Reassembly of the Tight Junction after Oxidative Stress Depends on Tyrosine Kinase Activity

Abstract

Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-d-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavengerN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress. Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-d-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavengerN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress. hydrogen peroxide tight junction transepithelial resistance 2-deoxyglucose Madin-Darby canine kidney zonula occludens Dulbecco's modified Eagle's medium phosphate-buffered saline N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine Many disease states of the kidney such as ischemia/reperfusion, inflammation, or toxic injury to the kidney and gut lead to loss of the epithelial barrier. Reactive oxygen species are directly involved in the pathophysiology of some of these diseases. Targets for hydrogen peroxide (H2O2)1include DNA, proteins, and lipids. Reported effects of hydrogen peroxide on renal epithelial cell lines include DNA damage with induction of apoptosis or necrosis (1Ueda N. Shah S.V. J. Clin. Invest. 1992; 90: 2593-2597Crossref PubMed Scopus (180) Google Scholar, 2Filipovic D.M. Meng X. Reeves W.B. Am. J. Physiol. 1999; 277: F428-436PubMed Google Scholar, 3Takeda M. Shirato I. Kobayashi M. Endou H. Nephron. 1999; 81: 234-238Crossref PubMed Scopus (57) Google Scholar), decreased activity of membrane transporters (4Andreoli S.P. McAteer J.A. Seifert S.A. Kempson S.A. Am. J. Physiol. 1993; 265: F377-384PubMed Google Scholar), and membrane lipid peroxidation (5Meng X. Reeves W.B. Am. J. Physiol. 2000; 278: F83-F90Crossref PubMed Google Scholar). Hydrogen peroxide decreases transepithelial resistance (TER) and increases transcellular permeability of MDCK cell monolayers (6Welsh M.J. Shasby D.M. Husted R.M. J. Clin. Invest. 1985; 76: 1155-1168Crossref PubMed Scopus (112) Google Scholar, 7Winter M. Wilson J.S. Bedell K. Shasby D.M. Am. J. Respir. Cell Mol. Biol. 1990; 2: 355-363Crossref PubMed Scopus (17) Google Scholar), which is also well documented in non-kidney epithelial cell lines (8Yamaya M. Sekizawa K. Masuda T. Morikawa M. Sawai T. Sasaki H. Am. J. Physiol. 1995; 268: L284-293PubMed Google Scholar, 9Rao R. Baker R.D. Baker S.S. Biochem. Pharmacol. 1999; 57: 685-695Crossref PubMed Scopus (79) Google Scholar, 10Rao R.K. Li L. Baker R.D. Baker S.S. Gupta A. Am. J. Physiol. 2000; 279: G332-340Crossref PubMed Google Scholar, 11Baker R.D. Baker S.S. LaRosa K. Dig. Dis. Sci. 1995; 40: 510-518Crossref PubMed Scopus (33) Google Scholar) and endothelial cell lines (12Shasby D.M. Lind S.E. Shasby S.S. Goldsmith J.C. Hunninghake G.W. Blood. 1985; 65: 605-614Crossref PubMed Google Scholar, 13Wilson J. Winter M. Shasby D.M. Blood. 1990; 76: 2578-2582Crossref PubMed Google Scholar, 14Hinshaw D.B. Burger J.M. Armstrong B.C. Hyslop P.A. J. Surg. Res. 1989; 46: 339-349Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 15Kevil C.G. Oshima T. Alexander B. Coe L.L. Alexander J.S. Am. J. Physiol. 2000; 279: C21-30Crossref PubMed Google Scholar). However, the biochemical and subcellular effects of hydrogen peroxide on tight junction (TJ) proteins have not been studied, nor have they been distinguished from effects due to partial ATP depletion. Short-term depletion and repletion of intracellular ATP in cultured cells has been used to study the disassembly and/or reassembly of junctions as a model of organ ischemia and reperfusion (16Tsukamoto T. Nigam S.K. Am. J. Physiol. 1999; 276: F737-750PubMed Google Scholar, 17Tsukamoto T. Nigam S.K. J. Biol. Chem. 1997; 272: 16133-16139Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 18Molitoris B.A. Leiser J. Wagner M.C. Pediatr. Nephrol. 1997; 11: 761-767Crossref PubMed Scopus (49) Google Scholar, 19Mandel L.J. Doctor R.B. Bacallao R. J. Cell Sci. 1994; 107: 3315-3324Crossref PubMed Google Scholar, 20Bacallao R. Garfinkel A. Monke S. Zampighi G. Mandel L.J. J. Cell Sci. 1994; 107: 3301-3313Crossref PubMed Google Scholar). However, little is known about the reassembly of the tight junction after exposure to reactive oxygen species due to the lack of a reproducible model system. We have now developed and analyzed a model of reversible hydrogen peroxide-induced disassembly and reassembly of the TJ in vitro and show that the reassembly pathway, as monitored by physiological, biochemical, and immunocytochemical parameters, depends upon tyrosine kinase activity. Furthermore, the cellular and biochemical consequences of H2O2 exposure are distinct from those caused by ATP depletion-repletion. Madin-Darby canine kidney cells (MDCK, type II) were obtained from American Type Tissue Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum, 50 IU/ml penicillin, and 50 μg/ml streptomycin. Cells were incubated at 37 °C in 5% CO2 and were passaged weekly. DMEM was from Cellgro (Herndon, VA), streptomycin, penicillin, and fetal calf serum from Sigma. Plasticware was from Falcon (Lincoln Park, NJ). Transwells were obtained from Costar (Cambridge, MA), the ohm meter (Millicell ERS) from Millipore (Bedford, MA). Anti-ZO-1 rat monoclonal antibody (R40.76) was kindly supplied by Dr. D. Goodenough (Harvard Medical School, Boston). Anti-E-cadherin antibodies were isolated from hybridoma cells (rr-1) kindly provided by Barry Gumbiner (University of California, San Francisco). Anti-ZO-2 and anti-occludin polyclonal rabbit antibodies were from Zymed Laboratories Inc. (San Francisco, CA), anti-phosphotyrosine antibody was from Upstate (4G10), Upstate Biotechnology, Inc., Lake Placid, NY. All other reagents used in these experiments were of analytical grade. Confluent monolayers were cultured in DMEM without fetal calf serum 24 h prior to the exposure with specific concentrations of hydrogen peroxide and throughout the experiment. To terminate the exposure to hydrogen peroxide, catalase was added into the media at a final concentration of 5000 units/ml. Control experiments were performed with monolayers in serum free DMEM with and without catalase or Me2SO. After treatment of MDCK monolayers with 5 mm H2O2 or 2-deoxy-d-glucose (dGlc) for various times, cells were washed with PBS twice, and exposed to 0.4% trypan blue in PBS supplemented with 1.5 mm CaCl2 and mm for 5 The cells were a and the of and cells was MDCK cells were to on and exposed to H2O2 with or without treatment with or After of the monolayers with in PBS for apoptosis was detected with the free of DNA to the cells were of intracellular ATP was by the MDCK monolayers were serum in DMEM for 24 washed with PBS times, and exposed to PBS 1.5 mm and mm 2-deoxy-d-glucose for of intracellular ATP levels was by the media to Control experiments were performed in DMEM MDCK cells were on at and maintained in serum media for h to tight 24 h cells were exposed to some known of hydrogen peroxide or of intracellular were in DMEM exposure to H2O2 on of the was at after treatment with these reagents and/or hydrogen peroxide with a Millipore resistance as A. M. Nigam S.K. J. Physiol. 1994; PubMed Scopus Google Scholar). were in ohm as a of the after of of monolayers were and ohm of cellular ATP were performed with a ATP After the with PBS times, of cell the Cells were and of by for 5 at The ATP of of was with of ATP in ATP levels are as of the after of Confluent monolayers of MDCK cells were on specific the the monolayers were with with for for and or for at anti-phosphotyrosine and in PBS at After 5% serum, serum for cells were incubated for h with After 5 in PBS the monolayers were incubated with or antibodies for The cells were in Inc., The were through a with a to a were San The are as S.E. to the of The was used to from were by of was to concentrations of H2O2 and on after treatment for h and mm H2O2 only by concentrations of caused a in to near concentrations and caused a loss of the h of MDCK monolayers with 5000 catalase reversed the of for H2O2 concentrations of to about h for cells with mm h for cells with 5 mm and did not h after exposure to However, h after catalase of the cells with mm H2O2 not media or exposure to catalase on on these a of 5 mm was for as decreased to of and recovery h in the of catalase a new and reproducible model for reversible disassembly and reassembly. the of recovery of after treatment with 5 mm H2O2 upon of MDCK monolayers with 5 for or treatment with 5000 catalase in a recovery with for to of was after h for a and h for a not To the recovery of after treatment with hydrogen peroxide and catalase is dependent on of new TJ or other proteins, MDCK monolayers were with the protein and 5 with catalase. of was in cells the h to in the the effects of protein for the recovery of new protein is not blue was observed in of the cells The of trypan blue cells to after exposure to 5 and after h After of H2O2 with catalase for the of trypan blue cells at the for of H2O2 exposure PP-2 and genistein did not the of trypan blue cells The of cells was and did not after of exposure to 5 mm H2O2 or after exposure to PP-2 or genistein for h with or without catalase After h exposure to hydrogen peroxide of the cells for After the which a and recovery of for or inhibitors of intracellular were used to the reduction was due to oxidative and to specific for hydrogen hydrogen peroxide by reduction of H2O2 to at the J. Clin. Invest. PubMed Scopus Google Scholar). is also by cells and as intracellular scavenger of of pyruvate with hydrogen peroxide the loss of indicating that pyruvate is intracellular H2O2 of experiments MDCK cells were with 5 mm pyruvate in DMEM for exposure to the cells were washed with PBS to with pyruvate reduced the in to 5% of to after of catalase was to 5 h did not were for h and with hydrogen peroxide for and the in was The reduced the of to a in the to 50 the membrane hydrogen peroxide and scavenger and the scavenger on the in for h exposure to with the cell heavy metal which as intracellular reduced the in at to Furthermore, the of was reduced and recovery from the exposure to H2O2 was and after h The cell was for h and reduced the in to Exposure to or on TJ is known to by a variety of mechanisms R. Baker R.D. Baker S.S. Biochem. Pharmacol. 1999; 57: 685-695Crossref PubMed Scopus (79) Google Scholar, T. Nigam S.K. Am. J. 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Physiol. 1999; 277: PubMed Google Scholar). a of inhibitors of known to the the were obtained with the tyrosine kinase inhibitors PP-2 or and genistein 50 or of which Src tyrosine kinases, genistein is also a tyrosine kinase MDCK cells were with these agents for h exposed to 5 mm The of was not from a but the recovery after 5000 catalase was and indicating a for tyrosine kinase in the reassembly of the TJ but not the Control experiments with or changes in not tyrosine kinase due to a Src to for the recovery of the TJ after exposure to hydrogen has also been to TJ in other L. K. M. J.M. J. Cell Biol. 1993; PubMed Scopus Google Scholar, Nigam S.K. Sci. S. A. 1995; PubMed Scopus Google Scholar, S.K. N. S. Biochem. Res. PubMed Scopus Google Scholar). inhibitors were for to the recovery of after exposure to hydrogen C and reduced at h by and with The agents did not in experiments ATP levels in various cell lines after exposure to H. Am. J. Physiol. 1999; 277: PubMed Google Scholar, S.P. McAteer J.A. 1990; Full Text PDF PubMed Scopus Google Scholar). MDCK ATP levels have been to with mm H2O2 M. Wilson J.S. Bedell K. Shasby D.M. Am. J. Respir. Cell Mol. Biol. 1990; 2: 355-363Crossref PubMed Scopus (17) Google Scholar). exposure of MDCK monolayers to 5 or for in a partial ATP depletion to and of Furthermore, the for the recovery of cellular ATP after treatment with 5000 catalase or repletion with media after was changes or catalase treatment on the intracellular ATP concentration and after after mm was used in the experiments to the of partial ATP depletion on and of TJ proteins. to the effects of 5 on treatment with mm was not with a in transepithelial the at a in of ATP monolayers to 5% the of monolayers was observed 5 with in in trypan blue was The that is in cells ATP to of levels is with that that the for ATP in occurs at of ATP levels (4Andreoli S.P. McAteer J.A. Seifert S.A. Kempson S.A. Am. J. Physiol. 1993; 265: F377-384PubMed Google Scholar, X. Reeves W.B. Am. 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Google Scholar). that H2O2 a partial of ZO-1 in the TJ after and exposure of MDCK cells to 5 in a ZO-1 staining throughout the for and a of and of the with 5000 catalase the of the TJ and after h a of proteins was and The exposure of MDCK cells to mm on the of or indicating that these changes, the in not by ATP levels a and for ZO-1 for The ZO-1 staining but of MDCK monolayers after staining with anti-phosphotyrosine and antibodies staining of the tight junction junction h after exposure to H2O2 and catalase or catalase a MDCK cell monolayers that were with PP-2 or genistein for h and exposed to the oxidative stress and catalase in the of the inhibitors a in ZO-1 staining after h that the without catalase with the indicating that these agents recovery inhibitors on the ZO-1 staining in the of catalase without oxidative stress and phosphorylation in the lateral membrane after short-term exposure to hydrogen peroxide with monolayers as R.K. Baker R.D. Baker S.S. Gupta A. M. Am. J. Physiol. 1997; Google J. Cell Biol. 76: PubMed Scopus Google Scholar). Furthermore, tyrosine phosphorylation in the lateral cell h after exposure to H2O2 and with catalase as that the staining for tyrosine phosphorylation and ZO-1 in the tight junction staining was observed in the lateral membrane and the of the cells PP-2 and genistein tyrosine phosphorylation and to a and staining which the tyrosine phosphorylation of monolayers with H2O2 treatment did not induce tyrosine phosphorylation in the cell junction These experiments that tyrosine possibly in the lateral membrane near or in the occurs TJ and that the of catalase to induce the recovery of the tight junction possibly these phosphorylation Oxidative stress is known to the permeability in renal epithelial cells in resistance and intracellular ATP levels after exposure to hydrogen peroxide, the permeability for the increases (6Welsh M.J. Shasby D.M. Husted R.M. J. Clin. 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Biol. 1990; 2: 355-363Crossref PubMed Scopus (17) Google Scholar, S.P. McAteer J.A. 1990; Full Text PDF PubMed Scopus Google Scholar). of have a of intracellular ATP levels to of oxidative stress in renal epithelial cell lines D.M. Meng X. Reeves W.B. Am. J. Physiol. 1999; 277: F428-436PubMed Google Scholar, X. Reeves W.B. Am. J. Physiol. 2000; 278: F83-F90Crossref PubMed Google Scholar, 7Winter M. Wilson J.S. Bedell K. Shasby D.M. Am. J. Respir. Cell Mol. Biol. 1990; 2: 355-363Crossref PubMed Scopus (17) Google Scholar). in ATP of of after with 5 mm ATP is ATP levels obtained with the ATP depletion model of TJ disassembly in which and oxidative phosphorylation are The used reagents for ATP 2-deoxy-d-glucose and intracellular ATP levels in MDCK cells to of the (16Tsukamoto T. Nigam S.K. Am. J. Physiol. 1999; 276: F737-750PubMed Google Scholar, 17Tsukamoto T. Nigam S.K. J. Biol. Chem. 1997; 272: 16133-16139Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, R.B. Bacallao R. Mandel L.J. Am. J. Physiol. 1994; PubMed Google Scholar). ATP depletion the disassembly of the tight junction, and increases the of TJ proteins with the T. Nigam S.K. J. Biol. Chem. 1997; 272: 16133-16139Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The of the effects of ATP depletion on the TJ of MDCK cells has been in (16Tsukamoto T. Nigam S.K. Am. J. Physiol. 1999; 276: F737-750PubMed Google Scholar, 17Tsukamoto T. Nigam S.K. J. Biol. Chem. 1997; 272: 16133-16139Abstract Full Text Full Text PDF PubMed Scopus (169) Google B.A. Am. J. Physiol. Google Scholar, Am. J. Physiol. 1994; Google Scholar). During the ATP repletion the reassembly of the tight junction depends on tyrosine phosphorylation of TJ proteins (16Tsukamoto T. Nigam S.K. Am. J. Physiol. 1999; 276: F737-750PubMed Google Scholar). To in the of the intracellular ATP is not for the disassembly of the ATP levels were reduced to the observed after exposure to hydrogen with 2-deoxy-d-glucose for decreased ATP to the as hydrogen partial ATP depletion to of the to a in transepithelial Although these that other are to to the disassembly of the tight junction, they not of the decreased ATP on the of the tight junction oxidative stress. of the tight junction after partial ATP depletion not monitored since treatment with did not the tight junction. is that the reassembly of the TJ after oxidative stress ATP depletion in a is well that hydrogen peroxide as a R.K. Li L. Baker R.D. Baker S.S. Gupta A. Am. J. Physiol. 2000; 279: G332-340Crossref PubMed Google Scholar, D. Biochem. Res. 1992; PubMed Scopus Google Scholar, A. R. G. G. G. G. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). epithelial exposure to hydrogen peroxide or inhibitors has been to and which by tyrosine kinase R.K. Baker R.D. Baker S.S. Gupta A. M. Am. J. Physiol. 1997; Google Scholar, J. Cell Biol. 76: PubMed Scopus Google Scholar). was that the in tyrosine phosphorylation induce the disassembly of the tight junction, specific phosphorylation in the tight junction were not genistein and PP-2 did not the disassembly of the tight junction in study but the for tyrosine phosphorylation also in the of TJ reassembly after oxidative stress. disassembly and reassembly of the tight junction are by to the of tight junction proteins the reassembly of the junction. Furthermore, the specific of PP-2 a for Src tyrosine in phosphorylation that with ZO-1 as well as the lateral membrane was observed disassembly and the recovery but not after h of exposure to tyrosine phosphorylation and recovery of staining of ZO-1 after recovery of the TJ were by PP-2 and that tyrosine phosphorylation of tight junction proteins or proteins is critical the recovery of the tight junction. However, a specific for tyrosine phosphorylation the tight junction was not phosphorylation of and is known to the reassembly of the TJ after ATP depletion by tyrosine phosphorylation was inhibited by genistein (16Tsukamoto T. Nigam S.K. Am. J. Physiol. 1999; 276: F737-750PubMed Google Scholar). a of into MDCK cells a of with a of and into the Goodenough Mol. Biol. 2000; 11: PubMed Scopus Google Scholar). After treatment of these cells with the protein kinase kinase occludin and ZO-1 but not were tyrosine proteins were to the cell ZO-1 was also tyrosine into the TJ with after treatment of cell lines with J.M. J. Cell Sci. 1995; Google Scholar). in tyrosine phosphorylation of ZO-1 was after of rat with The tyrosine phosphorylation with the of the epithelial with tight junctions H. J.M. Am. J. Physiol. 1995; 268: Google Scholar). 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Cell Biol. 1997; PubMed Scopus Google Scholar). the reversible model for the study of oxidative stress on the TJ in epithelial the of tyrosine the reassembly of the tight junction and from other of tight junction experiments with the model the study of the phosphorylation of specific TJ proteins after short-term oxidative stress. the specific of the reassembly of the tight junction to in We for with the of the tight junction after oxidative stress depends on tyrosine kinase of and and The of these is of these are PDF