Reassembly of the Tight Junction after Oxidative Stress Depends on Tyrosine Kinase Activity
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m011477200
ISSN1083-351X
AutoresTobias Meyer, Catherine Schwesinger, Jiuming Ye, Bradley M. Denker, Sanjay K. Nigám,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoOxidative 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 2 mm MgCl2 for 5 min. The cells were examined using a Nikon Diaphot inverted microscope, and the number of viable and nonviable cells was determined. MDCK cells were grown to confluency on glass coverslips and exposed to H2O2 with or without treatment with catalase, genistein, or PP-2. After fixation of the monolayers with 4% paraformaldehyde in PBS for 30 min, apoptosis was detected with the ApopTagTM Peroxidase kit (Intergen, Purchase, NY), labeling free 3′-OH termini of DNA strand breaks, according to the manufacturers instructions. Apoptotic cells were counted under an inverted microscope (Nikon). Depletion of intracellular ATP was achieved by using the glycolytic inhibitor 2-deoxy-d-glucose. In brief, confluent MDCK monolayers were serum starved in serum-free DMEM for 24 h, washed with PBS three times, and exposed to PBS containing 1.5 mmCaCl2, 2 mm MgCl2, and 12 mm 2-deoxy-d-glucose for different time periods. Repletion of intracellular ATP levels was achieved by changing the media to serum-free DMEM. Control experiments were performed in serum-free DMEM alone. MDCK cells were plated on polycarbonate filters (Transwell, Costar) at confluent density (roughly 2 × 105 cells/cm2) and maintained in serum containing media for 48 h to establish tight monolayers. 24 h before TER measurement cells were exposed to serum-free DMEM. In some experiments, known scavengers of hydrogen peroxide or modulators of intracellular signaling pathways were preincubated in serum-free DMEM before exposure to H2O2 on both sides of the transwell. TER was measured at different time points after treatment with these reagents and/or hydrogen peroxide with a Millipore electrical resistance system, as described previously (21Stuart R.O. Sun A. Panichas M. Hebert S.C. Brenner B.M. Nigam S.K. J. Cell. Physiol. 1994; 159: 423-433Crossref PubMed Scopus (105) Google Scholar). Measurements were expressed in ohm × cm2, as a mean of the original readings after subtraction of background values. Basal TER measurement of confluent monolayers were recorded between 150 and 300 ohm × cm2. Measurements of total cellular ATP content were performed with a luciferase-based ATP determination kit (Sigma). After rinsing the monolayer with PBS three times, 300 μl of somatic cell ATP-releasing agent solubilized the cells. Cells were immediately scraped, aspirated, and cleared of insoluble material by spinning for 5 min at 10,000 rpm. The ATP content of 100 μl of supernatant was measured with 100 μl of Sigmas diluted luciferase-containing ATP assay mixture in an automated chemiluminescence counter (Berthold, Bad Wildholz, Germany). ATP levels are expressed as percent of the initial value after subtraction of background readings. Confluent monolayers of MDCK cells were grown on clear coverslips. At specific time points during the experiment, the monolayers were rinsed twice with PBS, fixed with 100% methanol (−80 °C) for 10 min for ZO-1, occludin, and E-cadherin staining, or 4% paraformaldehyde for 20 min at room temperature (for anti-phosphotyrosine staining), and stored in PBS at 4 °C. After blocking (PBS, 5% (v/v) goat serum, 3% (w/v) bovine serum albumin) for 1 h, cells were incubated for 2 h with primary antibodies. After washing (4 × 5 min in PBS + 0,05 Triton X-100) the monolayers were incubated with Texas red or fluorescein isothiocyanate-conjugated secondary antibodies for 1 h. The cells were mounted in Fluoromount (Southern Biotechnology Associates Inc., Birmingham, AL). The filters were viewed through a ×100 oil immersion objective with a laser scanning confocal system (model MRC-1024/2p, Bio-Rad, Cambridge, MA) coupled to a Zeiss Axiovert microscope. Images were processed using Photoshop software (Adobe, San Jose, CA). The data are given as mean values ± S.E. (n), where n refers to the number of measurements performed. The paired Student's t test was used to compare mean values within one experimental series. Data from two groups were compared by unpaired t test. A p value of <0.05 was accepted to indicate statistical significance. Low concentrations of H2O2 (0.5 and 0.75 mm) had no detectable effect on TER even after treatment for 8 h and 1 mm H2O2 only lowered TER by 9 ± 2%. Intermediate concentrations of H2O2(2.5 mm) caused a gradual decrease in TER to values near zero over 8 h. Higher concentrations (5 and 10 mm) caused a more rapid loss of TER within the first 3 h (Fig.1 A). Treatment of MDCK monolayers with 5000 units/ml catalase reversed the decrease of TER for all H2O2 concentrations used. Recovery of TER to control values took about 3 h for cells treated with 2.5 mm H2O2, 4.5 h for cells treated with 5 mm H2O2, and did not occur within 8 h after exposure to 10 mmH2O2 (Fig. 1 B). However, 20 h after catalase treatment, TER values of the cells treated with 10 mm H2O2 reached baseline values (data not shown). Changing media or exposure to catalase alone had no effect on TER. Based on these results, a H2O2concentration of 5 mm was chosen for further experiments, as TER reproducibly decreased to 26 ± 3% (n = 35) of baseline within 30 min and complete recovery occurred within 4–6 h in the presence of catalase (Fig. 1 B). This represents a new and reproducible model for reversible H2O2-dependent disassembly and reassembly. In addition, the rate of recovery of TER after treatment with 5 mm H2O2 depended upon incubation time. Incubation of MDCK monolayers with 5 mmH2O2 for 45 or 60 min before treatment with 5000 units/ml catalase resulted in a slower recovery compared with incubation for 30 min (Fig. 1 C). Recovery to control values of TER was complete after ∼12 h for a 45-min incubation and 20 h for a 60-min incubation (data not shown). To test whether the recovery of TER after treatment with hydrogen peroxide and catalase is dependent on synthesis of new TJ or other proteins, MDCK monolayers were coincubated with the protein synthesis inhibitor cycloheximide (50 μg/ml) and 5 mmH2O2 before scavenging with catalase. Recovery of TER was nearly identical in cycloheximide-treated cells over the initial 6 h (Fig. 1 D). At later time points, TER began to drop in the cycloheximide-treated monolayers, most likely reflecting the general effects of inhibiting protein synthesis. This result indicates that, for the early recovery of barrier function, new protein synthesis is not necessary. Trypan blue uptake was observed in 0.1 ± 0.1% of the cells under baseline conditions. The number of trypan blue positive cells increased to 0.7 ± 0.1% after 30 min exposure to 5 mmH2O2 and 2.5 ± 0.6% after 6 h (Fig.2 A). After scavenging of H2O2 with catalase for 5.5 h, the number of trypan blue positive cells remained at the same value for 30 min of H2O2 exposure (0.7 ± 0.3%,n = 10). PP-2 and genistein did not increase the number of trypan blue positive cells (Fig. 2 B). The number of apoptotic cells was 0.18 ± 0.1% under control conditions and did not increase significantly after 30 min of exposure to 5 mm H2O2 or after exposure to PP-2 or genistein for 6 h with or without catalase (0.2% ± 0.1%, Fig. 2 C). After 6 h exposure to hydrogen peroxide 1.1 ± 0.06% of the cells stained positive for apoptosis. After establishing the conditions under which a constant fall and recovery of TER occurred reproducibly (5 mmH2O2 for 30 min), different scavengers or inhibitors of intracellular signaling pathways were used to determine whether the TER reduction was due to oxidative stress, and to investigate specific targets for hydrogen peroxide. Pyruvate scavenges hydrogen peroxide by direct nonenzymatic reduction of H2O2 to water while undergoing decarboxylation at the 1-carbon position (22Salahudeen A.K. Clark E.C. Nath K.A. J. Clin. Invest. 1991; 88: 1886-1893Crossref PubMed Scopus (222) Google Scholar). Pyruvate is also readily taken up by cells and can thus serve as an intracellular scavenger of H2O2. Co-incubation of pyruvate (5 mm) with hydrogen peroxide completely abolished the loss of TER, indicating that pyruvate is effectively scavenging intracellular H2O2 (Fig.3 A, n = 4). In another set of experiments MDCK cells were preincubated with 5 mm pyruvate in serum-free DMEM for 16 h. Before exposure to H2O2, the cells were washed with PBS to eliminate extracellular pyruvate. This preincubation with pyruvate significantly reduced the drop in TER to 62 ± 5% of control. Recovery to baseline after addition of catalase was accelerated to ∼2 versus 5 h under control conditions (Fig. 3 A, n = 4). Pyruvate alone did not influence TER. Other scavengers were preincubated for 1 h and coincubated with hydrogen peroxide for 30 min and the fall in TER was recorded (Fig. 3 B). The reducing agent dithiothreitol reduced the initial fall of TER to 57 ± 7% (dithiothreitol, 0.5 mm, n = 7). Superoxide dismutase (30 units/ml, n = 7) had a similar effect in reducing the fall to 50 ± 4%. In marked contrast, the membrane permeable hydrogen peroxide and hydroxyl radical scavenger dimethylthiourea (10 mm, n = 7) and the hydroxyl radical scavenger 5,5-dimethylpyrroline N-oxide (10 mm,n = 7) had no effect on the initial fall in TER. Preincubation for 1 h before exposure to H2O2, with the cell permeable heavy metal chelator TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, 1 μm), which acts as an intracellular scavenger, reduced the fall in TER at 30 min to 58 ± 3% (Fig. 3 C). Furthermore, the subsequent decline of TER was significantly reduced and recovery from the exposure to H2O2 was faster and complete after ∼2 h (Fig. 3B, n = 6). The cell permeable iron chelator deferoxamine (0.1 mm) was preincubated for 16 h and subsequently reduced the initial fall in TER to 56 ± 7% (Fig. 3 C, n = 6). Exposure to TPEN or deferoxamine alone had no effect on TER. TJ assembly is known to be modulated by a variety of signaling mechanisms (9Rao R. Baker R.D. Baker S.S. Biochem. Pharmacol. 1999; 57: 685-695Crossref PubMed Scopus (79) Google Scholar, 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, 23Balda M.S. Gonzalez-Mariscal L. Contreras R.G. Macias-Silva M. Torres-Marquez M.E. Garcia-Sainz J.A. Cereijido M. J. Membr. Biol. 1991; 122: 193-202Crossref PubMed Scopus (244) Google Scholar, 24Balda M.S. 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In a broad screen of many inhibitors of signaling events known to modulate the TJ, the most consistent results were obtained with the two tyrosine kinase inhibitors PP-2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)-pyrazolo[3,4-d]pyrimidine, 1 or 10 μm) and genistein (4′,5,7-trihydroxyisoflavone, 50 or 100 μm), both of which inhibit Src family tyrosine kinases, although genistein is also a broad spectrum tyrosine kinase inhibitor. MDCK cells were preincubated with these agents for 1 h before being exposed to 5 mm H2O2. The drop of TER was not different from a control experiment, but the recovery after 5000 units/ml catalase was virtually abolished (Fig.4, A and B,n = 6), indicating a role for tyrosine kinase in the reassembly of the TJ but not the initial disassembly. Control experiments with PP-2, genistein, or Me2SO alone showed no changes in baseline TER (n = 4, data not shown). Thus, tyrosine kinase activity, most likely due to a Src family member, appears to be important for the recovery of the TJ after exposure to hydrogen peroxide. PKC has also been shown to regulate TJ assembly in other models (24Balda M.S. Gonzalez-Mariscal L. Matter K. Cereijido M. Anderson J.M. J. Cell Biol. 1993; 123: 293-302Crossref PubMed Scopus (348) Google Scholar, 29Stuart R.O. Nigam S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6072-6076Crossref PubMed Scopus (240) Google Scholar, 30Nigam S.K. Denisenko N. Rodriguez-Boulan E. Citi S. Biochem. Biophys. Res. Commun. 1991; 181: 548-553Crossref PubMed Scopus (70) Google Scholar). Three PKC inhibitors were tested for their ability to inhibit the recovery of TER after exposure to hydrogen peroxide. Calphostin C (500 nm, n = 7), bisindolylmaleimide I (500 nm, n = 7), and chelerythrine (1 μm, n = 7) reduced TER at 6 h by 73 ± 5, 55 ± 2, and 42 ± 5%, respectively, when compared with control (Fig. 4 C). The agents did not decrease TER in control experiments (n = 3). Intracellular ATP levels decrease in various cell lines after exposure to H2O2(31Aito H. Aalto T.K. Raivio K.O. Am. J. Physiol. 1999; 277: C878-883Crossref PubMed Google Scholar, 32Andreoli S.P. McAteer J.A. Kidney Int. 1990; 38: 785-794Abstract Full Text PDF PubMed Scopus (106) Google Scholar). In MDCK cells, ATP levels have been shown to decrease with 1 mm H2O2 (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 our experiments, exposure of MDCK monolayers to 5 mmH2O2 or 12 mm2-deoxy-d-glucose for 30 min resulted in a partial ATP depletion to 21.4 ± 4 and 20.7 ± 2% of control, respectively (Fig. 5, A andB, n = 4–8). Furthermore, the time course for the recovery of cellular ATP content after treatment with either 5000 units/ml catalase (H2O2 group) or repletion with control media (dGlc group) after 30 min was similar. Media changes or catalase treatment had no effect on the intracellular ATP concentration (98.7 ± 3 and 96.3 ± 4% after after 6 h, respectively). Therefore, 12 mm dGlc was used in the following experiments to examine the effect of partial ATP depletion on TER, extractability and localization of TJ proteins. In marked contrast to the effects of 5 mmH2O2 on TER (see Fig. 1), treatment with 12 mm dGlc was not associated with a detectable drop in transepithelial resistance. On the contrary, at later time points, a rise in TER of partially ATP depleted monolayers to 25.5 ± 5% above the TER of repleted monolayers was observed (Fig. 5 C,n = 4). Consistent with this finding, in dGlc-treated cells, no increase in trypan blue uptake was recorded (n = 10). The finding that TER is unchanged in cells ATP depleted to 20% of control levels is consistent with previous work that indicates that the threshold for ATP depletion-induced declines in TER occurs at around 5–10% of normal ATP levels (4Andreoli S.P. McAteer J.A. Seifert S.A. Kempson S.A. Am. J. Physiol. 1993; 265: F377-384PubMed Google Scholar, 5Meng 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, 17Tsukamoto T. Nigam S.K. J. Biol. Chem. 1997; 272: 16133-16139Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,33Canfield P.E. Geerdes A.M. Molitoris B.A. Am. J. Physiol. 1991; 261: F1038-1045PubMed Google Scholar). Confocal microscopy revealed that H2O2 induces a reversible, partial breakdown of ZO-1 in the TJ after 30 min (Fig.6 A, b, f, and j). Prolonged exposure of MDCK cells to 5 mmH2O2 resulted in a discontinuous ZO-1 staining pattern throughout the monolayer (Fig. 6 A, c, g,and k, n = 4 for each observation). Occludin and E-cadherin showed a similar pattern of redistribution and broadening of the staining. Treatment with 5000 units/ml catalase prevented the disruption of the TJ and after 6 h a normal distribution of all three junctional proteins was demonstrated (Fig.6 A, d, h, and l). The exposure of MDCK cells to 12 mm dGlc had no effect on the distribution of ZO-1, occludin, or E-cadherin, indicating that these changes, like the drop in TER, could not be explained by lower ATP levels (Fig. 6 B, a and b, data shown for ZO-1 only, n = 3 for each observation). The ZO-1 staining remained continuous but appeared slightly more jagged during later time points. Immunofluorescence of MDCK monolayers after double staining with anti-phosphotyrosine and anti-ZO-1 antibodies showed intact staining of the tight junction junction 6 h after exposure to H2O2 and catalase or catalase alone (Fig. 7, a andb). In contrast, MDCK cell monolayers that were pretreated with 10 μm PP-2 (Fig. 7 c) or 100 μm genistein (Fig. 7 e) for 1 h and exposed to the same oxidative stress and catalase in the presence of the inhibitors showed a marked disruption in ZO-1 staining after 6 h that resembled the disruption without catalase scavenging (see Fig.6 c), consistent with the data indicating that these agents inhibit TER recovery (Fig. 4). Both inhibitors had no effect on the ZO-1 staining in the presence of catalase without oxidative stress (Fig. 7, d and f). Tyrosine phosphorylation occurred in the lateral membrane after short-term exposure to hydrogen peroxide (Fig.8 A, b) when compared with untreated monolayers (Fig. 8 A, a), as described before (34Rao R.K. Baker R.D. Baker S.S. Gupta A. Holycross M. Am. J. Physiol. 1997; 273: G812-823PubMed Google Scholar,35Collares-Buzato C.B. Jepson M.A. Simmons N.L. Hirst B.H. Eur. J. Cell Biol. 1998; 76: 85-92Crossref PubMed Scopus (136) Google Scholar). Furthermore, strong tyrosine phosphorylation occurred in the lateral cell border 6 h after exposure to H2O2 and scavenging with catalase (Fig.8 A, f, same sample as Fig. 7 a). Analysis ofz-sections revealed that the staining for tyrosine phosphorylation and ZO-1 overlapped in the tight junction although additional phosphotyrosine staining was observed in the lateral membrane and the cytosol of the cells (Fig. 8 B). Both PP-2 and genistein prevented this tyrosine phosphorylation (Fig. 8 A, d and e), giving rise to a very weak and diffuse staining pattern which resembled the tyrosine phosphorylation of monolayers treated with H2O2 alone (Fig.8 A, c). Catalase treatment alone did not induce tyrosine phosphorylation in the cell junction (Fig. 8 A, g). These experiments imply that tyrosine phosphorylation, possibly in the lateral membrane near or in the TJ, occurs during TJ recovery, and that the ability of catalase to induce the recovery of the tight junction possibly requires these phosphorylation events. Oxidative stress is known to disturb the permeability barrier in renal epithelial cells in culture. Transepithelial resistance and intracellular ATP levels fall after exposure to hydrogen peroxide, whereas the permeability for small solutes across the monolayer increases (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). Initially, a reduced intracellular ATP level was thought to cause the disruption of the junction (36Hyslop P.A. Hinshaw D.B. Halsey Jr., W.A. Schraufstatter I.U. Sauerheber R.D. Spragg R.G. Jackson J.H. 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Here, we present a new model in which the disruptive effect of hydrogen peroxide on physiological, biochemical, and immunocytochemical parameters of TJ integrity is reversible. H2O2 (5 mm) consistently dropped transepithelial resistance to 23% of control. Thereafter, the epithelial cell monolayers completely recovered to baseline within 6 h. After recovery, the MDCK monolayers showed a stable TER for several days. The exposure time of 30 min used in our experiments was associated with only a small decrease in cell viability. This finding is in good agreement with previous studie
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