Department of Pharmacology, University of Illinois, Chicago, Illinois 60612
Submitted 14 March 2003 ; accepted in final form 2 May 2003
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ABSTRACT |
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vascular endothelial cadherin; isoelectric focusing
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 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,
-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.
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MATERIALS AND METHODS |
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Antibodies and reagents. Goat polyclonal for VEC was purchased
from Research Diagnostics, and monoclonal VEC was from Chemicon (Temecula,
CA). The monoclonal antibodies for -cadherin, plakoglobin, and p120 were
purchased from Transduction Laboratories (Lexington, KY). The rabbit
polyclonal for
-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
inhibitor or
cPKC
and cPKC
, depending on the concentration], and LY-379196
(cPKC
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,
-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 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,
-catenin,
-catenin, p120, plakoglobin), we calculated the
isoelectric point (pI), which ranges from 5 to 6.5 (i.e., VEC pI = 5.01,
-catenin pI = 5.43, plakoglobin pI = 5.68,
-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 47 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
-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.
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RESULTS |
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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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Localized dissociation of catenins from VEC in thin membrane
projections. VEC normally associates with -,
-, and
p120-catenin and plakoglobin (
-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
-catenin did not
change in 30 s or 5 or 15 min after thrombin exposure
(Fig. 3).
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To further visualize the pattern of colocalization of the VEC with
catenins, we doublestained the cells with red (VEC) and green (-,
-, 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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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 (Gö-6976;
Fig. 5, C and
D) or PKC
(LY-379196;
Fig. 5E) inhibitors.
PKC
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
(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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PKC-dependent alterations in phosphorylation of VEC,
-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,
-catenin,
plakoglobin, and
-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,
-catenin (mol wt
92,000, pI 5.43) exists in five distinct isoforms
(Fig. 6,
-catenin). At 15
min after thrombin treatment, new spots appeared on the basic side, indicating
that
-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
-catenin, plakoglobin (mol wt 82,000, pI 5.68) and
-catenin (mol
wt 102,000, pI 5.95) showed no distinct changes in their pattern after
thrombin (Fig. 7).
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DISCUSSION |
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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 and PKC
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
inhibition alone by LY-379196 had no effect on AJ disassembly.
In future studies, we plan to use dominant negative mutants specific to
PKC
and/or PKC
to study more precisely the specific role of these
PKC isoforms. These results extend previous findings showing PKC
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 -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 -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
-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 -catenin dephosphorylation. In any case,
the present study makes clear that PKC-dependent modifications of VEC, p120,
and
-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, -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
-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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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