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• W2154419594 abstract "Vascular endothelial-cadherin (VE-cadherin) controls endothelial cell-cell adhesion and preserves endothelial integrity. In order to maintain endothelial barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation and cytoskeletal dynamics. Here, we show that loss of VE-cadherin function results in intercellular gap formation and a drop in electrical resistance of monolayers of primary human endothelial cells. Detailed analysis revealed that loss of endothelial cell-cell adhesion, induced by VE-cadherin-blocking antibodies, is preceded by and dependent on a rapid activation of Rac1 and increased production of reactive oxygen species. Moreover, VE-cadherin-associated β-catenin is tyrosine-phosphorylated upon loss of cell-cell contact. Finally, the redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) is activated and recruited to cell-cell junctions following the loss of VE-cadherin homotypic adhesion. Conversely, the inhibition of Pyk2 activity in endothelial cells by the expression of CRNK (CADTK/CAKβ-related non-kinase), an N-terminal deletion mutant that acts in a dominant negative fashion, not only abolishes the increase in β-catenin tyrosine phosphorylation but also prevents the loss of endothelial cell-cell contact. These results implicate Pyk2 in the reduced cell-cell adhesion induced by the Rac-mediated production of ROS through the tyrosine phosphorylation of β-catenin. This signaling is initiated upon loss of VE-cadherin function and is important for our insight in the modulation of endothelial integrity. Vascular endothelial-cadherin (VE-cadherin) controls endothelial cell-cell adhesion and preserves endothelial integrity. In order to maintain endothelial barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation and cytoskeletal dynamics. Here, we show that loss of VE-cadherin function results in intercellular gap formation and a drop in electrical resistance of monolayers of primary human endothelial cells. Detailed analysis revealed that loss of endothelial cell-cell adhesion, induced by VE-cadherin-blocking antibodies, is preceded by and dependent on a rapid activation of Rac1 and increased production of reactive oxygen species. Moreover, VE-cadherin-associated β-catenin is tyrosine-phosphorylated upon loss of cell-cell contact. Finally, the redox-sensitive proline-rich tyrosine kinase 2 (Pyk2) is activated and recruited to cell-cell junctions following the loss of VE-cadherin homotypic adhesion. Conversely, the inhibition of Pyk2 activity in endothelial cells by the expression of CRNK (CADTK/CAKβ-related non-kinase), an N-terminal deletion mutant that acts in a dominant negative fashion, not only abolishes the increase in β-catenin tyrosine phosphorylation but also prevents the loss of endothelial cell-cell contact. These results implicate Pyk2 in the reduced cell-cell adhesion induced by the Rac-mediated production of ROS through the tyrosine phosphorylation of β-catenin. This signaling is initiated upon loss of VE-cadherin function and is important for our insight in the modulation of endothelial integrity. Vascular endothelial-cadherin (VE-cadherin, 1The abbreviations used are: VE-cadherin, vascular endothelial-cadherin; ROS, reactive oxygen species; CRNK, CADTK/CAKβ-related non-kinase; Pyk2, proline-rich tyrosine kinase 2; p-Pyk2, phospho-Pyk2; wt, wild type; pY, phosphotyrosine; mAb, monoclonal antibody; Ab, antibody; HUVEC, human umbilical vein endothelial cells; FITC, fluorescein isothiocyanate; fn, fibronectin; DHR, dihydrorodamine-1,2,3; N-AC, N-acetylcysteine; PBS, phosphate-buffered saline; SH2, Src homology 2. Cadherin-5) is a transmembrane, calcium-dependent homophilic adhesion molecule that connects adjacent endothelial cells. A loss of VE-cadherin function results in unstable endothelial junctions and a decrease in endothelial monolayer electrical resistance, despite the fact that several other adhesion proteins such as claudin, occludin, and platelet-endothelial cell adhesion molecule 1 are also concentrated at sites of endothelial cell-cell contact (1Dejana E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 261-270Crossref PubMed Scopus (913) Google Scholar, 2van Buul J.D. Hordijk P.L. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 824-833Crossref PubMed Scopus (135) Google Scholar). Thus, VE-cadherin function is indispensable for the maintenance of the endothelial barrier function. VE-cadherin is linked to the actin cytoskeleton via the armadillo family members β- and γ-catenin that bind the actin-binding protein α-catenin (3Dejana E. Bazzoni G. Lampugnani M.G. Exp. Cell Res. 1999; 252: 13-19Crossref PubMed Scopus (211) Google Scholar, 4Vestweber D. J. Pathol. 2000; 190: 281-291Crossref PubMed Scopus (114) Google Scholar). VE-cadherin function is controlled by cytoskeletal dynamics and by protein phosphorylation events. Lampugnani et al. (5Lampugnani M.G. Corada M. Andriopoulou P. Esser S. Risau W. Dejana E. J. Cell Sci. 1997; 110: 2065-2077Crossref PubMed Google Scholar) showed that tyrosine phosphorylation of VE-cadherin and associated catenins is increased in loosely confluent endothelial monolayers, whereas tyrosine phosphorylation is reduced in confluent cells. Recently, a novel VE-protein tyrosine phosphatase was shown to interact with VE-cadherin and to increase VE-cadherin-mediated barrier function (6Nawroth R. Poell G. Ranft A. Kloep S. Samulowitz U. Fachinger G. Golding M. Shima D.T. Deutsch U. Vestweber D. EMBO J. 2002; 21: 4885-4895Crossref PubMed Scopus (244) Google Scholar). In addition, Ukropec et al. (7Ukropec J.A. Hollinger M.K. Salva S.M. Woolkalis M.J. J. Biol. Chem. 2000; 275: 5983-5986Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) reported that the phosphatase SHP-2 (SH2-containing tyrosine phosphatase Shp2) interacts with β-catenin and thereby regulates thrombin-induced changes in the endothelial barrier function (7Ukropec J.A. Hollinger M.K. Salva S.M. Woolkalis M.J. J. Biol. Chem. 2000; 275: 5983-5986Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The specific association of VE-protein tyrosine phosphatase with VE-cadherin and SHP-2 with β-catenin provides further evidence that tyrosine phosphorylation of the VE-cadherin-catenin complex is important for the regulation of endothelial cell-cell adhesion. Recently, it became clear that Rac1-induced reactive oxygen species (ROS, e.g. H2O2) disrupt VE-cadherin-based cell-cell adhesion (8van Wetering S. van Buul J.D. Quik S. Mul F.P. Anthony E.C. ten Klooster J.P. Collard J.G. Hordijk P.L. J. Cell Sci. 2002; 115: 1837-1846Crossref PubMed Google Scholar, 9Alexander J.S. Zhu Y. Elrod J.W. Alexander B. Coe L. Kalogeris T.J. Fuseler J. Microcirculation. 2001; 8: 389-401Crossref PubMed Google Scholar). Moreover, Rac1-induced ROS regulate protein tyrosine phosphorylation by inhibiting tyrosine phosphatase activity (10Kim H.S. Song M.C. Kwak I.H. Park T.J. Lim I.K. J. Biol. Chem. 2003; 278: 37497-37510Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 11Rhee S.G. Bae Y.S. Lee S.R. Kwon J. Sci. STKE 2000. 2000; : PE1Google Scholar, 12Tai L.K. Okuda M. Abe J. Yan C. Berk B.C. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1790-1796Crossref PubMed Scopus (71) Google Scholar). In addition, Tai et al. (12Tai L.K. Okuda M. Abe J. Yan C. Berk B.C. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1790-1796Crossref PubMed Scopus (71) Google Scholar) reported recently that shear stress induces activation of the tyrosine kinase Pyk2 in a ROS-dependent manner in endothelial cells (12Tai L.K. Okuda M. Abe J. Yan C. Berk B.C. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1790-1796Crossref PubMed Scopus (71) Google Scholar). Pyk2, also known as CAKβ/RAFTK/CADTK and FAK2, is a redox-sensitive tyrosine kinase that can be dephosphorylated by SHP-2 (13Cheng J.J. Chao Y.J. Wang D.L. J. Biol. Chem. 2002; 277: 48152-48157Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 14Tang H. Zhao Z.J. Landon E.J. Inagami T. J. Biol. Chem. 2000; 275: 8389-8396Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In this study, the role of Pyk2 in VE-cadherin function was explored. To induce the loss of VE-cadherin-mediated cell-cell adhesion, we made use of antibodies that block interactions between the extracellular regions of the VE-cadherin protein (15Corada M. Liao F. Lindgren M. Lampugnani M.G. Breviario F. Frank R. Muller W.A. Hicklin D.J. Bohlen P. Dejana E. Blood. 2001; 97: 1679-1684Crossref PubMed Scopus (261) Google Scholar, 16Hordijk P.L. Anthony E. Mul F.P. Rientsma R. Oomen L.C. Roos D. J. Cell Sci. 1999; 112: 1915-1923Crossref PubMed Google Scholar, 17van Buul J.D. Voermans C. van den Berg V. Anthony E.C. Mul F.P. van Wetering S. van der Schoot C.E. Hordijk P.L. J. Immunol. 2002; 168: 588-596Crossref PubMed Scopus (84) Google Scholar). This strategy mimics the loss of endothelial integrity induced by pro-inflammatory cytokines or by leukocyte transendothelial migration. We found that the loss of VE-cadherin function activates the small GTPase Rac1 and increases production of ROS, which subsequently leads to the loss of cell-cell adhesion. This reduced cell-cell adhesion is accompanied by increased tyrosine phosphorylation of β-catenin, which depends on the activation of Pyk2. Together, these data provide novel information on the role of Pyk2 in the regulation of VE-cadherin-based endothelial cell-cell adhesion and endothelial integrity. Reagents and Antibodies—Monoclonal antibodies (mAbs) to VE-cadherin (cl75), β-catenin, α-catenin, Pyk2, and phosphotyrosine (PY-20) were from BD Transduction Laboratories. VE-cadherin mAb 7H1 was from Pharmingen (San Diego, CA). β-Catenin polyclonal Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal Ab to phosphotyrosine was from Zymed Laboratories Inc.. The polyclonal Ab to Pyk2 was a kind gift of Dr. L. Graves (University of North Carolina, Chapel Hill, NC). α-Myc monoclonal antibodies were purchased from Invitrogen. Texas Red phalloidin, Alexa 633 phalloidin, FITC-labeled 3000 Dextran, Alexa 488-labeled goat-α-mouse-Ig, Alexa 568-labeled goat-α-mouse-Ig, and Alexa 488-labeled goat-α-rabbit-Ig secondary Abs were from Molecular Probes (Leiden, The Netherlands). Horseradish peroxidase-labeled goat-α-mouse-Ig or goat-α-rabbit-Ig was from DAKO (Glostrup, Denmark). Fibronectin (fn) was obtained from the Sanguin Research (Amsterdam, The Netherlands). Fetal calf serum was from Invitrogen. Basic fibroblast growth factor was from Roche Applied Science. Phospho-pyk2 (pY402) was purchased from BIOSOURCE (Camarillo, CA). EDTA, EGTA, and α-FLAG monoclonal Ab (clone M2) were from Sigma. Cell Cultures and Treatments—Immortalized human umbilical vein endothelial cells (HUVEC) (16Hordijk P.L. Anthony E. Mul F.P. Rientsma R. Oomen L.C. Roos D. J. Cell Sci. 1999; 112: 1915-1923Crossref PubMed Google Scholar) and primary HUVEC isolated from umbilical cord or purchased from Cambrex (Baltimore, MD) were cultured in fn-coated culture flasks (Nunc, Invitrogen) in Medium 199 (Invitrogen) supplemented with 20% (v/v) pooled heat-inactivated fetal calf serum, 1 ng/ml basic fibroblast growth factor, 5 units/ml heparin, 300 μg/ml glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. After reaching confluency, the endothelial cells were passaged by treatment with trypsin/EDTA (Invitrogen). To disrupt VE-cadherin-based cell-cell adhesion, cells were treated with either 12.5 or 25 μg/ml anti-VE-cadherin cl75 antibody, as indicated in the figure legends. To study signaling effects during the induced loss of VE-cadherin-based cell-cell adhesion, cells were stimulated for only 10 min with cl75, as indicated in figure legends. All of the cell lines were cultured or incubated at 37 °C at 5% CO2. Peptide Synthesis—The Rac17-32 peptide, which inhibits Rac1 function (18Vastrik I. Eickholt B.J. Walsh F.S. Ridley A. Doherty P. Curr. Biol. 1999; 9: 991-998Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), was designed in conjunction with the protein transduction domain of the human immunodeficiency virus Tat protein (19Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (887) Google Scholar). The resulting peptide (YGRKKRRQRRRGTCLLISYTTNAFPGEY) was synthesized at the Netherlands Cancer Institute (Amsterdam, The Netherlands). Adenoviral Infection of CRNK Construct—The recombinant adenoviral vector CRNK was a kind gift of Dr. L. Graves (University of North Carolina) (20Sorokin A. Kozlowski P. Graves L. Philip A. J. Biol. Chem. 2001; 276: 21521-21528Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Endothelial cells were serum-starved for 30 min and subsequently infected with adenovirus containing the CRNK-construct in serum-free culture medium. After 3 h, medium was replaced by normal culture medium, and following 16–24 h of infection, cells were washed three times with phosphate-buffered saline and used for assays. Immunocytochemistry—HUVEC were cultured on fn-coated glass coverslips, fixed, and immunostained as described previously (16Hordijk P.L. Anthony E. Mul F.P. Rientsma R. Oomen L.C. Roos D. J. Cell Sci. 1999; 112: 1915-1923Crossref PubMed Google Scholar) with a mAb to VE-cadherin (7H1, 10 μg/ml) or to PY-20 (10 μg/ml). Polyclonal anti-phosphotyrosine (10 μg/ml), anti-α-catenin (10 μg/ml), and anti-β-catenin (10 μg/ml) were used when endothelial cells were pretreated with mAbs to VE-cadherin. Subsequent visualization was performed with fluorescently labeled secondary Abs (10 μg/ml). F-actin was visualized with Texas Red phalloidin (1 unit/ml). In some experiments, cells were pretreated for 30 min at 37 °C with 20 μg/ml Rac17-32 peptide followed by washing. Images were recorded with a ZEISS LSM510 confocal microscope with appropriate filter settings. Cross-talk between the green and red channel was avoided by use of sequential scanning. Electric Cell Substrate Impedance Sensing—Endothelial cells were seeded at 100,000 cells/well (0.8 cm2) on fn-coated electrode arrays and grown to confluency. After the electrode check of the array and when the basal electrical resistance of the endothelial monolayer reached a plateau, Abs to VE-cadherin were added and electrical resistance was monitored on-line at 37 °C and at 5% CO2 with the electric cell substrate impedance sensing Model-100 Controller from BioPhysics, Inc. (Troy, NY). After 8 h, the data were collected and changes in resistance of endothelial monolayer were analyzed. Permeability—Permeability of HUVEC monolayers cultured on 5-μm pore 6.5-mm Transwell filters (Costar, Cambridge, MA) was determined using FITC-labeled 3000 Dextran as described previously (8van Wetering S. van Buul J.D. Quik S. Mul F.P. Anthony E.C. ten Klooster J.P. Collard J.G. Hordijk P.L. J. Cell Sci. 2002; 115: 1837-1846Crossref PubMed Google Scholar). The permeability response to thrombin (1 unit/ml) after 30 min was used as a control. After the assay, filters were washed with ice-cold Ca2+- and Mg2+-containing PBS and then fixed with 2% paraformaldehyde and 1% Triton X-100-containing PBS and stained with Texas Red phalloidin to inspect the HUVEC monolayer by confocal laser-scanning microscopy. Immunoprecipitation and Western Blot Analysis—Cells were grown to confluency on fn-coated dishes (50 cm2), washed twice gently with ice-cold Ca2+- and Mg2+-containing PBS, and lysed in 1 ml of lysis buffer (25 mm Tris, 150 mm NaCl, 10 mm MgCl2, 2mm EDTA, 0.02% (w/v) SDS, 0.2% (w/v) deoxycholate, 1% Nonidet P-40, 0.5 mm orthovanadate with the addition of fresh protease-inhibitor-mixture tablets (Roche Applied Science), pH 7.4). After 10 min on ice, cell lysates were collected and precleared for 30 min at 4 °C with protein G-Sepharose (15 μl for each sample, Amersham Biosciences). The supernatant separated by centrifugation (14,000 × g, 15 s at 4 °C) was incubated with 15 μl of protein G-Sepharose that had been coated with 5 μg/ml β-catenin mAb for1hat 4 °C under continuous mixing. The beads were washed three times in lysis buffer, and proteins were eluted by boiling in SDS-sample buffer containing 4% 2-mercaptoethanol (Bio-Rad). The samples were analyzed by SDS-PAGE. Proteins were transferred to 0.45-μm nitrocellulose (Schleicher & Schüll), and the blots were blocked with blocking buffer (1% (w/v) low fat milk in Tris-buffered saline with Tween 20) for 1 h and subsequently incubated at room temperature with the appropriate Abs for 1 h followed by incubation with rabbit-α-mouse-Ig-horseradish peroxidase for 1 h at room temperature. Between the various incubation steps, the blots were washed three times with Tris-buffered saline with Tween 20 and finally developed with an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences). Rac1 Activity Assays—Cells were stimulated for the indicated times with cl75 (25 μg/ml) or 5 mm EDTA. Cells were kept on ice and washed with ice-cold PBS, lysed for 10 min in lysis buffer, and assayed for Rac activation with glutathione S-transferase-p21-activating kinase, as described by Sander et al. (21Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (587) Google Scholar). Finally, in both assays, the beads were washed four times with lysis buffer. After the fourth wash, the beads were put into new tubes and subsequently suspended in 2× sample buffer containing 4% 2-mercaptoethanol. Samples were analyzed by SDS-PAGE as described above. Cell Fractionation—Cells were grown to confluency on fn-coated dishes (50 cm2), washed twice gently with ice-cold Ca2+- and Mg2+-containing PBS, and lysed in 1 ml of lysis buffer, as described above, containing 1% Triton X-100 instead of 1% Nonidet P-40. After 10 min on ice, cell lysates were collected and separated by centrifugation (14,000 × g, 1 min at 4 °C). The pellet fraction contained Triton X-100-insoluble proteins associated to the actin cytoskeleton, and the supernatant contained Triton X-100-soluble cytosolic proteins. Samples were boiled in SDS sample buffer containing 4% 2-mercaptoethanol and were immediately analyzed by SDS-PAGE and continued as described above. Measurement of ROS—To measure generation of reactive oxygen species (ROS) in endothelial cells, primary HUVEC cultured on fibronectin-coated glass coverslips were loaded with dihydrorodamine-1,2,3 (DHR, 30 μm; Molecular Probes) for 30 min, washed, and subsequently treated with the VE-cadherin Ab cl75, control Ab IgG, or medium. Fluorescence of DHR was quantified by time lapse confocal microscopy. Intensity values are shown as the percentage increase relative to the basal DHR values at the start of the experiment. VE-cadherin is an essential homotypic adhesion molecule that specifically localizes to adherens junctions and controls endothelial integrity. Using Abs that recognize distinct epitopes in the extracellular domain of VE-cadherin, we and others (15Corada M. Liao F. Lindgren M. Lampugnani M.G. Breviario F. Frank R. Muller W.A. Hicklin D.J. Bohlen P. Dejana E. Blood. 2001; 97: 1679-1684Crossref PubMed Scopus (261) Google Scholar, 17van Buul J.D. Voermans C. van den Berg V. Anthony E.C. Mul F.P. van Wetering S. van der Schoot C.E. Hordijk P.L. J. Immunol. 2002; 168: 588-596Crossref PubMed Scopus (84) Google Scholar, 22Gotsch U. Borges E. Bosse R. Boggemeyer E. Simon M. Mossmann H. Vestweber D. J. Cell Sci. 1997; 110: 583-588Crossref PubMed Google Scholar) have described differential effects on the permeability of endothelial cells. However, these studies were in part based on diffusion of a fluorescently labeled high molecular weight marker over the endothelial monolayer to measure the endothelial integrity. We now used a more sensitive approach, which is based on real-time analysis of the electrical resistance of endothelial monolayers. This analysis shows that the VE-cadherin-blocking Ab cl75 rapidly reduces the transendothelial resistance, whereas the non-blocking Ab 7H1 did not have any effect on the resistance (Fig. 1A). Previous reports from our group and from others (15Corada M. Liao F. Lindgren M. Lampugnani M.G. Breviario F. Frank R. Muller W.A. Hicklin D.J. Bohlen P. Dejana E. Blood. 2001; 97: 1679-1684Crossref PubMed Scopus (261) Google Scholar, 17van Buul J.D. Voermans C. van den Berg V. Anthony E.C. Mul F.P. van Wetering S. van der Schoot C.E. Hordijk P.L. J. Immunol. 2002; 168: 588-596Crossref PubMed Scopus (84) Google Scholar, 22Gotsch U. Borges E. Bosse R. Boggemeyer E. Simon M. Mossmann H. Vestweber D. J. Cell Sci. 1997; 110: 583-588Crossref PubMed Google Scholar) have shown that blocking VE-cadherin Abs are able to disrupt endothelial junctions and induce a redistribution of VE-cadherin over the endothelial cell surface. We also found that β-catenin became diffusely distributed when the endothelial cells were treated with the cl75 Ab (Fig. 1B). To study whether a loss of VE-cadherin-mediated cell-cell adhesion induced the dissociation of the VE-cadherin complex from the actin cytoskeleton, we fractionated the cl75-treated endothelial cells and analyzed the cytoskeletal and the cytosol/membrane fractions for the presence of α-catenin, which links VE-cadherin and β-catenin to the actin cytoskeleton. These experiments showed that α-catenin shifted from the cytoskeleton to the membrane and cytosol fraction when VE-cadherin-mediated cell-cell contacts were disrupted (Fig. 1C). Also, β-catenin translocated to the same fraction as α-catenin upon the loss of cell-cell contact (see Fig. 8B). These data indicate that “outside-in” signaling induced by the loss of VE-cadherin-mediated homotypic interaction dissociates the entire VE-cadherin complex from the cytoskeleton.Fig. 8Pyk2 is involved in endothelial permeability and phosphorylation of β-catenin upon the loss of VE-cadherin function. A, expression of CRNK prevents cl75-induced permeability. HUVEC were cultured and grown to confluency on Transwell filters and infected with Ad CRNK as described under “Materials and Methods” (open bars) or left untreated (closed bars). Subsequently, cells were treated with cl75 or left untreated as described under “Materials and Methods” and FITC-labeled 3000 Dextran was added to the upper compartment of the Transwell. After 3 h, the level of fluorescence in the lower compartment was measured and calculated as the percentage increase compared with control levels. Control levels represent basal leakage of the endothelial monolayer incubated with medium alone. The experiment is performed twice in triplicate. CRNK significantly reduces cl75-induced increase of endothelial monolayer permeability (p < 0.01). B, expression of CRNK prevents cl75-induced loss of endothelial cell-cell adhesion. Cells were cultured on glass covers, infected with Ad CRNK, and treated with cl75. Fixed and permeabilized cells were stained for CRNK expression in green (α-FLAG; a and e), β-catenin in red (b and f), and F-actin in grayscale (d and h). Merge shows CRNK and β-catenin localization (c and g). Bar, 20 μm. C, Pyk2 mediates β-catenin phosphorylation. Cells were treated as described in the legend of Fig. 4B with the exception of the expression of CRNK (lower panel). Upper panel shows increased tyrosine phosphorylation of β-catenin after 30 min of incubation with cl75, which is inhibited by CRNK. The middle panel shows equal amounts of β-catenin per immunoprecipitation (I.P.). The experiment was done three times with similar results. D, Pyk2 mediates β-catenin association with the cytoskeleton. Cells were cultured as described in the legend of Fig. 1C. Immunoblotting for β-catenin shows that cl75 treatment induced a loss of β-catenin from the cytoskeletal fraction (upper panel), which is prevented by CRNK. The lower panel shows CRNK expression. A representative of three independent experiments is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previously, we have shown by protein transduction that an active mutant of the small GTPase Rac, RacV12, induced the loss of cell-cell contacts of confluent endothelial cells (8van Wetering S. van Buul J.D. Quik S. Mul F.P. Anthony E.C. ten Klooster J.P. Collard J.G. Hordijk P.L. J. Cell Sci. 2002; 115: 1837-1846Crossref PubMed Google Scholar), in agreement with findings by others (24Braga V.M. Del Maschio A. Machesky L. Dejana E. Mol. Biol. Cell. 1999; 10: 9-22Crossref PubMed Scopus (226) Google Scholar, 25Vouret-Craviari V. Boquet P. Pouyssegur J. Obberghen-Schilling E. Mol. Biol. Cell. 1998; 9: 2639-2653Crossref PubMed Scopus (212) Google Scholar, 26Wojciak-Stothard B. Potempa S. Eichholtz T. Ridley A.J. J. Cell Sci. 2001; 114: 1343-1355Crossref PubMed Google Scholar). In addition, Waschke et al. (27Waschke J. Drenckhahn D. Adamson R.H. Curry F.E. Am. J. Physiol. 2004; 287: H704-H711Crossref PubMed Scopus (55) Google Scholar) showed recently that Rac1 inhibition leads to a loss of VE-cadherin-adhesive capacity. To test whether “outside-in” signaling induced by loss of VE-cadherin function involves Rac1, Rac1-GTP loading was measured by pull-down assays with glutathione S-transferase-p21-activating kinase. These experiments showed that cl75 indeed induces Rac1 activation (Fig. 2A). The response was highest at 5 min, after which it declined somewhat, although Rac1 activation remained elevated up to 30 min (Fig. 2A). Rac1 activation results in increased ROS production in neutrophils as well as in endothelial cells (8van Wetering S. van Buul J.D. Quik S. Mul F.P. Anthony E.C. ten Klooster J.P. Collard J.G. Hordijk P.L. J. Cell Sci. 2002; 115: 1837-1846Crossref PubMed Google Scholar, 28Park H.S. Lee S.H. Park D. Lee J.S. Ryu S.H. Lee W.J. Rhee S.G. Bae Y.S. Mol. Cell. Biol. 2004; 24: 4384-4394Crossref PubMed Scopus (201) Google Scholar, 29Price M.O. Atkinson S.J. Knaus U.G. Dinauer M.C. J. Biol. Chem. 2002; 277: 19220-19228Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Therefore, we tested the possibility that Rac activation, effected by the loss of VE-cadherin function, induces ROS production. Consistently, cl75-induced loss of VE-cadherin-mediated cell-cell contacts promoted a rapid increase in ROS production (Fig. 2B). Additional experiments showed that irrelevant antibodies such as isotype control IgG1 or medium changes did not mimic the induction of ROS. Moreover, the ROS production is followed by loss of cell-cell contacts, as is observed with real-time phase-contrast microscopy imaging (Fig. 2C, and supplemental movies). These observations indicate that activation of Rac1 and the production of ROS are rapidly induced following the loss of VE-cadherin function. To define whether the cellular response to VE-cadherin-mediated outside-in signaling, i.e. the formation of intercellular gaps, in fact depends on Rac1 signaling, we used a cell-permeable peptide inhibitor of Rac1, Tat-Rac17-32. This peptide represents part of the effector loop of Rac1 and competes with Rac1-effector interactions, thus preventing down-stream signaling (18Vastrik I. Eickholt B.J. Walsh F.S. Ridley A. Doherty P. Curr. Biol. 1999; 9: 991-998Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 30van Wetering S. van den Berk N. van Buul J.D. Mul F.P. Lommerse I. Mous R. ten Klooster J.P. Zwaginga J.J. Hordijk P.L. Am. J. Physiol. 2003; 285: C343-C352Crossref PubMed Scopus (184) Google Scholar). After 30 min, cl75 had maximally reduced the endothelial resistance of untreated control monolayers (Fig. 2D). Tat-Rac17-32-incubated monolayers showed a reduced response to the cl75 treatment (∼50% reduction in the loss of resistance, Fig. 2D). This indicates that the loss of cell-cell contact depends, at least in part, on Rac1 activity. Interestingly, under control conditions, we observed a small decrease in the electrical resistance of the endothelial monolayer when the confluent endothelial monolayers were incubated with Tat-Rac17-32 (data not shown). This finding suggests that, to maintain stable endothelial junctions, a low level of active Rac1 is required (27Waschke J. Drenckhahn D. Adamson R.H. Curry F.E. Am. J. Physiol. 2004; 287: H704-H711Crossref PubMed Scopus (55) Google Scholar). In conclusion, these findings indicate that VE-cadherin-mediated outside-in signaling, resulting from a loss of cadherin function, requires Rac1 to induce loss of cell-cell adhesion. Scavenging ROS by incubating the cells with N-acetylcysteine (N-AC) prevented the cl75-induced redistribution of VE-cadherin (Fig. 3A). In line with this result, the Ab-induced increase in endothelial monolayer permeability was prevented by N-AC (Fig. 3B). Interestingly, we found that N-AC decreased the permeability of 7H1-treated endothelial monolayers (Fig. 3B) and medium-treated monolayers (data not shown). The effect of thrombin on the permeability seemed to act independently from ROS, because N-AC was not able to prevent thrombin-induced permeability. These findings indicate that Rac-mediated production of ROS regulates the endothelial barrier function and that ROS are important mediators of VE-cadherin outside-in signaling. Low levels of cell-cell contact are associated with tyrosine phosphorylation of the VE-cadherin complex (5Lampugnani M.G. Corada M. Andriopoulou P. Esser S. Risau W. Dejana E. J. Cell Sci. 1997; 110: 2065-2077Crossref PubMed Google Scholar). Since cl75 induces the loss of endothelial cell-cell contacts, we studied its effects on the tyrosine phosphorylation of junctional proteins in confluent monolayers. Immunocytochemical analys" @default.
• W2154419594 created "2016-06-24" @default.
• W2154419594 creator A5007988336 @default.
• W2154419594 creator A5036411640 @default.
• W2154419594 creator A5060278537 @default.
• W2154419594 creator A5070642099 @default.
• W2154419594 creator A5087169555 @default.
• W2154419594 date "2005-06-01" @default.
• W2154419594 modified "2023-10-15" @default.
• W2154419594 title "Proline-rich Tyrosine Kinase 2 (Pyk2) Mediates Vascular Endothelial-Cadherin-based Cell-Cell Adhesion by Regulating β-Catenin Tyrosine Phosphorylation" @default.
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