Protein kinase C modifications of VE-cadherin, p120, and {beta}-catenin contribute to endothelial barrier dysregulation induced by thrombin

Maria Konstantoulaki, Panos Kouklis, and Asrar B. Malik

Department of Pharmacology, University of Illinois, Chicago, Illinois 60612

Submitted 14 March 2003 ; accepted in final form 2 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The adherens junction is a multiprotein complex consisting of the transmembrane vascular endothelial cadherin (VEC) and cytoplasmic catenins (p120, {beta}-catenin, plakoglobin, {alpha}-catenin) responsible for the maintenance of endothelial barrier function. Junctional disassembly and modifications in cadherin/catenin complex lead to increased paracellular permeability of the endothelial barrier. However, the mechanisms of junctional disassembly remain unclear. In this study, we used the proinflammatory mediator thrombin to compromise the barrier function and test the hypothesis that phosphorylation-induced alterations of VEC, {beta}-catenin, and p120 regulate junction disassembly and mediate the increased endothelial permeability response. The study showed that thrombin induced dephosphorylation of VEC, which is coupled to disassembly of cell-cell contacts, but VEC remained in aggregates at the plasma membrane. The cytoplasmic catenins dissociated from the VEC cytoplasmic domain in thin membrane projections formed in interendothelial gaps. We also showed that thrombin induced dephosphorylation of {beta}-catenin and phosphorylation of p120. Thrombin-induced interendothelial gap formation and increased endothelial permeability were blocked by protein kinase C inhibition using chelerythrine and Gö-6976 but not by LY-379196. Chelerythrine also prevented thrombin-induced phosphorylation changes of the cadherin/catenin complex. Thus the present study links posttranslational modifications of VEC, {beta}-catenin, and p120 to the mechanism of thrombin-induced increase in endothelial permeability.

vascular endothelial cadherin; isoelectric focusing


ENDOTHELIAL CELLS SERVE the fundamental role of creating a continuous cell monolayer that is semipermeable to plasma constituents and is also highly regulated by blood-borne mediators. The barrier function is regulated by a number of cell-cell adhesive structures such as tight junctions and adherens junctions (AJ) (49). AJ is the primary site of action of endothelial permeability-modifying agents, such as thrombin and sphingosine-1-phosphate, that either increase or decrease permeability, respectively (11, 27, 29-31, 41). The proinflammatory mediator thrombin acts through its transmembrane heptahelical G-coupled receptors (proteolytically activated receptors) to induce AJ disassembly and barrier dysfunction (10, 12, 29, 30, 41). VE-cadherin (VEC), a type II classic cadherin, is expressed exclusively in endothelial cells and is responsible for endothelial cell-cell adhesion (35). VEC extracellular domain forms homotypic adhesive interactions in a Ca2+-dependent manner (25, 46). The same domain was recently shown to interact with an endothelial-specific transmembrane phosphatase (vascular endothelial protein tyrosine phosphatase; VE-PTP) (36). The cytoplasmic domain of VEC interacts with catenins p120, {beta}-catenin, and plakoglobin (also known as {gamma}-catenin). {beta}-catenin and plakoglobin bind in the COOH terminus of cadherins in a mutually exclusive manner (19, 24, 45). Both can associate with {alpha}-catenin, an actin-binding protein, which mediates the association between cadherin complex and actin cytoskeleton (20). This association is necessary for adhesion and phosphorylation of catenins, and cadherin cytoplasmic domain can regulate the strength of cell-cell adhesion (17, 19). Tyrosine phosphorylation of {beta}-catenin induced by Src or VEGF receptor negatively regulates adhesion, leading to AJ disruption (13, 37). However, serine/threonine phosphorylation induced by casein kinase II increases the strength of adhesion between cadherin and {beta}-catenin (28). Tyrosine phosphorylation of catenins can be regulated by tyrosine phosphatases such as the transmembrane leukocyte common antigen-related protein tyrosine phosphatase (LARPTP), PTPµ, and the cytoplasmic Src homology phosphatase-2 (SHP-2), shown to interact with {beta}-catenin (9, 33, 34, 48, 50). In endothelial cells, studies have shown that increased tyrosine phosphorylation of {beta}-catenin and downregulation of phosphatase activity are linked to increased endothelial permeability (7, 18, 23, 36).

p120 binds at the juxtamembrane domain of VEC, but its function in adhesion strength is controversial because it was shown to be both a positive and negative regulator of adhesion in epithelial cells (5). p120 can be phosphorylated by tyrosine kinases such as Src and associates with phosphatases such as LAR-PTP, PTPµ, and SHP-1 (4, 22, 33, 55). In addition, protein kinase C (PKC) isoforms can affect p120 dephosphorylation (39).

Thrombin-induced activation of PKC is an important determinant of the increased endothelial permeability response (29, 30, 44). Inhibition of PKC activation prevented the thrombin-induced increase in endothelial permeability (41). Increase in intracellular Ca2+ concentration, activation, and translocation of the conventional PKC{alpha} is also involved in AJ disassembly (41). In the present study, we addressed the molecular basis of thrombin-induced AJ disassembly. We identified for the first time the posttranslational modifications of AJ proteins during thrombin, which include phosphorylation and dephosphorylation of VEC, {beta}-catenin, and p120 and showed that these events were PKC dependent. Our results imply that in addition to kinases, phosphatases play an important role in thrombin-induced endothelial cell shape changes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial cell culture. Human pulmonary arterial endothelial cells (HPAE) purchased from BioWhittaker/Clonetics (Walkersville, MD) were grown in endothelial growth medium supplemented with bullets (EBM-2) medium supplemented with 10% FBS (Hyclone, Logan, UT), 0.1% VEGF, R3-IGF-I, ascorbic acid, human endothelial growth factor, GA-1000, heparin, 0.4% human fibroblast growth factor, and 0.04% hydrocortisone at 37°C in a humidified 5% CO2 incubator. The cells were used at passages 4–8. Confluent cells were treated with thrombin (4 U/ml; Enzyme Research Laboratories, South Bend, IN) in serum-free MCDB-131 medium (GIBCO, Grand Island, NY) or EBM-2 containing 1% FBS.

Antibodies and reagents. Goat polyclonal for VEC was purchased from Research Diagnostics, and monoclonal VEC was from Chemicon (Temecula, CA). The monoclonal antibodies for {beta}-cadherin, plakoglobin, and p120 were purchased from Transduction Laboratories (Lexington, KY). The rabbit polyclonal for {alpha}-catenin was purchased from Sigma Chemical. Anti-mouse horseradish peroxidase (HRP), anti-rabbit HRP, anti-goat HRP (Jackson Laboratories, West Grove, PA), donkey anti-goat/mouse/rabbit, and goat anti-mouse/rabbit (Alexa) were purchased from Molecular Probes. Phalloidin-A568 (Alexa) was purchased from Molecular Probes. Chelerythrine (a pan-PKC inhibitor), Gö-6976 [conventional (c)PKC{alpha} inhibitor or cPKC{alpha} and cPKC{beta}, depending on the concentration], and LY-379196 (cPKC{beta} inhibitor) were purchased from Calbiochem (San Diego, CA).

Immunofluorescence staining. Control cells or cells treated with thrombin or with inhibitors were washed three times with PBS and fixed with 3.7% formaldehyde for 10 min at room temperature (RT). After being washed with PBS, cells were soaked first in permeabilization buffer (PBS and 0.4% Triton X-100) and then in blocking solution (20 mM HEPES, pH 7.9, 250 mM KCl, 1% BSA, 0.4% gelatin, 0.05% NaN3) for 10 min at RT. The first antibody was incubated for 1 h at RT, usually in dilution 1:50 (except plakoglobin 1:20, {alpha}-catenin 1:100), and the secondary antibody for 1 h, in dark and at RT, usually in dilution 1:150. The secondary antibodies used were goat anti-mouse Alexa 488 (green), goat anti-rabbit Alexa 568 (red), donkey anti-goat Alexa 594 (red; Molecular Probes, Eugene, OR), donkey anti-mouse FITC, and donkey anti-rabbit FITC (Jackson Immunoresearch Laboratories, West Grove, PA). Pictures were captured with a Zeiss Pascal confocal microscope and assembled in Adobe Photoshop.

In vivo labeling of VEC. Confluent HPAE cells grown on coverslips were incubated for 1 h before thrombin treatment with 1 mg/ml of monoclonal VEC antibody (Chemicon) that recognizes the extracellular domain of the molecule. The cells were washed with PBS and serum-free medium and incubated with thrombin. Upon fixation and permeabilization, the cells were stained with a secondary anti-mouse antibody. The cells were then processed with a Zeiss Pascal confocal microscope where different sections from the basal to the apical surface (Z-stack images) were captured.

Transendothelial electrical resistance measurement. Endothelial cell retraction, an indication of increased endothelial permeability, was measured (47). HPAE cells were seeded on a gelatin-coated gold electrode (5.0 x 104 cm2) and grown to confluence. The small electrode and the larger counterelectrode were connected to a phase-sensitive lock-in amplifier. A constant current of 1 µA was supplied by a 1-V, 4,000-Hz alternate current signal connected serially to a 1-M{Omega} resistor between the small electrode and the larger counterelectrode. The voltage between the small electrode and the large counterelectrode was monitored by a lock-in amplifier, stored, and processed by a personal computer. The same computer controlled the output of the amplifier and switched the measurement to different electrodes in the course of an experiment. Before the experiment, confluent endothelial monolayers were washed with serum-free growth medium and were then used for measuring the thrombin-induced change in endothelial monolayer electrical resistance. The data are presented as the change in resistive portions of the resistance normalized to its value at time 0.

Two-dimensional electrophoresis. Control confluent endothelial cells or confluent endothelial cells stimulated with thrombin (4.4 U/ml) or chelerythrine-pretreated cells (for 30 min at a concentration of 10 µM), which were then treated with thrombin, were washed with PBS and removed from the dishes by scraping, centrifuged (Eppendorf microcentrifuge, 4°C, 2,500 rpm, 5 min), and resuspended in two-dimensional sample buffer containing 9.8 M urea, 2% [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 0.2% ampholyte 5-7 (Bio-Lyte; Bio-Rad, Hercules, CA), 100 mM DTT, and traces of bromphenol blue (similar to rehydration buffer provided by Bio-Rad). The cell extracts were vortexed, placed in a rehydration chamber, and absorbed by the immobilized polyacrylamide gradient gels (IPGs; Bio-Rad). Membranes were prepared to obtain the profile of the membrane-bound VEC and catenins. The cells after treatment were washed with PBS, lysed in a hypotonic buffer (250 mM sucrose, 1 mM MgCl2, 10 mM Tris, pH 7.5, protease inhibitors), broken in a Dounce homogenizer, and pelleted to remove debris and the unbroken cells, and the supernatant was ultracentrifuged (Ti90 rotor; 1 h, 45,000 rpm). The pellet is the membrane fraction and is resuspended in the urea buffer and processed as the total extract. From the sequence of the proteins (VEC, {alpha}-catenin, {beta}-catenin, p120, plakoglobin), we calculated the isoelectric point (pI), which ranges from 5 to 6.5 (i.e., VEC pI = 5.01, {beta}-catenin pI = 5.43, plakoglobin pI = 5.68, {alpha}-catenin pI = 5.95, and p120 pI = 6.49). Thus we used IPG strips that form a pH gradient between 3 and 10. For p120, we observed better focusing with 4–7 IPG strips. Both the rehydration and the isoelectric focusing were carried out in the Protean IEF cell (Bio-Rad). The conditions were set up in passive rehydration for 12 h in 20°C and immediately after the three-stepped isoelectric focusing started (250 V for 20 min, 2 h in 4,000 V and 12,000 volt hours). The temperature was maintained at 20°C. Immediately thereafter, the gels were equilibrated for 20 min in buffers containing either 6 M urea, 2% SDS, 0.375 M Tris, pH 8.8, 20% glycerol, and 130 mM DTT (buffer I) or 6 M urea, 2% SDS, 0.375 M Tris pH 8.8, 20% glycerol, and 135 mM iodoacetamide (buffer II). After the equilibration step, the gels were transferred to 7.5% SDS-PAGE gels (second dimension). After SDS-PAGE, the proteins were transferred to a 0.45-µm nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were blocked either overnight in 4°C or for 1 h at RT with 20 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 with 1% gelatin (from cold-water fish skin; Sigma) and stained for catenins (dilution 1:3,000, except plakoglobin 1:1,000 and {alpha}-catenin 1:5,000) and VEC (goat polyclonal; 1:3,000, Research Diagnostics). All the secondary antibodies (anti-mouse, anti-rabbit, anti-goat) conjugated with HRP were purchased from Jackson Immunoresearch Laboratories. Antibody-positive bands were visualized by an enhanced chemiluminescence assay (Super Signal West Pico; Pierce, Rockford, IL).

Immunoprecipitation. Cells were washed with PBS, pelleted at a low speed (Eppendorf microcentrifuge, 4°C, 2,500 rpm, 5 min), lysed with immunoprecipitation buffer (10 mM Tris, pH 7.5, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, and protease inhibitors), vortexed, and incubated for 30 min on ice. At the end of the incubation, the lysates were centrifuged (15 min, 14,000 rpm, 4°C) and precleared with mouse IgG and protein A/G. The precleared lysate was incubated with the catenin antibodies and protein A/G for 3 h. The beads were washed with immunoprecipitation buffer, resuspended in 2x Laemmli buffer, boiled, electrophoresed/transferred, and Western blotted with VEC and the corresponding catenins.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thrombin-induced AJ disruption in endothelial cells. HPAE cells were grown to confluence on glass coverslips as shown by VEC staining (Fig. 1A, untreated). Upon addition of thrombin (4 U/ml), the cells established a rounded morphology and formed intercellular gaps secondary to disruption of cell-cell adhesive structures. This effect in AJ was evident at 5 min with small intracellular gaps that increase in size at 15 min (Fig. 1A). Cell-cell interactions upon disruption of the junctions were maintained by thin membrane projections (Fig. 1A, arrowhead, 15-min thrombin). Disruption of cadherin-mediated junctions was reversible; that is, at 2 h (Fig. 1A), the cell junctions reassembled in the majority of cadherin junctions, although some small gaps persisted. The same phenomenon was evident using the measurement of electrical resistance of HPAE cell monolayers grown on gold electrodes, a measure of transendothelial permeability. Transendothelial electrical resistance decreased on addition of thrombin (arrowhead) and returned to baseline within 2 h (Fig. 1, B and C). Hence, thrombin modified the VEC junctions in a rapid and reversible manner.



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Fig. 1. Transient and reversible effects of thrombin on adherens junctions of endothelial cells. A: vascular endothelial cadherin (VEC) staining on confluent human pulmonary arterial endothelial (HPAE) cells at different time points before (untreated) and after thrombin (5 min, 15 min, 2 h). Thrombin induces junction disruption that is evident in 5 min with small gaps (arrow) and more pronounced in 15 min, when gaps are bigger (arrow). The junctions are reassembled in 2 h, although some gaps are still visible (arrow). In 15 min, the arrowhead shows the thin membrane projections between 2 neighboring cells. Bar, 10 µm. Results represent 3 experiments. B: measurement of electrical resistance in untreated and thrombin-treated cells. Cells were plated on golden electrodes, grown to confluency, and stimulated with thrombin. Thrombin decreases the resistance of the endothelial barrier by inducing cell shape change. Results represent 3 experiments. Thr, thrombin. C: bar graph of the change in resistance in 15 min and 1 and 2 h after thrombin. The untreated cells had no change in resistance. The graphs summarize 5 experiments. Results are shown as means ± SE.

 

VEC-cadherin aggregates in plasma membrane during AJ disassembly. We stained living confluent HPAE cells grown on glass coverslips with VEC antibody recognizing the extracellular domain. These labeled cells were then treated with thrombin for 15 min before formaldehyde fixation. Cells were permeabilized, stained with secondary FITC-conjugated antibody, and analyzed with Z-stack sectioning in a Zeiss Pascal confocal microscope (each micrograph depicts a level from basal to apical). Staining was detected solely in the plasma membrane, indicating that VEC remained at the plasma membrane (Fig. 2). At higher magnification, we observed areas of aggregates of VEC and other areas where VEC staining was absent.



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Fig. 2. VEC remains in plasma membrane but in aggregated form after thrombin exposure. HPAE were incubated with VEC antibody before thrombin treatment and were photographed in different levels (Z-stack). A: HPAE cells 15 min after thrombin stimulation, photographed from the basal to the apical. Each photograph shows a different level of the cells. VEC exclusively stains the membrane, where it aggregates in some areas but is absent from others. Bar, 10 µm. B: higher magnification of one of the middle sections in which the arrow points to an area of aggregated VEC molecules. The arrowhead points to an area where VEC is absent from the plasma membrane. This is a strong indication that aggregation and not internalization of VEC is of major importance in the disassembly of adherens junctions upon thrombin stimulation. Bar, 10 µm. C: control endothelial cells before thrombin stimulation. Bar, 20 µm. Results represent 3 experiments.

 

Localized dissociation of catenins from VEC in thin membrane projections. VEC normally associates with {alpha}-, {beta}-, and p120-catenin and plakoglobin ({gamma}-catenin). By immunoprecipitation, we did not detect any changes in the association of the catenins with VEC after thrombin; that is, the amount of VEC pulled down by {beta}-catenin did not change in 30 s or 5 or 15 min after thrombin exposure (Fig. 3).



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Fig. 3. Coimmunoprecipitation of {beta}-catenin with VEC. VEC was coimmunoprecipitated with {beta}-catenin before thrombin (0, lane 2), immediately after thrombin stimulation (30 s, lane 3), after 5 min (lane 4), and after 15 min (lane 5). The first lane shows the mouse IgG (mIgG) control where nonspecific proteins are not detected. {beta}-catenin antibody was used for immunoprecipitation, and the nitrocellulose membrane was blotted against VEC and {beta}-catenin in 2 different Western blots without stripping the membrane. The numbers indicate the molecular weight sizes (mol wt of VEC 135,000, mol wt of {beta}-catenin 92,000). Results represent 5 experiments.

 

To further visualize the pattern of colocalization of the VEC with catenins, we doublestained the cells with red (VEC) and green ({alpha}-, {beta}-, and p120-catenin and plakoglobin) fluorescence and studied the alterations by confocal microscopy. The cadherin and catenin merged images (yellow) showed marked colocalization in the plasma membrane of unstimulated confluent HPAE cells (Fig. 4). Thrombin stimulation induced AJ disassembly, as evident by formation of numerous intercellular gaps. However, the association of VEC with the catenins remained intact in the plasma membrane, as shown by the merged images (Fig. 4). Also, we observed that the catenins were locally dissociated from VEC in the membrane extensions formed after thrombin stimulation.



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Fig. 4. Association of catenins with VEC but localized disruption on thin membrane projections. VEC colocalizes with catenins ({alpha}-catenin, {beta}-catenin, plakoglobin, p120) before and during thrombin treatment on the plasma membrane (merged color, red: VEC, green: catenins). Upon thrombin administration to the HPAE cells, gaps are formed and adherens junctions are disrupted, forming thin membrane projections that keep contact between adjacent cells. On these thin membrane projections, catenins dissociate from VEC. The third column shows higher magnification of the membrane projections where VEC stains the projections without associating with the catenins. Bar, 10 µm in untreated and 15-min thrombin-treated cells and 5 µm in higher magnification. Results represent 5 experiments.

 

PKC involvement in disassembly of AJ. Because the localized dissociation of catenins from VEC in the membrane extensions may be the result of phosphorylation/dephosphorylation of AJ complex proteins, we addressed the involvement of PKC in modifying AJ. We first used chelerythrine, a pan-PKC inhibitor. Chelerythrine pretreatment alone did not affect AJ or the colocalization of VEC with catenins (Fig. 5A). In chelerythrine-pretreated cells, thrombin stimulation failed to disrupt AJ and induce formation of intercellular gaps (Fig. 5A), indicating an important role of PKC in the mechanism of AJ disruption. We also showed that chelerythrine prevented the thrombin-induced decrease in transendothelial electrical resistance (Fig. 5B). We also used PKC{alpha} (Gö-6976; Fig. 5, C and D) or PKC{beta} (LY-379196; Fig. 5E) inhibitors. PKC{beta} inhibition did not have any effect on AJ disassembly induced by thrombin (Fig. 5E). However, Gö-6976, at 100 nM concentration, which inhibits the activation of PKC{alpha} (A. Rahman, personal communication), prevented the thrombin-induced VEC junction disassembly (Fig. 5C). Gö-6976 also prevented the thrombin-induced decrease in transendothelial electrical resistance (Fig. 5D). Figure 5F summarizes the effects of Gö-6976 and chelerythrine on the thrombin-induced resistance changes.



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Fig. 5. Protein kinase C (PKC) inhibition prevents adherens junction disassembly. Chelerythrine, a broad-range PKC inhibitor, prevents the disruption of adherens junctions upon thrombin stimulation as judged by immunofluorescence (A) and measurement of electrical resistance (B). The same effect on the adherens junctions is achieved with treatment with 100 nM of the conventional (c)PKC{alpha} and cPKC{beta} inhibitor Gö-6976 (C and D). The above data indicate that cPKC{alpha} can modulate VEC-mediated junctions. PKC{beta} inhibition does not have any effect on the thrombin-induced adherens junction disruption (E). A: doublestaining with VEC and {beta}-catenin in chelerythrine alone or chelerythrine and thrombin-treated cells. B: cells were plated on golden electrodes, grown to confluency, and stimulated with thrombin, chelerythrine, or chelerythrine + thrombin. Thrombin decreases the resistance of the endothelial barrier, whereas chelerythrine and chelerythrine + thrombin fail to change cell shape and attenuate the barrier function. C: double staining with VEC and {beta}-catenin in Gö-6976 or Gö-6976 + thrombin. D: cells were plated on golden electrodes and stimulated with thrombin, Gö-6976, or Gö-6976 + thrombin. E: double staining with VEC and p120 in LY-379196 or LY-379196 + thrombin. Bar, 10 µm. Results in A–E represent 5 experiments. F: graph summarizing 5 different transendothelial resistance measurements for each group showing % change of resistance. Values are shown as means ± SE.

 

PKC-dependent alterations in phosphorylation of VEC, {beta}-catenin, and p120. To address the thrombin-induced posttranslational modifications of VEC and the associated catenins, we prepared cell extracts from untreated or thrombin-stimulated cells (for 15 min, 4 U/ml), which were ultracentrifuged to isolate the membrane fractions. We dissolved the pellet in urea buffer and isoelectrically focused the membrane pellet proteins using two-dimensional electrophoresis. The proteins in the first dimension were run in a gradient formed by immobilized ampholytes and were separated according to their charges. In the second dimension (SDS-PAGE), the proteins were separated according to their molecular weights. Posttranslational modifications leading to charge changes shift the isoelectric point toward the acidic or the basic side of the ampholyte gradient. Phosphorylation increases the negative charge and shifts the spot toward the positive or acidic end of the strip, whereas dephosphorylation increases the positive charge and induces the shift toward the negative or basic side. Extracts from untreated or thrombin-treated cells were isoelectrically focused, electrophoresed in a SDS-PAGE, transferred in nitrocellulose, and Western blotted with VEC, p120, {beta}-catenin, plakoglobin, and {alpha}-catenin (Figs. 6 and 7). VEC (mol wt 130,000, pI 5.01) shows the pattern in Fig. 6. Upon thrombin stimulation, a cluster of spots disappeared, and new spots appeared, in the basic side of the gel, indicative of dephosphorylation. Chelerythrine (which blocked AJ disassembly as shown above) prevented these alterations. In the basal state, {beta}-catenin (mol wt 92,000, pI 5.43) exists in five distinct isoforms (Fig. 6, {beta}-catenin). At 15 min after thrombin treatment, new spots appeared on the basic side, indicating that {beta}-catenin is also dephosphorylated after 15 min of thrombin stimulation. Chelerythrine prevented this response. p120 (mol wt 100,000 and 120,000, pI 6.49) shows five distinct isoforms in the basal state (Fig. 6, p120). After thrombin, new spots appeared in the acidic position, indicating phosphorylation. The response was prevented by chelerythrine. In contrast to VEC, p120, and {beta}-catenin, plakoglobin (mol wt 82,000, pI 5.68) and {alpha}-catenin (mol wt 102,000, pI 5.95) showed no distinct changes in their pattern after thrombin (Fig. 7).



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Fig. 6. Isoelectric focusing of VEC/catenin complex: response to thrombin and effects of PKC inhibition. Untreated: profile of VEC, {beta}-catenin, and p120 in the basal situation. The numbers indicate the different isoforms. +, acidic side; -, basic side of the gel. 15-min thrombin: VEC and {beta}-catenin are dephosphorylated upon thrombin stimulation (left shift, arrowheads) and p120 is phosphorylated. 15-min thrombin and chelerythrine: chelerythrine- and thrombin-treated cells. Chelerythrine prevents the dephosphorylation of VEC and {beta}-catenin and the phosphorylation of p120. Results represent 5 experiments.

 


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Fig. 7. Isoelectric focusing of {alpha}-catenin and plakoglobin. Untreated: profile of {alpha}-catenin and plakoglobin in the basal situation. +, acidic side; -, basic side of the gel. 15-min thrombin: profile of {alpha}-catenin and plakoglobin after thrombin stimulation. Results represent 5 experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A fine balance between tethering and contractile forces serves to maintain the barrier function in endothelial cells. Thrombin is a serine protease with proinflammatory properties that induce the activation of the actinomyosin-driven endothelial contractile machinery (10, 16, 31, 47). Thrombin binds to a heterotrimeric G protein-coupled receptor, proteolytically activated receptor-1, in endothelial cells and activates the second messengers Ca2+ and PKC. Endothelial cells change their shape to a rounded morphology (12, 29, 51). This response is the result of increased contractile force induced by actin stress fiber formation and myosin light chain phosphorylation as well as disruption of the cell-to-cell junctions leading to intercellular gaps (12, 29, 30, 41, 51). It has been shown in a series of inhibitor experiments that PKC and myosin light chain kinase inhibition prevented the cell retraction and rounding induced by thrombin (15, 38). Other studies also showed that inhibitors of cAMP-dependent protein kinase, phosphatidylinositol 3-kinase, MAPK/ERK, and Src kinases had no effect on these responses (51).

We observed that the thrombin-induced AJ disassembly was transient and that reformation of junctions was largely complete within 2 h. We also observed that VEC at 15 min after thrombin stimulation formed aggregates in some areas of the plasmalemma and was absent from others. It was recently shown that plate-let/endothelial cell adhesion molecule-1, VEC, and CD99 localized both in the plasma membrane and in reticular membrane structures called subjunctional reticulum, just below the plasmalemma, connected to the junctional surface when endothelial monolayers were labeled with specific FITC-conjugated monoclonal antibodies in vivo (32). These molecules seemed to rapidly recycle between the surface and the subjunctional reticulum. At this point, we cannot conclude whether, upon thrombin treatment, VEC aggregates are at the plasma membrane only or are also moving to specific subjunctional compartments. Interestingly, in endothelial cells treated with Ca2+ chelators, which interfere with homotypic interaction of cadherin trans-mers, VEC appears diffuse (14) and is probably endocytosed (3). VEC behavior during thrombin treatment differs from preconfluent or extracellular Ca2+-depleted epithelial cells, in which E-cadherin shuttles between the membrane and cytosol, and as such its endocytosis and recycling are markedly increased in the absence of stable cell-cell contacts (2, 21, 26).

Coimmunoprecipitation studies showed that catenins immunoprecipitated with VEC following thrombin. This finding is in agreement with morphological observations showing that the components of the VEC/catenin complex clearly colocalized in areas of the plasmalemma. Evidence of catenin dissociation from VEC was seen only in the membrane extensions formed between cells. It was shown previously that p120 and plakoglobin dissociate, at least to some extent, from the VEC complex during thrombin stimulation in the cell line EA.hy926, which is a hybrid between human umbilical vein endothelial cells (HUVEC) and nonendothelial lung carcinoma A549/8 (38). It is possible that these cells behave differently compared with HPAE during thrombin treatment. Another possibility could be that our immunoprecipitation assay did not detect small quantitative changes in the association between VEC and catenins during thrombin stimulation. Our finding is consistent with observations of histamine effects in HUVEC, where no dissociation of the catenins from VEC was observed but instead decreased the amount of these proteins in the detergent-insoluble fraction (6).

PKC is a key signaling kinase responsible for regulating endothelial permeability (12, 29, 38, 41, 44, 51). We observed that PKC inhibition, and more specifically Gö-6976, a PKC{alpha} and PKC{beta} inhibitor, at the concentration used, prevented the thrombin-induced AJ disassembly. At lower concentrations (5 nM), formation of thrombin-induced gaps was reduced but not completely blocked (data not shown). On the other hand, PKC{beta} inhibition alone by LY-379196 had no effect on AJ disassembly. In future studies, we plan to use dominant negative mutants specific to PKC{alpha} and/or PKC{beta} to study more precisely the specific role of these PKC isoforms. These results extend previous findings showing PKC{alpha} translocation to the plasma membrane upon thrombin activation, leading to VEC junction discontinuities (41).

In the present study, we addressed mechanisms of AJ destabilization induced by thrombin and specifically by activated PKC. It is established that phosphorylation or dephosphorylation of cadherin residues and associated catenins can result in conformational changes in these molecules (17). VEC/catenin proteins are substrates for the action of kinases and phophatases (9, 20, 28, 33, 36, 41, 48, 54). Phosphorylation of the AJ proteins has been shown to influence cell-cell adhesion (6, 7, 13, 23, 42, 43) and thus may regulate endothelial permeability. For example, tyrosine phosphorylation and dephosphorylation of {beta}-catenin is associated with loss or gain, respectively, of AJ integrity (23, 28, 42, 48).

Changes in phosphorylation status of VEC/catenin complex and resulting alterations in conformation and binding partner interactions may influence cadherin adhesive interactions and contribute to the thrombin-induced AJ disassembly. We used two-dimensional electrophoresis to assess posttranslational modifications, such as phosphorylation/dephosphorylation of VEC/catenin complex, during thrombin treatment. Our data are consistent with this concept. From these experiments, we assume that p120 is phosphorylated in a PKC-dependent manner. A study on endothelial permeability using EA.hy926 treated with histamine and lysophosphatidic acid showed mobility shift of bands of p100/120, indicating dephosphorylation on serine-threonine residues in a PKC-dependent and -independent manner (39). Although these seem to be conflicting results, the explanation could lie in the different methods used. Two-dimensional gel pattern reveals the entire spectrum of protein posttranslational modifications, although it does not provide evidence for residue-specific phosphorylation events. Since thrombin activation unleashes a complex signal cascade, it is possible that both phosphorylation and dephosphorylation events take place in the same molecule.

Plasma membrane-associated VEC and {beta}-catenin are also subject to changes in their phosphorylation status, which may affect the association of catenins with VEC (48). We showed that the two-dimensional electrophoresis pattern of these proteins was consistent with predominant dephosphorylation after thrombin. It was shown previously that, upon thrombin treatment, SHP-2 nonreceptor tyrosine phosphatase is removed from the VEC/catenin complex, leading to increased tyrosine phosphorylation of {beta}-catenin in endothelial cells (48). This study was performed in HUVEC, but there was no physiological or morphological evidence that these cells established mature AJ. We noticed that confluence determined by light microscopy does not always correspond to AJ formation in endothelial cells. Indirect immunofluorescence combined with transendothelial resistance measurements are crucial assays to determine the integrity of AJ.

Our results imply, for the first time, that phosphatases play a crucial role in thrombin activation of endothelial cells by directly regulating cadherin/catenin function. It is known that a number of tyrosine phosphatases (SHP-1, SHP-2, PTPµ, VE-PTP, LARPTP, PTP1B) interact with the cadherin complex (1, 9, 22, 36, 48, 54). Since PKC can regulate the activity of tyrosine phosphatases (8, 40), it could activate these phosphatases to induce VEC and {beta}-catenin dephosphorylation. In any case, the present study makes clear that PKC-dependent modifications of VEC, p120, and {beta}-catenin are important in the mechanism of AJ disassembly and the resultant increased endothelial permeability response induced by thrombin.

We showed that posttranslational modifications of VEC, {beta}-catenin, and p120 occur during thrombin stimulation. The direct role of these modifications in VEC/catenin complex stability is unknown. These changes may modify the conformation of cadherin complex, resulting in modulation of adhesion strength. There is the possibility, however, that these changes might reflect major changes at the endothelial membrane cytoskeleton level beyond cell-cell adhesion. Upon thrombin stimulation, actin stress fiber formation and actomyosin contractile machinery activation result in cell shape changes followed by loss of cell adhesion. It is possible that cadherin is involved not only in cell-cell adhesion regulation but also in rearrangement of cortical cytoskeleton through catenins known to associate with actin and actin-associated proteins such as {alpha}-actinin and vinculin (52, 53). In both cases, cadherin/catenin complex changes contribute to adhesion changes either directly or indirectly by participating in transient remodeling of the cortical cytoskeleton.

In summary, the present study provides evidence of PKC-activated modifications of the cadherin/catenin complex that may lead to destabilization and disruption of AJ. We showed that there is a specific signature of PKC-dependent phosphorylation and dephosphorylation in the cadherin/catenin complex induced by thrombin, which may promote conformational changes in the complex leading to junctional instability and increased endothelial permeability response.


    ACKNOWLEDGMENTS
 
We thank Dr. Meiling Chen for invaluable help with the confocal microscope. We also thank Drs. Arshad Rahman and Raudel Sandoval for input and discussion.

P. Kouklis is a Fellow of Parker B. Francis Foundation.

DISCLOSURES

This work has been supported by National Heart, Lung, and Blood Institute Grant HL-20352.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Kouklis, Dept. of Pharmacology, Univ. of Illinois, 835 S. Wolcott Ave., M/C 868, Chicago, IL 60612 (E-mail: pkouklis{at}uic.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.


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