Chemical anoxia of tubular cells induces activation of c-Src and its translocation to the zonula adherens

Diviya Sinha, Zhiyong Wang, Valerie R. Price, John H. Schwartz, and Wilfred Lieberthal

Renal Section, Evans Biomedical Research Center, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts 02118


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cyanide (CN)-induced chemical anoxia of cultured mouse proximal tubular (MPT) cells increased the kinase activity of c-Src by approximately threefold. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a specific inhibitor of c-Src, prevented Src activation. CN also increased the permeability of MPT cell monolayers, an event ameliorated by PP2. During CN treatment, the proteins of the zonula adherens (ZA; E-cadherin and the catenins) disappeared from their normal location at cell-cell borders and appeared within the cytosol. CN also resulted in the appearance of c-Src at cell-cell borders. PP2 prevented these CN-induced alterations in the distribution of ZA proteins and c-Src. CN also increased the association of c-Src with beta -catenin and p120 and induced a substantial increase in tyrosine phosphorylation of both catenins. PP2 prevented the CN-induced phosphorylation of these catenins. In summary, we show that CN-induced chemical anoxia activates c-Src and induces its translocation to cell-cell junctions where it binds to and phosphorylates beta -catenin and p120. Our findings suggest that these events contribute to the loss of the epithelial barrier function associated with chemical anoxia.

ischemia; tight junction; Src kinase; E-cadherin; catenins; p120ctn; renal tubular cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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CHEMICAL ANOXIA, A TERM THAT refers to the depletion of cell energy stores using mitochondrial 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 chemical anoxia 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 complex comprises at least three structures: the zonula occludens (ZO), the zonula adherens (ZA), and desmosomes (11, 18, 21, 22). The ZO (also called the tight junction) is the component of the junctional complex that represents the physical barrier to paracellular flux of molecules 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).

The ZA consists of a complex of proteins consisting of E-cadherin and members of the catenin family of proteins. E-cadherin is a transmembrane 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 catenin family consists of a number of isoforms including alpha -, beta -, gamma - and p120 catenin (p120ctn) (1, 2). The beta - and gamma -catenins compete for binding to a single site on the cytoplasmic tail of E-cadherin called the "catenin-binding domain"(CBD), so each E-cadherin molecule binds to either beta - or gamma -catenin in a mutually exclusive manner (1, 19). Both beta - and gamma -catenin bind to alpha -catenin, which, in turn, binds to the actin cytoskleleton either directly (42) or indirectly via the actin binding protein alpha -actinin (29, 38). By contrast, p120ctn (p120) binds to the juxtamembrane (JMB) domain of the cytoplasmic tail of E-cadherin and is present in all E-cadherin complexes containing either beta - or gamma -catenin (49, 53). Thus the cytoplasmic tail of E-cadherin has at least two distinct protein-binding epitopes in its cytoplasmic tail: one, the CBD, that associates with either a beta - or gamma -catenin, and another, the JMB, that binds to p120 (2).

We have previously reported that sublethal injury induced in tubular cells by CN-induced chemical anoxia leads to tyrosine phosphorylation of beta -catenin and that phosphorylation of components of the ZA contributes to the disruption of ZA and the loss of the epithelial permeability barrier associated with sublethal injury (44). These findings are consistent with several lines of evidence that have implicated protein tyrosine kinases and protein tyrosine 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 proteins and cell-cell adhesion in keratinocytes and other nonrenal epithelial cells (9, 28, 48).

In this study, we provide entirely novel evidence that CN-induced sublethal injury to renal tubular cells activates c-Src and induces its translocation to the ZA. In addition, activated c-Src tyrosine binds to, and tyrosine phosphorylates, beta -catenin and p120. Finally, we demonstrate that these events contribute to the reversible loss of cell-cell adhesion and the increase in epithelial permeability associated with sublethal injury.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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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 (Santa Cruz Biotechnology, Cambridge, MA); a mouse monoclonal anti-p120 antibody; a rabbit polyclonal anti-beta -catenin antibody (Sigma, St. Louis, MO); a rabbit polyclonal anti-beta -catenin antibody (Sigma); a mouse monoclonal anti-focal adhesion kinase (FAK) antibody (Upstate Biotechnology, Lake Placid, NY); and a mouse monoclonal anti-phosphotyrosine antibody (PY20; Transduction Laboratories, Lexington, KY). Secondary antibodies for immunoblotting were rabbit or mouse IgG conjugated with horseradish peroxidase (Sigma). Secondary antibodies used for immunofluoresence were donkey anti-mouse or goat anti-rabbit IgG conjugated with indocarbocyanine (CY3; Sigma). The Src kinase assay kit was purchased from Upstate Biotechnology, and the Src kinase inhibitor 4-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.

Isolation and characterization of a conditionally immortalized mouse proximal tubular cell line. We have established a conditionally immortalized mouse proximal tubular cell line using the "immortoMouse," a transgenic mouse containing the H-2Kb-tsA58 transgene (47). This transgene is a temperature-sensitive mutant of the SV40 large T antigen oncogene. The presence of gamma -interferon in the medium together with a low incubation temperature (33°C) provides the permissive conditions necessary for expression of the H-2Kb-tsA58 transgene. The expression of the gene is almost completely inhibited (by >95%) under nonpermissive conditions in which gamma -interferon is not added to the medium and cells are incubated at 37°C (47). This study is the first in which we use this novel cell line.

We established the conditionally immortalized cell line by breeding a homozygous male ImmortoMouse with a wild-type female mouse (both purchased from Charles River Laboratories). We used the F1 generation of mice containing a single copy of the H-2Kb-tsA58 transgene to culture proximal tubular cells. Primary cultures of proximal tubular cells were grown from tubular segments obtained from kidneys of the F1 mice using techniques well established in our laboratory (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. Six hours later, single cells were identified and isolated with cloning rings. One day later, cells that had proliferated within each ring were trypsinized 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 in these studies and named the cell line "Boston University mouse proximal tubular cell-clone 306 cells" (BUMPT-306 cells). BUMPT-306 cells form cobblestone monolayers typical of epithelial cells and, when grown on permeable cell culture supports (Millipore), have a transepithelial resistance of ~300 Omega  · cm2. The clone also expresses the Na+/glucose transporter and megalin, both specific markers of proximal tubular cells. Immunofluorescence studies demonstrated that both the Na+/glucose transporter and megalin were expressed by all the cells and were localized to the apical surface. In addition, E-cadherin was present in these cell monolayers at the basolateral surface. All these features indicate that the BUMPT-306 cell line, which is derived from a single cultured tubular cell, has structural and functional features of a homogenous population of differentiated proximal tubular cells.

Passaging of BUMPT-306 cells. The BUMPT-306 cells were passaged under permissive conditions in P100 culture dishes. For all experimental studies, cells were grown to confluence on culture dishes coated with rat tail collagen under permissive conditions. When close to confluence, monolayers were then incubated under nonpermissive conditions for 2 days before experimental studies 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 KH2PO4, 15 HEPES, 1 CaCl2, and 1 MgCl2. Dextrose (10 mM) was added to the medium of control cells. ATP depletion was induced with medium containing sodium cyanide (NaCN; 5 mM) to inhibit mitochondrial function and an absence of dextrose (to inhibit glycolysis), as described previously (30, 46). ATP depletion followed by recovery was achieved by incubating the cells in CN/no dextrose for 45 min and then incubating the cells in medium containing 10 mM dextrose without CN for an additional 30 min (the CN washout period) (30, 46). In some studies, Src was inhibited in dextrose- and CN-treated cells using PP2 (10 µM), a specific inhibitor of Src (24).

Measurement of cellular ATP content. Cellular ATP was measured using the luciferase assay as previously described (46). ATP levels were measured in control (dextrose-treated) cell monolayers and after 5, 10, 15, 30, and 45 min of incubation with CN. ATP levels at each of these time points are expressed as the percentage of control values.

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, and 10 NaPiPO4, as well as 0.5% Na-deoxycholate, 0.1% SDS, 1% Triton X-100, and 10% glycerol. Sodium vanadate [1 mM, a tyrosine phosphatase inhibitor and the protease inhibitors contained in Complete tablets (Boeringer Mannheim)] were added to the lysis buffer just before use. The lysate was harvested and centrifuged at 4°C for 15 min at 2,000 rpm, and the supernatants were saved and kept on ice. Protein G-Sepharose beads were prepared for use by washing with washing buffer [(in mM) 20 Tris · HCl, pH 7.5, 140 NaCl, 1 EDTA, pH 7.5, 1 vanadate, 1 NaF, 1 PMSF, and 10 NaPiPO4, 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 this precleared sample using a polyclonal antibody to c-Src (Santa Cruz Biotechnology) conjugated to the protein G beads. Immunoprecipitates were washed with washing buffer and used to measure Src kinase activity or to immunoblot for proteins associated with Src.

Src kinase assay. The immunoprecipitates were suspended in a reaction buffer containing a synthetic peptide as the Src kinase substrate peptide (150 µM/assay) and [gamma -32P]ATP and incubated for 10 min at 30°C. The reactions were stopped by incubating the immunoprecipitates with 40% TCA (20 µl/tube) at room temperature for 5 min. The beads were pelleted, and the supernatant (10 µl/tube) was transferred to p81 paper assay squares. The assay squares were washed six times for 5 min each with 40 ml of 0.75% phosphoric acid and once with acetone. Assay squares were then transferred to scintillation vials each containing 5 ml scintillation cocktail, and counts incorporated were read on a scintillation counter. The kinase activity was expressed as picomoles of phosphate incorporated per minute per milligram protein.

Western blotting. Lysates immunoprecipitated with c-Src antibody were boiled in 2× SDS sample buffer for 10 min. The beads were then spun down, and the supernatant containing the immunoprecipitated proteins were subjected to SDS-PAGE and immunoblotted with appropriate antibodies to E-cadherin, pp120, alpha -, beta -, and gamma -catenin, p120, or PY20. Immunoblots were probed with horseradish peroxidase-conjugated secondary antibodies using chemiluminiscence. Each immunoblot was stripped and reprobed with the antibody used for immunoprecipitation to confirm that equal amounts of immunoprecipitated protein were loaded in each lane.

Immunofluorescence studies. BUMPT-306 cells were grown to confluence on collagen-coated coverslips and treated with NaCN or dextrose for 45 min at 37°C. The cells were fixed in 3.7% paraformaldehyde for 10 min at room temperature (RT). Then, monolayers were washed three times for 5 min each in Tris-buffered saline (TBS; 50 mM Tris, pH 7.6, 150 mM NaCl) and incubated with 1% BSA, prepared in TBS, for 15 min at RT. For visualization of c-Src, E-cadherin, and catenins, cells were then incubated with the appropriate primary antibody diluted in 1% BSA for 45 min at RT. After a washing with TBS, the cells were incubated with Cy3-conjugated secondary antibody for 45 min at RT. Unbound secondary antibody was removed by washing. The F-actin filaments were stained directly with rhodamine phalloidin (Molecular Probes) as described previously (30). After being stained, the cells were mounted on glass slides and photographed using an immunofluorescence microscope and a digital camera.

Measurement of epithelial permeability. The permeability of epithelial monolayers was determined with methods previously 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 with warm Krebs-Henseleit buffer [KHB; (in mM) 115 NaCl, 3.6 KCl, 1.3 KH2PO4, 25 NaHCO3, 1 CaCl2, and 1 MgCl2]. Then, KHB containing [3H]inulin was gently layered on the apical aspect of the monolayers, and the inserts were placed in 24-well plates containing KHB without added inulin. We determined that the volume of added KHB exerted no hydrostatic pressure across the monolayer. All experiments were performed in a humidified 95% air-5% CO2 incubator at 37°C. The amount of inulin [counts/min (cpm)] in the apical and basal 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 × Delta C) where F is the rate of flux of inulin from apical to basal compartments per second, Delta C is the inulin concentration gradient, and S is the surface area of the Millipore insert.

Initially, the KHB in the apical and basal compartments contained dextrose (5 mM) in all experiments. After a 1-h equilibration period, basal epithelial permeability was measured in all inserts during a further four periods by moving the inserts every 5 min into fresh wells containing KHB+dextrose. Basal permeability was calculated by averaging the four values obtained. Then, the KHB in the apical and basal compartments 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.

Statistics. Data are presented as means ± SE. All statistical comparisons were 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 cell protein. After treatment of cells with CN in the absence of dextrose for 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 < 0.01 vs. control levels). These values are comparable to those we obtained in response to CN treatment of primary cultures of mouse proximal tubular cells (30, 46) and of Madin-Darby canine kidney cells (46).

ATP depletion inceases c-Src kinase activity. The activity of c-Src increased within 5 min of ATP depletion and remained elevated for the duration of the 45-min period of ATP depletion (n = 5) (Fig. 1). PP2 (10 µM), a specific inhibitor of Src kinase (43), completely inhibited the increase in c-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: chemical anoxia induced by cyanide (CN) resulted in an ~3-fold increase in c-Src activity by 5 min of CN treatment (CN 5'). Src activity remained elevated throughout the 45 min of CN treatment (CN 10' and CN 45'). *P < 0.01 vs. dextrose. B: the increase in c-Src activity induced by 10 min of CN treatment was prevented by the specific Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). *P < 0.01 vs. dextrose. dagger P < 0.01 vs. CN.

Inhibition of c-Src kinase activity ameliorates the increase in epithelial permeability associated with ATP depletion. The permeability of epithelial monolayers was assessed by measuring the flux of tritiated inulin across monolayers of cells grown on permeable supports using methods previously described by our group (30, 40) (see MATERIALS AND METHODS for details). Cells grown to confluence on permeable supports were incubated with dextrose or CN in the presence and absence of 10 µM PP2. The basal permeability of dextrose-treated monolayers was 8.3 ± 1.5 cm/s × 10-5 and remained constant for the duration of the experiment (60 min; n = 5) (Fig. 2). The permeability of monolayers treated with dextrose and PP2 (25 µM) was comparable to those treated with dextrose alone (n = 5) (Fig. 2). In monolayers treated with CN, permeability rose from the basal value of 8.3 ± 1.5 to 27.3 ± 3.1 cm/s × 10-5 within 10 min and remained relatively constant at that value for the duration of the experiment (P < 0.01 vs. dextrose, ANOVA for repeated measures; n = 8) (Fig. 2). In the presence of CN and PP2, permeability rose from 10.5 ± 1.7 to 14.5 ± 2.4 cm/s × 10-5 by 10 min and remained relatively unchanged for 60 min (P < 0.02 vs. CN alone and dextrose+PP2). Thus inhibition of c-Src kinase activity with PP2 ameliorated, but did not completely prevent, the CN-induced loss of epithelial barrier function. We conclude that impaired barrier function of BUMPT-306 cells, induced by CN-induced chemical anoxia, is partly mediated 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 control levels (Fig. 2), we infer that factors in addition to Src activation must contribute to the loss of permeability associated with CN.


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Fig. 2.   Effect of chemical anoxia on the permeability of mouse proximal tubular (MPT) monolayers in the presence and absence of Src inhibition. MPT cell monolayers were grown to confluence on permeable supports and treated with dextrose or CN in the presence or absence of PP2. Epithelial permeability was measured using [3H]inulin as a marker. CN treatment increased the permeability of the cell monolayers by 5-fold. The increase in permeability induced by CN was markedly ameliorated, but not completely prevented, by the presence of PP2. *P < 0.01 vs. CN+PP2. dagger P < 0.02 vs. dextrose and dextrose+PP2.

Immunofluoresence studies of the effects of CN on ZA proteins, actin, and c-Src. Immunofluoresence techniques were used to examine the distribution of E-cadherin, as well as alpha - and beta -catenin, in BUMPT-306 cells subjected to chemical anoxia in the presence and absence of PP2. In control, dextrose-treated cells, E-cadherin was localized predominantly at cell-cell borders as expected for a normal epithelial monolayer. CN-induced chemical anoxia caused a marked decrease in the amount of E-cadherin at cell-cell junctions and an increase in the protein within cytosolic aggregates (Fig. 3). PP2 largely prevented the disruption of normal E-cadherin localization induced by CN (Fig. 3). Comparable results were obtained when the effect of CN and CN+PP2 on the distribution of other ZA proteins was studied (alpha - and beta -catenin; data not shown).


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Fig. 3.   Immunofluoresence microscopy of E-cadherin. In control monolayers (top), E-cadherin was distributed predominantly at cell-cell borders (arrows). CN treatment (middle) resulted in a redistribution of E-cadherin with loss of staining at cell-cell borders (arrows) and the presence of aggregates of E-cadherin in the cytosol. Inhibition of PP2 (bottom) inhibited the redistribution of E-cadherin induced by ATP depletion.

CN treatment also resulted in the disruption of actin stress fibers without affecting the ring of cortical actin (Fig. 4). PP2 did not ameliorate the CN-induced disruption of the actin stress fibers. These data suggest that activation of Src contributes to the loss of the structural integrity of the ZA associated with ATP depletion but appears to have no role in modulating the disruption of the actin stress fibers associated with chemical anoxia.


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Fig. 4.   Fluorescence microscopy of the actin cytoskeleton. Control cells (top) demonstrate the normal appearance of the actin cytoskeleton, with stress fibers present within the cell (arrows). In CN-treated cells (middle), the stress fibers are severely disrupted while the actin ring, present at cell-cell borders (arrows), appears unaffected by ATP depletion. PP2 does not ameliorate the disruption of stress fibers associated with CN.

We also examined the effect of CN on the distribution of Src kinase in the BUMPT-306 cells. In control cells, c-Src was present within the cytosol, predominantly in a perinuclear distribution but also peripherally in a speckled pattern (Fig. 5). There was no evidence of c-Src at cell-cell borders in control cells, whereas in cells treated with CN for 45 min c-Src was present at cell-cell borders (Fig. 5). PP2 prevented the appearance of c-Src at cell-cell borders associated with CN treatment (Fig. 5).


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Fig. 5.   Immunofluorescence microscopy of c-Src. In control cells (top), c-Src was present predominantly within the cytosol in a perinuclear distribution. However, there was also Src staining peripherally, in a speckled pattern, consistent with localization of Src in adhesion plaques. There was no c-Src at cell-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-cell borders associated with chemical anoxia.

Effects of CN on coimmunoprecipitation with ZA- and focal adhesion-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 monolayer were subjected to coimmunoprecipitation using a monoclonal antibody to v-Src, and the immunoprecipitates were subjected to SDS-PAGE and then probed by Western blotting for p120, beta -catenin, E-cadherin, or FAK. ATP depletion increased the association of Src with all three ZA proteins (p120, beta -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, beta -catenin, E-cadherin, and FAK were immunoprecipitated from lysates obtained from dextrose-, CN-, and CN+PP2-treated cells and probed for Src (data not shown). In contrast to the effects of CN on Src association with the ZA proteins, CN had no effect on the degree of association of Src with FAK (Fig. 6).


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Fig. 6.   Effect of chemical anoxia on the amount of zona adherens (ZA) proteins that coimmunoprecipitates with c-Src. Lysates from cells treated with either dextrose or CN for 5, 10, 15, and 45 min, dextrose+PP2 for 10 min, or CN+PP2 for 10 min were immunoprecipitated with c-Src antibody, subjected to SDS electrophoresis, and immunoblotted with antibodies to beta -catenin, E-cadherin, p120, and focal adhesion kinase (FAK). ATP depletion with CN markedly increased the association of c-Src with both catenins and E-cadherin, an effect prevented by PP2. However, CN did not alter the association with FAK. The figure shows a representative blot from 5 experiments. WB, Western blot.

Effect of CN on tyrosine phosphorylation of beta -catenin, p120, E-cadherin, and FAK. Finally, we examined the effect of ATP depletion on the degree of tyrosine phosphorylation of p120, beta -catenin, E-cadherin, and FAK. We immunoprecipitated each of these proteins from lysates of monolayers treated with dextrose, CN, dextrose+PP2, and CN+PP2 and immunoblotted the precipitates with a tyrosine phospho-specific antibody (PY20). CN treatment increased the degree of tyrosine phosphorylation of both p120 and beta -catenin, an effect that was completely prevented by pretreatment 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 beta -catenin and p120, CN induced a fall in tyrosine phosphorylation of FAK, an effect prevented by PP2 (Fig. 7).


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Fig. 7.   Effect of chemical anoxia on the state of tyrosine phosphorylation of p120, beta -catenin, E-cadherin, and FAK. Lysates from cells treated with either dextrose or CN in the presence and absence of PP2 were immunoprecipitated with an antibody to p120, beta -catenin, E-cadherin, or FAK and then immunoblotted with PY20 antibody to detect tyrosine phosphorylation. CN treatment resulted in marked hyperphosphorylation of both p120 and beta -catenin. PP2 prevented the hyperphosphorylaton of the 2 catenins induced by CN. E-cadherin was not tyrosine phosphorylated under either basal or CN-treated conditions. FAK, which was tyrosine phosphorylated under control conditions, became dephosphorylated during CN treatment, an effect prevented by PP2. The figure shows a representative blot from 4 experiments.


    DISCUSSION
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An important functional consequence of sublethal ischemic injury to renal tubular cells is loss of the normal barrier function of the tubular epithelium (4, 7, 10, 30, 35, 36). To elucidate 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 focused instead 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 to the functional integrity of the ZO. The importance of the ZA in tight junctional function has been demonstrated in two ways. Removal of extracellular calcium impairs ZO function (12, 23, 45). Because formation of the ZO itself does not require calcium, these data demonstrate that a structurally intact ZO cannot maintain the epithelial permeability barrier in the absence of a ZA. Also, direct inhibition of ZO assembly using neutralizing antibodies to E-cadherin has also been shown to impair tight junctional integrity and function (11, 21, 22).

We have previously demonstrated in a series of articles that CN-induced injury to tubular cells increases the permeability of the tubular cell monolayer and results in the disassociation of the ZA from its normal location at cell-cell junctions, as evidenced by the withdrawal of E-cadherin as well as alpha -, beta -, and gamma -catenin to the basolateral membrane of tubular cells (30, 40, 44). Similar alterations in the distribution of ZA proteins have been shown by other investigators after ischemic injury to tubular cells (8, 31, 35). Our group has also reported that chemical anoxia is associated with increased tyrosine phosphorylation of beta -catenin and that the loss of ZA integrity and of tight junctional function is due, at least in part, to the changes in regulation of tyrosine phosphorylation induced by ATP depletion (44). Our findings are consistent with the role of tyrosine phosphorylation of beta -catenin as a regulator of cell-cell adhesion under physiological conditions (5, 6, 9, 13).

Some evidence suggests that alterations in ZA phosphorylation and cell-cell adhesion in nonrenal epithelial cells may be due to increased activity of a nonreceptor member of the Src kinase family (9, 48). The purpose of this study was to determine whether Src activation and phosphorylation of ZA proteins contribute to the loss of integrity of the ZA that we (44) and others (8, 31, 35) have previously observed in response to chemical anoxia.

We provide entirely novel evidence that chemical anoxia activates c-Src in tubular cells (Fig. 1). Our data showing that PP2 partly inhibits the loss of epithelial permeability and withdrawal of ZA proteins from cell-cell borders associated with chemical anoxia suggest a participatory 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 Src inhibition in ameliorating the disruption of actin stress fibers suggests that this alteration in the actin cytoskeleton is not an important event in the loss of integrity of the ZA associated with chemical anoxia. These data are consistent with the notion that the actin ring, a component of the actin cytoskleleton that is relatively preserved during chemical anoxia (Fig. 4), is more relevant to the stability of the ZA than actin stress fibers.

We also provide entirely novel evidence that chemical anoxia induces translocation of c-Src to the ZA. Immunofluoresence studies show that CN induces the appearance of cSrc at cell-cell borders, an effect prevented by Src inhibition with PP2 (Fig. 5). In addition, using immunopreciptation and immunoblotting we demontrate that CN induces an increased association of c-Src with the ZA proteins beta -catenin, p120, and E-cadherin (Fig. 6). However, CN does not change the degree of association of c-Src with FAK, a protein localized in adhesion plaques (Fig. 6).

We also show that CN treatment increases tyrosine phosphorylation of p120 and beta -catenin (Fig. 7), both of which are known substrates of Src (2, 13, 41). The tyrosine phosphorylation of p120 and beta -catenin during ATP depletion is inhibited by PP2 (Fig. 7). These findings are consistent with a role for c-Src in the tyrosine phosphorylation of beta -catenin and p120 during ATP depletion. However, E-cadherin, unlike p120 and beta -catenin, is not tyrosine phosphorylated under control or CN treatment conditions (Fig. 7). Taken together, our data suggest that Src binds to and phosphorylates both p120 and beta -catenin during chemical anoxia. The apparent increase in the association of E-cadherin with Src during CN treatment (shown by immunoprecipitation and immunoblotting studies in Fig. 6) is probably not due to the direct binding of Src to E-cadherin but rather to the presence of E-cadherin bound to catenins in complexes of ZA proteins.

In previous studies, beta -catenin and p120 have both been shown to be readily phosphorylated by protein tyrosine kinases (2, 13, 41). It is also well established that alterations in the degree of tyrosine phosphorylation of beta -catenin, which binds to the CBD of the cytoplasmic tail of cadherins (5, 13), is an important event in modulating cell-cell adhesion. The process of ZA formation and cell-cell adhesion requires tyrosine dephosphorylation of beta -catenin (5), whereas loss of cell-cell adhesion is associated with an increase in the tyrosine phosphorylation of beta -catenin (13). Thus it is likely that c-Src-induced phosphorylation of beta -catenin observed in this study contributes to the loss of tubular cell-cell adhesion associated with sublethal injury induced by chemical anoxia.

While there is also evidence to support a role for p120 in modulating cell-cell adhesion, the mechanisms involved are less well defined than for beta -catenin (2). There is evidence that serine/threonine phosphorylation as well as tyrosine phosphorylation of p120 plays a role in regulating p120 function and cell-cell adhesion (3, 39). It is well established that Src is able to tyrosine 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 p120 as well as of beta -catenin (17). However, it has not as yet been possible to distinguish the functional consequences of tyrosine phosphorylation of p120 from the effects of phosphorylation of the many other targets of activated Src, including beta -catenin (2). Given the present uncertainty of the effects of tyrosine phosphorylation of p120 on cell adhesion, we cannot as yet speculate on the significance of our observation that p120 is tyrosine phosphorylated by c-Src during chemical anoxia (Fig. 7).

Interestingly, we show that CN decreases tyrosine phosphorylation of adhesion plaque protein FAK, an effect that is the opposite of what happens to phosphorylation of the catenins (Fig. 7). Other investigators have also demonstrated that FAK becomes tyrosine dephosphorylated during ATP depletion (52). While hyperphosphorylation of the catenins is known to be associated with loss 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 opposite effects on the degree of tyrosine phosphorylation of ZA and adhesion plaque proteins, these effects are consistent with the loss of both cell-cell and cell-matrix adhesion, well-known functional effects of sublethal injury (10, 30).

In summary, we have shown for the first time that CN-induced chemical anoxia activates c-Src and leads to the translocation of the activated form of c-Src to the ZA. There, c-Src binds to and phosphorylates beta -catenin and p120. We also show that these effects of c-Src contribute to the structural and functional loss of the ZA and to the increase in epithelial permeability associated with sublethal injury. Further studies are necessary to define in more detail the effects of Src-induced phosphorylation that occur in response to chemical anoxia.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-385101, DK-59793, and DK-58306.


    FOOTNOTES

Address for reprint requests and other correspondence: W. Lieberthal, Renal Section, Evans Biomedical Research Ctr., Rm. 537, 650 Albany St., Boston, MA 02118 (E-mail: wliebert{at}bu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 5, 2002;10.1152/ajprenal.00172.2002

Received 1 May 2002; accepted in final form 23 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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