Chemical anoxia of tubular cells induces activation of c-Srcand its translocati - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:50 |只看该作者 |倒序浏览 |打印

作者：DiviyaSinha, ZhiyongWang, Valerie R.Price, John H.Schwartz,  WilfredLieberthal作者单位：Renal Section, Evans Biomedical Research Center, Departmentof Medicine, Boston Medical Center and Boston University School ofMedicine, Boston, Massachusetts 02118
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   Cyanide(CN)-induced chemical anoxia of cultured mouse proximal tubular (MPT)cells increased the kinase activity of c-Src by approximatelythreefold.4-Amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4- d ]pyrimidine (PP2), a specific inhibitor of c-Src, prevented Src activation. CN alsoincreased the permeability of MPT cell monolayers, an event amelioratedby PP2. During CN treatment, the proteins of the zonula adherens (ZA;E-cadherin and the catenins) disappeared from their normal location atcell-cell borders and appeared within the cytosol. CN also resulted inthe appearance of c-Src at cell-cell borders. PP2 prevented theseCN-induced alterations in the distribution of ZA proteins and c-Src. CNalso increased the association of c-Src with -catenin and p120 andinduced a substantial increase in tyrosine phosphorylation of bothcatenins. PP2 prevented the CN-induced phosphorylation of thesecatenins. In summary, we show that CN-induced chemical anoxia activatesc-Src and induces its translocation to cell-cell junctions where itbinds to and phosphorylates -catenin and p120. Our findings suggestthat these events contribute to the loss of the epithelial barrierfunction associated with chemical anoxia.
      【关键词】 ischemia tight junction Src kinase Ecadherin catenins p ctn renal tubular cells- w5 T8 j+ t# @0 ]; c' l
   INTRODUCTION

CHEMICAL ANOXIA, A TERM THAT refers to the depletion of cell energy stores usingmitochondrial inhibitors such as antimycin ( 10, 51 ),rotenone ( 16 ), or cyanide (CN) ( 37, 46 ), is widely used as a model of reversible injury to cultured cells ( 34 ). A number of investigators have shown that chemicalanoxia can reversibly impair the integrity of the junctional complex, an effect that manifests functionally as loss of transepithelial electrical resistance and an increase in permeability of the epithelial monolayer ( 7, 10, 30, 44 ). The junctional complexcomprises at least three structures: the zonula occludens (ZO), thezonula adherens (ZA), and desmosomes ( 11, 18, 21, 22 ). TheZO (also called the tight junction) is the component of the junctional complex that represents the physical barrier to paracellular flux ofmolecules and ions across the epithelium ( 11 ). However,the integrity of the ZO is dependent on the formation of an intact ZA,which lies immediately basal to the ZO and mediates cadherin-dependent cell-cell adhesion ( 22 ).$ F, I; R- F" p. x$ o0 Y
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The ZA consists of a complex of proteins consisting of E-cadherin andmembers of the catenin family of proteins. E-cadherin is atransmembrane cell-cell adhesion molecule that mediates cell-cell adhesion by the homophilic binding of the extracellular domains expressed on adjacent cells ( 1, 19, 33 ). The cateninfamily consists of a number of isoforms including -, -, - andp120 catenin (p120 ctn ) ( 1, 2 ). The - and -catenins compete for binding to a single site on the cytoplasmictail of E-cadherin called the "catenin-binding domain"(CBD), soeach E-cadherin molecule binds to either - or -catenin in amutually exclusive manner ( 1, 19 ). Both - and -catenin bind to -catenin, which, in turn, binds to the actincytoskleleton either directly ( 42 ) or indirectly via the actin binding protein -actinin ( 29, 38 ). By contrast,p120 ctn (p120) binds to the juxtamembrane (JMB) domain ofthe cytoplasmic tail of E-cadherin and is present in all E-cadherincomplexes containing either - or -catenin ( 49, 53 ).Thus the cytoplasmic tail of E-cadherin has at least two distinctprotein-binding epitopes in its cytoplasmic tail: one, the CBD, thatassociates with either a - or -catenin, and another, the JMB,that binds to p120 ( 2 ).3 b8 m  T8 V5 c! F

We have previously reported that sublethal injury induced in tubularcells by CN-induced chemical anoxia leads to tyrosine phosphorylationof -catenin and that phosphorylation of components of the ZAcontributes to the disruption of ZA and the loss of the epithelialpermeability barrier associated with sublethal injury( 44 ). These findings are consistent with several lines ofevidence that have implicated protein tyrosine kinases and proteintyrosine phosphatases in the normal modulation of E-cadherin-catenin complex formation and disassociation ( 5, 6, 9, 11, 13, 32 ). Furthermore, activation of the Src family of tyrosine kinases has been implicated in tyrosine phosphorylation of ZA proteinsand cell-cell adhesion in keratinocytes and other nonrenal epithelialcells ( 9, 28, 48 ).6 S* [8 B7 O9 `1 v; y
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In this study, we provide entirely novel evidence that CN-inducedsublethal injury to renal tubular cells activates c-Src and induces itstranslocation to the ZA. In addition, activated c-Src tyrosine bindsto, and tyrosine phosphorylates, -catenin and p120. Finally, wedemonstrate that these events contribute to the reversible loss ofcell-cell adhesion and the increase in epithelial permeabilityassociated with sublethal injury.

MATERIALS AND METHODS2 k- d# V( P0 U/ l& L
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Reagents. Protein G-Sepharose beads were obtained from Pierce (Rockford, IL).Primary antibodies were a rabbit polyclonal anti-c-Src antibody (SantaCruz Biotechnology, Cambridge, MA); a mouse monoclonal anti-p120antibody; a rabbit polyclonal anti- -catenin antibody (Sigma, St.Louis, MO); a rabbit polyclonal anti- -catenin antibody (Sigma); amouse monoclonal anti-focal adhesion kinase (FAK) antibody (UpstateBiotechnology, Lake Placid, NY); and a mouse monoclonal anti-phosphotyrosine antibody (PY20; Transduction Laboratories, Lexington, KY). Secondary antibodies for immunoblotting were rabbit ormouse IgG conjugated with horseradish peroxidase (Sigma). Secondary antibodies used for immunofluoresence were donkey anti-mouse or goatanti-rabbit IgG conjugated with indocarbocyanine (CY3; Sigma). The Srckinase assay kit was purchased from Upstate Biotechnology, and the Srckinase inhibitor4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4- d ]pyrimidine (PP2) was from Calbiochem (La Jolla, CA). Rhodamine phalloidin was purchased from Molecular Probes.
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Isolation and characterization of a conditionally immortalizedmouse proximal tubular cell line. We have established a conditionally immortalized mouse proximal tubularcell line using the "immortoMouse," a transgenic mouse containingthe H-2K b -tsA58 transgene ( 47 ).This transgene is a temperature-sensitive mutant of the SV40 large Tantigen oncogene. The presence of -interferon in the medium togetherwith a low incubation temperature (33°C) provides the permissiveconditions necessary for expression of the H-2K b -tsA58 transgene. The expression of thegene is almost completely 95%) under nonpermissiveconditions in which -interferon is not added to the medium and cellsare incubated at 37°C ( 47 ). This study is the first inwhich we use this novel cell line.$ H7 e: v+ _7 B

We established the conditionally immortalized cell line by breeding ahomozygous male ImmortoMouse with a wild-type female mouse (bothpurchased from Charles River Laboratories). We used the F1 generationof mice containing a single copy of the H-2K b -tsA58 transgene to culture proximal tubular cells. Primary cultures ofproximal tubular cells were grown from tubular segments obtained fromkidneys of the F1 mice using techniques well established in ourlaboratory ( 30, 46 ). The first passage of the primary cultures of MPT cells were then trypsinized and cloned under permissive conditions at a limiting dilution in ten 150-mm culture dishes. Sixhours later, single cells were identified and isolated with cloningrings. One day later, cells that had proliferated within each ring weretrypsinized again, and each clone was plated in separate dishes,expanded under permissive conditions, and then characterized.

We chose the cell line obtained from clone 306 for use inthese studies and named the cell line "Boston University mouseproximal tubular cell-clone 306 cells" (BUMPT-306 cells). BUMPT-306cells form cobblestone monolayers typical of epithelial cells and, when grown on permeable cell culture supports (Millipore), have atransepithelial resistance of ~300 · cm 2. The clone also expressesthe Na /glucose transporter and megalin, both specificmarkers of proximal tubular cells. Immunofluorescence studiesdemonstrated that both the Na /glucose transporter andmegalin were expressed by all the cells and were localized to theapical surface. In addition, E-cadherin was present in these cellmonolayers at the basolateral surface. All these features indicate thatthe BUMPT-306 cell line, which is derived from a single culturedtubular cell, has structural and functional features of a homogenouspopulation of differentiated proximal tubular cells.. E. x: ~8 p5 K4 j- g: c! h. [
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Passaging of BUMPT-306 cells. The BUMPT-306 cells were passaged under permissive conditions in P100culture dishes. For all experimental studies, cells were grown toconfluence on culture dishes coated with rat tail collagen underpermissive conditions. When close to confluence, monolayers were thenincubated under nonpermissive conditions for 2 days before experimentalstudies were begun.

Experimental model of ATP depletion. Cells were incubated in a HEPES-buffered solution (15 mM, pH 7.4)containing (in mM) 134 NaCl, 3.6 KCl, 1.3 KH 2 PO 4, 15 HEPES, 1 CaCl 2, and 1 MgCl 2. Dextrose (10 mM) was added to the medium of controlcells. ATP depletion was induced with medium containing sodium cyanide(NaCN; 5 mM) to inhibit mitochondrial function and an absence ofdextrose (to inhibit glycolysis), as described previously ( 30, 46 ). ATP depletion followed by recovery was achieved byincubating the cells in CN/no dextrose for 45 min and then incubatingthe cells in medium containing 10 mM dextrose without CN for anadditional 30 min (the CN washout period) ( 30, 46 ). Insome studies, Src was inhibited in dextrose- and CN-treated cells usingPP2 (10 µM), a specific inhibitor of Src ( 24 ).
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Measurement of cellular ATP content. Cellular ATP was measured using the luciferase assay as previouslydescribed ( 46 ). ATP levels were measured in control(dextrose-treated) cell monolayers and after 5, 10, 15, 30, and 45 minof incubation with CN. ATP levels at each of these time points areexpressed as the percentage of control values.
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Immunoprecipitation of c-Src. Cells were lysed in RIPA buffer comprising (in mM) 20 Tris · HCl, pH 7.5, 140 NaCl, 1 NaF, 1 PMSF, and10 NaP i PO 4, as well as 0.5% Na-deoxycholate,0.1% SDS, 1% Triton X-100, and 10% glycerol. Sodium vanadate [1mM, a tyrosine phosphatase inhibitor and the protease inhibitorscontained in Complete tablets (Boeringer Mannheim)] were added to thelysis buffer just before use. The lysate was harvested and centrifugedat 4°C for 15 min at 2,000 rpm, and the supernatants were saved andkept on ice. Protein G-Sepharose beads were prepared for use by washingwith washing buffer [(in mM) 20 Tris · HCl, pH7.5, 140 NaCl, 1 EDTA, pH 7.5, 1 vanadate, 1 NaF, 1 PMSF, and 10 NaP i PO 4, as well as 2% Nonidet P-40 and protease inhibitors] and then diluted in the same buffer. A 500-µg protein aliquot of this supernatant was "precleared"( 3 ) by incubation with non-immune (normal rabbit serum)and beads. We immunoprecipitated c-Src in the supernatant of thisprecleared sample using a polyclonal antibody to c-Src (Santa CruzBiotechnology) conjugated to the protein G beads. Immunoprecipitateswere washed with washing buffer and used to measure Src kinase activityor to immunoblot for proteins associated with Src.0 q1 {4 B( `; i+ Y7 Q& \
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Src kinase assay. The immunoprecipitates were suspended in a reaction buffer containing asynthetic peptide as the Src kinase substrate peptide (150 µM/assay)and [ - 32 P]ATP and incubated for 10 min at 30°C. Thereactions were stopped by incubating the immunoprecipitates with 40%TCA (20 µl/tube) at room temperature for 5 min. The beads werepelleted, and the supernatant (10 µl/tube) was transferred to p81paper assay squares. The assay squares were washed six times for 5 mineach with 40 ml of 0.75% phosphoric acid and once with acetone. Assaysquares were then transferred to scintillation vials each containing 5 ml scintillation cocktail, and counts incorporated were read on ascintillation counter. The kinase activity was expressed as picomolesof phosphate incorporated per minute per milligram protein.

Western blotting. Lysates immunoprecipitated with c-Src antibody were boiled in 2× SDSsample buffer for 10 min. The beads were then spun down, and thesupernatant containing the immunoprecipitated proteins were subjectedto SDS-PAGE and immunoblotted with appropriate antibodies toE-cadherin, pp120, -, -, and -catenin, p120, or PY20.Immunoblots were probed with horseradish peroxidase-conjugated secondary antibodies using chemiluminiscence. Each immunoblot wasstripped and reprobed with the antibody used for immunoprecipitation toconfirm that equal amounts of immunoprecipitated protein were loaded ineach lane.7 D$ x+ U8 D* ~! L+ d8 ~- i

Immunofluorescence studies. BUMPT-306 cells were grown to confluence on collagen-coated coverslipsand treated with NaCN or dextrose for 45 min at 37°C. The cells werefixed in 3.7% paraformaldehyde for 10 min at room temperature (RT).Then, monolayers were washed three times for 5 min each inTris-buffered saline (TBS; 50 mM Tris, pH 7.6, 150 mM NaCl) andincubated with 1% BSA, prepared in TBS, for 15 min at RT. Forvisualization of c-Src, E-cadherin, and catenins, cells were thenincubated with the appropriate primary antibody diluted in 1% BSA for45 min at RT. After a washing with TBS, the cells were incubated withCy3-conjugated secondary antibody for 45 min at RT. Unbound secondaryantibody was removed by washing. The F-actin filaments were staineddirectly with rhodamine phalloidin (Molecular Probes) as describedpreviously ( 30 ). After being stained, the cells weremounted on glass slides and photographed using an immunofluorescencemicroscope and a digital camera.9 W5 K/ A+ y4 I( \, m: y( d8 M+ E

Measurement of epithelial permeability. The permeability of epithelial monolayers was determined with methodspreviously described by our group ( 30, 44 ) by assessing the paracellular flux of tritiated inulin across monolayers of BUMPT-306 cells. BUMPT-306 cells were grown to confluence on 12-mm permeable nitrocellulose inserts (Millicel-HA, Millipore, Bedford, MA).At the start of all experiments, the inserts were carefully washed withwarm Krebs-Henseleit buffer [KHB; (in mM) 115 NaCl, 3.6 KCl, 1.3 KH 2 PO 4, 25 NaHCO 3, 1 CaCl 2, and 1 MgCl 2 ]. Then, KHB containing[ 3 H]inulin was gently layered on the apical aspect of themonolayers, and the inserts were placed in 24-well plates containingKHB without added inulin. We determined that the volume of added KHBexerted no hydrostatic pressure across the monolayer. All experiments were performed in a humidified 95% air-5% CO 2 incubatorat 37°C. The amount of inulin [counts/min (cpm)] in the apical andbasal compartments was determined at the end of each 5-min period using a Packard scintillation counter. Epithelial permeability ( P in cm/s ×10 5 ) was calculated by the standard formula P = F/( S × C) where F is the rateof flux of inulin from apical to basal compartments per second, C isthe inulin concentration gradient, and S is the surface areaof the Millipore insert.9 n9 ~4 ~, x1 Z* y8 C1 J& I, H
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Initially, the KHB in the apical and basal compartments containeddextrose (5 mM) in all experiments. After a 1-h equilibration period,basal epithelial permeability was measured in all inserts during afurther four periods by moving the inserts every 5 min into fresh wellscontaining KHB dextrose. Basal permeability was calculated by averagingthe four values obtained. Then, the KHB in the apical and basalcompartments was changed to contain either dextrose alone, dextrose PP2(10 µM), KHB CN, or CN PP2. Permeability was then measured every 5 min for a total of 1 h.! ]1 D# h. h; M3 c
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Statistics. Data are presented as means ± SE. All statistical comparisonswere done using ANOVA, followed by the Bonferroni correction.

RESULTS
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Effect of CN on cell ATP content. The ATP content of control BUMPT-306 cells was 27 ± 2 ng/mg cellprotein. After treatment of cells with CN in the absence of dextrosefor 5, 10, 15, and 45 min, cellular ATP content fell to 71 ± 4, 45 ± 1, 21 ± 1, and 5 ± 1% of control levels,respectively (all P to those we obtained in response to CN treatmentof primary cultures of mouse proximal tubular cells ( 30, 46 ) and of Madin-Darby canine kidney cells ( 46 ).2 l! |: f. G/ ]7 y1 n
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ATP depletion inceases c-Src kinase activity. The activity of c-Src increased within 5 min of ATP depletion andremained elevated for the duration of the 45-min period of ATPdepletion ( n = 5) (Fig. 1 ). PP2 (10 µM), a specific inhibitor of Src kinase ( 43 ), completely inhibited the increase inc-Src activity observed after 10 min of CN treatment ( n = 6) (Fig. 1 ).
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Fig. 1. c-Src kinase is activated by ATP depletion. A : chemicalanoxia induced by cyanide (CN) resulted in an ~3-fold increase inc-Src activity by 5 min of CN treatment (CN 5'). Src activity remainedelevated throughout the 45 min of CN treatment (CN 10' and CN 45').* P B : the increase inc-Src activity induced by 10 min of CN treatment was prevented by thespecific Src kinase inhibitor4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4- d ]pyrimidine(PP2). * P P0 w+ }- D, o. A- p+ t- g. A
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Inhibition of c-Src kinase activity ameliorates the increase inepithelial permeability associated with ATP depletion. The permeability of epithelial monolayers was assessed by measuring theflux of tritiated inulin across monolayers of cells grown on permeablesupports using methods previously described by our group ( 30, 40 ) (see MATERIALS AND METHODS for details). Cellsgrown to confluence on permeable supports were incubated with dextroseor CN in the presence and absence of 10 µM PP2. The basalpermeability of dextrose-treated monolayers was 8.3 ± 1.5 cm/s × 10 5 and remained constant for the durationof the experiment (60 min; n = 5) (Fig. 2 ). The permeability of monolayerstreated with dextrose and PP2 (25 µM) was comparable to those treatedwith dextrose alone ( n = 5) (Fig. 2 ). In monolayerstreated with CN, permeability rose from the basal value of 8.3 ± 1.5 to 27.3 ± 3.1 cm/s × 10 5 within 10 minand remained relatively constant at that value for the duration of theexperiment ( P for repeatedmeasures; n = 8) (Fig. 2 ). In the presence of CN andPP2, permeability rose from 10.5 ± 1.7 to 14.5 ± 2.4 cm/s × 10 5 by 10 min and remained relativelyunchanged for 60 min ( P vs. CN alone anddextrose PP2). Thus inhibition of c-Src kinase activity with PP2ameliorated, but did not completely prevent, the CN-induced loss ofepithelial barrier function. We conclude that impaired barrier functionof BUMPT-306 cells, induced by CN-induced chemical anoxia, is partlymediated by activation of a member of the c-Src family of kinases.However, because PP2 completely inhibited activation of c-Src activity(Fig. 1 ) without completely reducing epithelial permeability to controllevels (Fig. 2 ), we infer that factors in addition to Src activation must contribute to the loss of permeability associated with CN.

Fig. 2. Effect of chemical anoxia on the permeability of mouseproximal tubular (MPT) monolayers in the presence and absence of Srcinhibition. MPT cell monolayers were grown to confluence on permeablesupports and treated with dextrose or CN in the presence or absence ofPP2. Epithelial permeability was measured using[ 3 H]inulin as a marker. CN treatment increased thepermeability of the cell monolayers by 5-fold. The increase inpermeability induced by CN was markedly ameliorated, but not completelyprevented, by the presence of PP2. * P P
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Immunofluoresence studies of the effects of CN on ZA proteins,actin, and c-Src. Immunofluoresence techniques were used to examine the distribution ofE-cadherin, as well as - and -catenin, in BUMPT-306 cellssubjected to chemical anoxia in the presence and absence of PP2. Incontrol, dextrose-treated cells, E-cadherin was localized predominantlyat cell-cell borders as expected for a normal epithelial monolayer.CN-induced chemical anoxia caused a marked decrease in the amount ofE-cadherin at cell-cell junctions and an increase in the protein withincytosolic aggregates (Fig. 3 ). PP2largely prevented the disruption of normal E-cadherin localizationinduced by CN (Fig. 3 ). Comparable results were obtained when theeffect of CN and CN PP2 on the distribution of other ZA proteins was studied ( - and -catenin; data not shown).

Fig. 3. Immunofluoresence microscopy of E-cadherin. In controlmonolayers ( top ), E-cadherin was distributed predominantlyat cell-cell borders (arrows). CN treatment ( middle )resulted in a redistribution of E-cadherin with loss of staining atcell-cell borders (arrows) and the presence of aggregates of E-cadherinin the cytosol. Inhibition of PP2 ( bottom ) inhibited theredistribution of E-cadherin induced by ATP depletion.7 U) R6 U+ N2 I, V2 h+ B, r* R
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CN treatment also resulted in the disruption of actin stress fiberswithout affecting the ring of cortical actin (Fig. 4 ). PP2 did not ameliorate the CN-induceddisruption of the actin stress fibers. These data suggest thatactivation of Src contributes to the loss of the structural integrityof the ZA associated with ATP depletion but appears to have no role inmodulating the disruption of the actin stress fibers associated withchemical anoxia.$ s1 W# w  R$ r0 I3 l$ ^8 [

Fig. 4. Fluorescence microscopy of the actin cytoskeleton.Control cells ( top ) demonstrate the normal appearance of theactin cytoskeleton, with stress fibers present within the cell(arrows). In CN-treated cells ( middle ), the stress fibersare severely disrupted while the actin ring, present at cell-cellborders (arrows), appears unaffected by ATP depletion. PP2 does notameliorate the disruption of stress fibers associated with CN.

We also examined the effect of CN on the distribution of Src kinase inthe BUMPT-306 cells. In control cells, c-Src was present within thecytosol, predominantly in a perinuclear distribution but alsoperipherally in a speckled pattern (Fig. 5 ). There was no evidence of c-Src atcell-cell borders in control cells, whereas in cells treated with CNfor 45 min c-Src was present at cell-cell borders (Fig. 5 ). PP2prevented the appearance of c-Src at cell-cell borders associated withCN treatment (Fig. 5 ).
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Fig. 5. Immunofluorescence microscopy of c-Src. In control cells( top ), c-Src was present predominantly within thecytosol in a perinuclear distribution. However, there was also Srcstaining peripherally, in a speckled pattern, consistent withlocalization of Src in adhesion plaques. There was no c-Src atcell-cell borders of control cells. In cells treated with CN( middle ), c-Src was present at cell-cell borders. PP2( bottom ) prevented the appearance of c-Src at cell-cellborders associated with chemical anoxia.
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Effects of CN on coimmunoprecipitation with ZA- and focaladhesion-associated proteins with c-Src. BUMPT-306 cells were treated with dextrose, dextrose PP2, and CN for 5, 10, 15, and 45 min, and CN (for 10 min) PP2. Lysates of each monolayerwere subjected to coimmunoprecipitation using a monoclonal antibody tov-Src, and the immunoprecipitates were subjected to SDS-PAGE and thenprobed by Western blotting for p120, -catenin, E-cadherin, or FAK.ATP depletion increased the association of Src with all three ZAproteins (p120, -catenin, and E-cadherin) (Fig. 6 ). The increased association of Src with all the ZA proteins was prevented by pretreatment of cells with PP2(Fig. 6 ). Similar results were obtained when p120, -catenin, E-cadherin, and FAK were immunoprecipitated from lysates obtained fromdextrose-, CN-, and CN PP2-treated cells and probed for Src (data notshown). In contrast to the effects of CN on Src association with the ZAproteins, CN had no effect on the degree of association of Src with FAK(Fig. 6 ).

Fig. 6. Effect of chemical anoxia on the amount of zona adherens (ZA)proteins that coimmunoprecipitates with c-Src. Lysates from cellstreated with either dextrose or CN for 5, 10, 15, and 45 min,dextrose PP2 for 10 min, or CN PP2 for 10 min were immunoprecipitatedwith c-Src antibody, subjected to SDS electrophoresis, andimmunoblotted with antibodies to -catenin, E-cadherin, p120, andfocal adhesion kinase (FAK). ATP depletion with CN markedly increasedthe association of c-Src with both catenins and E-cadherin, an effectprevented by PP2. However, CN did not alter the association with FAK.The figure shows a representative blot from 5 experiments. WB, Westernblot.5 H9 O9 _8 ]! i0 K  d- S5 O2 t5 x

Effect of CN on tyrosine phosphorylation of -catenin, p120,E-cadherin, and FAK. Finally, we examined the effect of ATP depletion on the degree oftyrosine phosphorylation of p120, -catenin, E-cadherin, and FAK. Weimmunoprecipitated each of these proteins from lysates of monolayerstreated with dextrose, CN, dextrose PP2, and CN PP2 and immunoblottedthe precipitates with a tyrosine phospho-specific antibody (PY20). CNtreatment increased the degree of tyrosine phosphorylation of both p120and -catenin, an effect that was completely prevented bypretreatment of cells with PP2 (Fig. 7 ). However, we found no tyrosine phosphorylation of immunoprecipitated E-cadherin under control conditions or after exposure of cells to CN(Fig. 7 ). In contrast to the effects of CN on phosphorylation of -catenin and p120, CN induced a fall in tyrosine phosphorylation ofFAK, an effect prevented by PP2 (Fig. 7 ).% ^! s7 h( ?! i
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Fig. 7. Effect of chemical anoxia on the state of tyrosine phosphorylationof p120, -catenin, E-cadherin, and FAK. Lysates from cells treatedwith either dextrose or CN in the presence and absence of PP2 wereimmunoprecipitated with an antibody to p120, -catenin, E-cadherin,or FAK and then immunoblotted with PY20 antibody to detect tyrosinephosphorylation. CN treatment resulted in marked hyperphosphorylationof both p120 and -catenin. PP2 prevented the hyperphosphorylaton ofthe 2 catenins induced by CN. E-cadherin was not tyrosinephosphorylated under either basal or CN-treated conditions. FAK, whichwas tyrosine phosphorylated under control conditions, becamedephosphorylated during CN treatment, an effect prevented by PP2. Thefigure shows a representative blot from 4 experiments.9 e' y- t# ?9 p+ f1 A6 j

DISCUSSION8 R6 F( t9 I1 w" w; d
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An important functional consequence of sublethal ischemicinjury to renal tubular cells is loss of the normal barrier function ofthe tubular epithelium ( 4, 7, 10, 30, 35, 36 ). Toelucidate the mechanisms responsible for this phenomenon, many investigators have focused their attention on the effects of ATP depletion on the ZO, which represents the physical barrier to paracellular ion flux ( 15, 20, 50 ). We have focusedinstead on the potential contribution of changes in the ZA to the loss of epithelial barrier function associated with ATP depletion( 40 ). The presence of an intact ZA is a prerequisite tothe functional integrity of the ZO. The importance of the ZA in tightjunctional function has been demonstrated in two ways. Removal ofextracellular calcium impairs ZO function ( 12, 23, 45 ).Because formation of the ZO itself does not require calcium, these datademonstrate that a structurally intact ZO cannot maintain theepithelial permeability barrier in the absence of a ZA. Also, directinhibition of ZO assembly using neutralizing antibodies to E-cadherinhas also been shown to impair tight junctional integrity and function( 11, 21, 22 ).+ b2 u- ^$ ^! Z3 v! E

We have previously demonstrated in a series of articles that CN-inducedinjury to tubular cells increases the permeability of the tubular cellmonolayer and results in the disassociation of the ZA from its normallocation at cell-cell junctions, as evidenced by the withdrawal ofE-cadherin as well as -, -, and -catenin to the basolateralmembrane of tubular cells ( 30, 40, 44 ). Similaralterations in the distribution of ZA proteins have been shown by otherinvestigators after ischemic injury to tubular cells ( 8, 31, 35 ). Our group has also reported that chemical anoxia isassociated with increased tyrosine phosphorylation of -catenin andthat the loss of ZA integrity and of tight junctional function is due,at least in part, to the changes in regulation of tyrosinephosphorylation induced by ATP depletion ( 44 ). Our findings are consistent with the role of tyrosine phosphorylation of -catenin as a regulator of cell-cell adhesion under physiological conditions ( 5, 6, 9, 13 ).& R, c' n+ m% r1 @+ Y/ K" B/ t

Some evidence suggests that alterations in ZA phosphorylation andcell-cell adhesion in nonrenal epithelial cells may be due to increasedactivity of a nonreceptor member of the Src kinase family ( 9, 48 ). The purpose of this study was to determine whether Srcactivation and phosphorylation of ZA proteins contribute to the loss ofintegrity of the ZA that we ( 44 ) and others ( 8, 31, 35 ) have previously observed in response to chemical anoxia.! O9 _' ~: j( Z1 R! J  c
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We provide entirely novel evidence that chemical anoxia activates c-Srcin tubular cells (Fig. 1 ). Our data showing that PP2 partly inhibitsthe loss of epithelial permeability and withdrawal of ZA proteins fromcell-cell borders associated with chemical anoxia suggest aparticipatory role for Src activation in these events( 24 ). Interestingly, PP2 has no effect on the disruption of actin stress fibers (Fig. 4 ), a well-documented effect of sublethal injury ( 25, 27, 30 ). The lack of efficacy of Srcinhibition in ameliorating the disruption of actin stress fiberssuggests that this alteration in the actin cytoskeleton is not animportant event in the loss of integrity of the ZA associated withchemical anoxia. These data are consistent with the notion that theactin ring, a component of the actin cytoskleleton that is relatively preserved during chemical anoxia (Fig. 4 ), is more relevant to thestability of the ZA than actin stress fibers.
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We also provide entirely novel evidence that chemical anoxia inducestranslocation of c-Src to the ZA. Immunofluoresence studies show thatCN induces the appearance of cSrc at cell-cell borders, an effectprevented by Src inhibition with PP2 (Fig. 5 ). In addition, usingimmunopreciptation and immunoblotting we demontrate that CN induces anincreased association of c-Src with the ZA proteins -catenin, p120,and E-cadherin (Fig. 6 ). However, CN does not change the degree ofassociation of c-Src with FAK, a protein localized in adhesion plaques(Fig. 6 ).  W  [0 b; N9 a& z  P
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We also show that CN treatment increases tyrosine phosphorylation ofp120 and -catenin (Fig. 7 ), both of which are known substrates ofSrc ( 2, 13, 41 ). The tyrosine phosphorylation of p120 and -catenin during ATP depletion is inhibited by PP2 (Fig. 7 ). Thesefindings are consistent with a role for c-Src in the tyrosinephosphorylation of -catenin and p120 during ATP depletion. However,E-cadherin, unlike p120 and -catenin, is not tyrosine phosphorylatedunder control or CN treatment conditions (Fig. 7 ). Taken together, ourdata suggest that Src binds to and phosphorylates both p120 and -catenin during chemical anoxia. The apparent increase in theassociation of E-cadherin with Src during CN treatment (shown byimmunoprecipitation and immunoblotting studies in Fig. 6 ) is probablynot due to the direct binding of Src to E-cadherin but rather to thepresence of E-cadherin bound to catenins in complexes of ZA proteins.' i* R% R! `; Y8 d( ?/ J' o
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In previous studies, -catenin and p120 have both been shown to bereadily phosphorylated by protein tyrosine kinases ( 2, 13, 41 ). It is also well established that alterations in the degreeof tyrosine phosphorylation of -catenin, which binds to the CBD ofthe cytoplasmic tail of cadherins ( 5, 13 ), is an importantevent in modulating cell-cell adhesion. The process of ZAformation and cell-cell adhesion requires tyrosine dephosphorylation of -catenin ( 5 ), whereas loss of cell-cell adhesion isassociated with an increase in the tyrosine phosphorylation of -catenin ( 13 ). Thus it is likely that c-Src-inducedphosphorylation of -catenin observed in this study contributes tothe loss of tubular cell-cell adhesion associated with sublethal injuryinduced by chemical anoxia.  p2 G* h! v' N3 L1 Q& @: a1 \

While there is also evidence to support a role for p120 in modulatingcell-cell adhesion, the mechanisms involved are less well defined thanfor -catenin ( 2 ). There is evidence that serine/threonine phosphorylation as well as tyrosine phosphorylation ofp120 plays a role in regulating p120 function and cell-cell adhesion( 3, 39 ). It is well established that Src is able totyrosine phosphorylate p120 in vitro and in vivo ( 41 ).Also, growth factors, such as EGF and hepatocyte growth factor (scatter factor), whose receptors have intrinsic tyrosine-specific protein kinase activity, increase the level of tyrosine phosphorylation of p120as well as of -catenin ( 17 ). However, it has not as yetbeen possible to distinguish the functional consequences of tyrosinephosphorylation of p120 from the effects of phosphorylation of the manyother targets of activated Src, including -catenin ( 2 ).Given the present uncertainty of the effects of tyrosine phosphorylation of p120 on cell adhesion, we cannot as yet speculate onthe significance of our observation that p120 is tyrosinephosphorylated by c-Src during chemical anoxia (Fig. 7 ).5 E3 w7 j& f; K, n
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Interestingly, we show that CN decreases tyrosine phosphorylation ofadhesion plaque protein FAK, an effect that is the opposite of whathappens to phosphorylation of the catenins (Fig. 7 ). Other investigators have also demonstrated that FAK becomes tyrosine dephosphorylated during ATP depletion ( 52 ). Whilehyperphosphorylation of the catenins is known to be associated withloss of cell-cell adhesion ( 5, 6, 9, 11, 13, 32 ),dephosphorylation of FAK causes loss of cell-matrix adhesion ( 14, 26 ). It is intriguing that while ATP depletion has oppositeeffects on the degree of tyrosine phosphorylation of ZA and adhesionplaque proteins, these effects are consistent with the loss of bothcell-cell and cell-matrix adhesion, well-known functional effects ofsublethal injury ( 10, 30 ).
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In summary, we have shown for the first time that CN-induced chemicalanoxia activates c-Src and leads to the translocation of the activatedform of c-Src to the ZA. There, c-Src binds to and phosphorylates -catenin and p120. We also show that these effects of c-Srccontribute to the structural and functional loss of the ZA and to theincrease in epithelial permeability associated with sublethal injury.Further studies are necessary to define in more detail the effects ofSrc-induced phosphorylation that occur in response to chemical anoxia.% F6 I/ b. E  p0 H, H6 N. W
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ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes andDigestive and Kidney Diseases Grants DK-385101, DK-59793, and DK-58306.. t# B! w& i  m4 k# H# S0 {
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