C. For determination of mucosal-to-serosal flux rates of inulin, Caco-2–plated filters with an epithelial resistance of 400 to 550 Ω·cm2 were used. All of the permeability experiments were repeated three to six times in triplicate. To study the time-course effect of LPS on MLCK protein expression, Caco-2 monolayers were treated with LPS (0.3 ng/mL) from 1 to 5 days. At the end of the experimental period, Caco-2 monolayers were immediately rinsed with ice-cold PBS. Cells were lysed 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. The cell lysates were placed in microfuge tubes and centrifuged at 10,000 × g for 10 minutes in an Eppendorf centrifuge (5417R; Eppendorf, Hauppauge, NY) to obtain a clear lysate. The supernatant was collected, and protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Laemmli gel loading buffer (catalog number 161-0737; Bio-Rad Laboratories) was added to the lysate containing 10 to 20 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 in tris-buffered saline–Tween 20 buffer). The membrane was then incubated with MLCK antibody (catalog number M7905; Sigma-Aldrich) in blocking solution. After a wash in tris-buffered saline–1% Tween buffer, the membrane was incubated in horseradish peroxidase–goat anti-mouse IgG (catalog number ab-20043; Abcam, Cambridge, MA) and developed using the Santa Cruz Western Blotting Luminol Reagents (Santa Cruz Biotechnology, Dallas, TX) on the Kodak BioMax MS film (Fisher Scientific, Pittsburgh, PA). The films were exposed between 5 seconds and 10 minutes. Biotinylated MLC was diluted in PBS and coated on streptavidin 96-well plates at 37°C for 1 hour. The plates were washed three times with PBS, incubated with blocking solution (1 mg/mL bovine serum albumin in PBS) at 37°C for 1 hour, and then washed three times with PBS. The kinase reaction buffer (90 μL), provided by the manufacturer (MBL International, Woburn, MA), and the treated samples (10 μL) were added to the wells, and the kinase reaction was performed at 37°C for 30 to 60 minutes. The reaction was stopped by removing the reaction mixtures and washing the plates three times with buffer (20 mmol/L Tris-HCl, pH 7.4, 0.5 mol/L NaCl, and 0.05% Tween 20). The washed plates were incubated with the anti–phosphorylated (phospho)–MLC-S19 antibody (5 ng/mL; catalog number M6068; Sigma-Aldrich) at room temperature for 1 hour, after which the plates were washed four times with buffer. Horseradish peroxidase–goat anti-rabbit IgG (diluted at 1:2000 in washing buffer; catalog number ab-6721; Abcam) was added to the wells, and the plates were incubated at 37°C for 1 hour. The plates were washed four times and then incubated with 100 μL substrate solution (tetramethylbenzidine) for 5 to 15 minutes at 37°C. The reaction was stopped by adding 100 μL of 0.5N H2SO4. The absorbance at 450 nm was determined using the SpectraMax 190 (Molecular Devices, Sunnyvale, CA). Targeted siRNA of MLCK was obtained from Dharmacon. Caco-2 monolayers were transiently transfected using DharmaFect transfection reagent (Dharmacon). Briefly, cells (5 × 105 per filter) were seeded into a 12-well transwell plate and grown to confluency. Caco-2 monolayers were then washed with PBS twice. Then, 0.5 mL Accell medium (Thermo Scientific, Lafayette, CO) was added to the apical compartment of each filter, and 1.5 mL was added to the basolateral compartment of each filter. MLCK siRNA or TLR-4 siRNA (5 ng) and DharmaFect reagent (2 μL) were preincubated in Accell medium. After 5 minutes of incubation, the two solutions were mixed, and the mixture was added to the apical compartment of each filter. The LPS experiments were conducted 24 hours after transfection. The silencing was confirmed by Western blot analysis. Caco-2 cells (5 × 105 cells per filter) were seeded into 12-well Transwell permeable inserts and grown to confluency. Filter-grown Caco-2 cells were then treated with LPS from 1 to 5 days. At the end of the experimental period, cells were washed wit

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