src-induced tyrosine phosphorylation of ve-cadherin is not

Src-induced Tyrosine Phosphorylation of VE-cadherinIs Not Sufficient to Decrease Barrier Function ofEndothelial Monolayers*□S �

Received for publication, October 27, 2009, and in revised form, December 23, 2009 Published, JBC Papers in Press, January 4, 2010, DOI 10.1074/jbc.M109.079277

Alejandro P. Adam‡, Amy L. Sharenko‡, Kevin Pumiglia§, and Peter A. Vincent‡1

From the ‡Center for Cardiovascular Sciences and §Center for Cell Biology and Cancer Research, Albany Medical College,Albany, New York 12208

Activation of Src family kinases (SFK) and the subsequentphosphorylation of VE-cadherin have been proposed as majorregulatory steps leading to increases in vascular permeability inresponse to inflammatory mediators and growth factors. Toinvestigate Src signaling in the absence of parallel signalingpathways initiated by growth factors or inflammatory media-tors, we activated Src and SFKs by expression of dominant neg-ative Csk, expression of constitutively active Src, or knockdownof Csk. Activation of SFK by overexpression of dominant nega-tive Csk inducedVE-cadherin phosphorylation at tyrosines 658,685, and 731. However, dominant negative Csk expression wasunable to induce changes in the monolayer permeability. Incontrast, expression of constitutively active Src decreased bar-rier function and promoted VE-cadherin phosphorylation ontyrosines 658 and 731, although the increase in VE-cadherinphosphorylation preceded the increase in permeability by 4–6h. Csk knockdown induced VE-cadherin phosphorylation atsites 658 and 731 but did not induce a loss in barrier function.Co-immunoprecipitation and immunofluorescence studiessuggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and �-catenin or the ability ofthese proteins to localize at the plasma membrane. Takentogether, our data show that Src-induced tyrosine phosphoryla-tion of VE-cadherin is not sufficient to promote an increase inendothelial cellmonolayer permeability and suggest that signal-ing leading to changes in vascular permeability in response toinflammatorymediators or growth factors may require VE-cad-herin tyrosine phosphorylation concurrently with other signal-ing pathways to promote loss of barrier function.

Endothelial cells line the wall of all blood vessels, where theyplay a critical role in a number of physiological responsesincluding regulation of vasoreactivity, hemostasis, and leuko-cyte recruitment. Vascular endothelial cells also act as a selec-tive barrier that regulates the passage of fluid, macromolecules,and white cells from the vascular space to the interstitium. Theproper regulation of fluid and protein flux is critical for main-

taining normal tissue function. This is accomplished by a num-ber of transmembrane cell-cell adhesion proteins that, whencoupled with their binding partners, contribute to the adhesionof one endothelial cell to another. The adherens junction com-plex, comprised of cadherins and the catenins, is a major adhe-sion structure that connects to the actin cytoskeleton (1, 2).VE-cadherin is found specifically in the endothelial cell adher-ens junction and has been implicated in playing a fundamentalrole in controlling the transport across the endothelial barrierand in regulating angiogenesis (3, 4). The cytoplasmic domainof VE-cadherin binds to �-catenin and plakoglobin, both ofwhich bind to�-catenin, a protein that supports the interactionof the VE-cadherin-catenin complex with the actin cytoskele-ton. In addition to catenins, VE-cadherin has been found tointeract with other signaling molecules and to serve as a scaf-folding molecule that participates in a signaling network thatcontrols endothelial cell-cell adhesion (5).The inflammatory response associated with ischemia-reper-

fusion during stroke and myocardial infarction results in a lossof endothelial barrier function that contributes to infarct sizeand loss of tissue function. Src inhibition prior to the ischemicperiod showed reduced infarct size when compared with con-trol animals due to inhibition of edema formation in bothmod-els ofmyocardial infarction and stroke (6, 7). In addition, infarctsize was reduced by 50% in Src knock-out mice (6). In vitrostudies have also implicated Src as a major signaling proteinleading to a loss of barrier function (8–11) with Src and othermembers of the Src kinase family being shown to play a role inlipopolysaccharide (10) and VEGF2 (8, 11)-induced loss ofendothelial integrity.A number of studies have implicated tyrosine phosphoryla-

tion of VE-cadherin in the regulation of the trans-vascular fluxof fluid and protein (12, 13). Indeed, previous studies have dem-onstrated an association of VE-cadherin phosphorylation andendothelial barrier function in response to inflammatorymedi-ators and growth factors (10, 15, 17, 37). In addition, both the

* This work was supported, in whole or in part, by National Institutes of HealthGrant RO1 HL77870 (to P. A. V.).

� This article was selected as a Paper of the Week.□S The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. 1–7.1 To whom correspondence should be addressed: 47 New Scotland Ave.,

Albany, NY 12208. Tel.: 518-262-6296; Fax: 518-262-8101; E-mail:[email protected].

2 The abbreviations used are: VEGF, vascular endothelial growth factor; DN-Csk, dominant negative C-terminal Src kinase; caSrc, constitutively activeSrc; SFK, Src family kinases; Erk, extracellular signal-regulated kinase; FAK,focal adhesion kinase; ICAM, intercellular adhesion molecule; HDMEC,human dermal microvascular endothelial cell; ECIS, electrical cell-substrateimpedance sensor; TEER, trans-endothelial electric resistance; siRNA, smallinterfering RNA; CHO, Chinese hamster ovary; GFP, green fluorescent pro-tein; PBS, phosphate-buffered saline; pfu, plaque-forming unit; DMSO,dimethyl sulfoxide; PIPES, 1,4-piperazinediethanesulfonic acid; pTyr,phosphotyrosine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 10, pp. 7045–7055, March 5, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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phosphorylation of VE-cadherin and monolayer permeabilitywere dependent on activation of Src family kinases (SFKs).More recently, studies have begun to use mutations of specifictyrosine residues to investigate the role of VE-cadherin phos-phorylation in regulating barrier function. Overexpression ofVE-cadherin in CHO cells allows for these cells to form arestrictive barrier to protein flux, giving them an epithelial phe-notype. Potter et al. (14) reported that overexpression ofrecombinant forms of VE-cadherin that mimic phosphoryla-tion of either Tyr-658 or Tyr-731 did not develop a restrictivemonolayer inCHOcells. Thesemutations also affected the abil-ity of VE-cadherin to bind other adherens junction compo-nents, p120, and �-catenin. In addition, expression of activatedSrc increased phosphorylation on both Tyr-658 and Tyr-731 ofVE-cadherin. In contrast, Wallez et al. (15) showed that Srcoverexpression in CHO cells induced VE-cadherin phosphory-lation exclusively in Tyr-685. This site was confirmed to be adirect Src target using an in vitro kinase assay. Furthermore,they could also detect this phosphorylation site in humanumbilical vein endothelial cells after VEGF stimulation. Recentstudies have found that tyrosine phosphorylation of VE-cad-herin is required for regulating leukocyte trans-endothelial cellmigration (16, 17) and that this requires activation of SFKs. Inaddition, it has also been shown that mutation of Tyr-658 orTyr-731 will attenuate VEGF-induced decreases in barrierfunction (37). Similar to studies investigating permeability, var-ious tyrosines have also been implicated in trans-endothelialcell migration (16, 17).Although the literature points to an important role of VE-

cadherin phosphorylation in the regulation endothelial func-tion, including barrier formation, further investigations areneeded to fully understand themechanisms of this process. Theexperiments presented here are a direct examination of the roleof Src-mediated VE-cadherin phosphorylation in the regula-tion of endothelial barrier function and junctional assembly. Tolimit the activation of other confounding signaling pathwaysknown to be initiated by growth factors or inflammatory medi-ators, SFKswere activated by expression of a dominant negativeC-terminal Src kinase (DN-Csk), constitutively active Src(caSrc), or knockdown of Csk. In the studies that follow, wedemonstrate that changes in endothelial permeability and thephosphorylation of specific tyrosine residues in VE-cadherinare dependent on the method used to increase active Src. Moreimportantly, our data demonstrate that although SFK activitymay be required for the loss of barrier function, SFK activationand the subsequent VE-cadherin tyrosine phosphorylation arenot sufficient to increase endothelial cell permeability and junc-tional disassembly.

EXPERIMENTAL PROCEDURES

Cell Culture—Human dermal microvascular endothelialcells (HDMECs) were isolated from neonatal foreskin by incu-bating the obtained cells with magnetic beads coated with anantibody to CD31 (DYNAL) as described previously (18). Cellswere then assessed for VE-cadherin and lectin binding to con-firm their endothelial origin and then frozen and stored in liq-uid nitrogen. Cells were grown in EGM-2MV (Lonza GroupLtd.) containing 5% fetal bovine serumandwere used for exper-

iments between passages 6 and 11. For all experimental proto-cols, cells were seeded at 1 � 105 cells/cm2 and incubated for48–72 h to obtain mature cell-cell junctions.Adenoviral Constructs—Dominant negative Csk adenovirus

containing a lysine to arginine substitution at position 222(DN-Csk) was a generous gift from Dr. S. Tanaka (Faculty ofMedicine, University of Tokyo) (19). A pUSEamp vector con-taining cDNA encoding caSrc containing an Y527A mutationwas purchased from Upstate Biotechnology. The coding se-quence was removed from pUSEamp using NotI and EcoRVand inserted into the pShuttle-IRES-hrGFP-1 vector (Strat-agene). Adenovirus was then produced using the pAdEasy sys-tem as described by He et al. (20). All infections were accom-panied by a controlGFP and/or�-galactosidase infectionwith amultiplicity of infection at or above the greatest multiplicity ofinfection used in the experimental groups.VEGF Treatments—HDMECs were seeded at confluence

(105 cells/cm2) and incubated for 3 days to allow the formationof a mature monolayer. Then, cells were washed and serum-starved for 18 h (in EBM-2 medium with 0.3% fetal bovineserumwith no additional factors). Then, cells were treated with50 ng/ml VEGF-165 (R&D Systems) or vehicle only (0.1%bovine serum albumin in PBS) for the time indicated for theparticular experiment.Electrical Cell-Substrate Impedance Sensor (ECIS)—Mono-

layer permeability was determined by measuring changes inelectrical resistance using an electric cell-substrate imped-ance sensor (ECIS, Applied BioPhysics, Inc.) (21) as pub-lished previously (3, 22). Briefly, HDMECs (80,000 cells)were seeded onto ECIS 10E cultureware (0.8 cm2/well) pre-coated with 0.1% gelatin and incubated for 3 days. The elec-trical impedance across the monolayer was measured at 1 V,4000 Hz with current flowing through 10 small gold elec-trodes per well plus one large counterelectrode, using theculture medium as the source of electrolytes. Impedance wasmonitored by the lock-in amplifier, stored, and then used tocalculate resistance and capacitance by the manufacturer’ssoftware. Data are presented as a plot of resistance versustime as this best represents the barrier provided by endothe-lial cell-cell junctions.Gel Electrophoresis and Immunoblotting—Cells growing on

6-well plates were scraped after lysis with 250 �l of Laemmlibuffer containing Complete protease inhibitor mixture (RocheApplied Science), PhosSTOP phosphatase inhibitor mixture(RocheApplied Science), and 0.1mMpervanadate and boiled. Atotal of 20 �l of cell lysate per lane was loaded on standardSDS-PAGE gels and transferred to nitrocellulose membranes.Immunoblotswere performedby blocking themembraneswith3% skim milk in PBS containing calcium and magnesium(PBS�) and incubating overnight at 4 °C with any of thefollowing antibodies: anti-VE-cadherin (intracellular domain,Santa Cruz Biotechnology), anti-VE-cadherin (extracellulardomain, Strategic Diagnostics Inc.), anti-pTyr-658 VE-cad-herin (Chemicon), anti-p-Tyr-685 VE-cadherin (ECM Bio-sciences), anti-p-Tyr-731 VE-cadherin (Chemicon), anti-p120(S-19, Santa Cruz Biotechnology), anti-�-catenin (BD Bio-sciences), anti-Src (GD11, Upstate Biotechnology), anti-p-Tyr-416 Src (Cell Signaling), anti-p-Tyr-527 Src (Cell Signaling),

Src Activity in Endothelial Barrier Function

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anti-Csk (C-20, Santa Cruz Biotechnology), anti-paxillin (349,BD Biosciences), anti-p-Tyr-118 paxillin (Cell Signaling), anti-FAK (A-17, Santa Cruz Biotechnology), anti-p-Tyr-861 FAK(BIOSOURCE), or anti-pTyr (4G10, Upstate Biotechnol-ogy). Secondary anti-mouse, anti-rabbit, or anti-goat anti-bodies conjugated with peroxidase (Jackson ImmunoResearchLaboratories) were incubated for 1 h at room temperature.Mem-branes were developed using SuperSignal West Pico or Femtochemiluminescent substrate (Pierce)andFujifilmLAS-3000 imag-ing system (Fujifilm).Immunoprecipitation—Cells were scraped from 10-cm

plates in 1 ml of immunoprecipitation buffer (0.1 M NaCl, 0.3 M

Sucrose, 30mMMgCl2, 10mMPIPES, 0.5mMEDTA, 0.1%Non-idet P-40) containing complete protease inhibitor mixture(Roche Applied Science), PhosSTOP phosphatase inhibitormixture (Roche Applied Science), and 0.5 mM pervanadate.After clearing by centrifugation, an aliquot was stored for totalcell lysis control, and the rest was divided evenly into two tubesfor incubation with the specific antibody (5 �g/tube, 30 min at4 °C, sources described above) and proteinA- or proteinG-aga-rose beads (50 �l/tube, 90 min at 4 °C, Santa Cruz Biotechnol-ogy) or beads alone. After washes with immunoprecipitationbuffer, the tubes were centrifuged and boiled in 60 �l of 2�Laemmli buffer containing Complete protease inhibitor mix-ture (Roche Applied Science), PhosSTOP phosphatase inhibi-tor mixture (Roche Applied Science), and 0.1 mM pervanadate.The samples were then processed for immunoblotting asdescribed above.Immunofluorescence Microscopy—Immunofluorescence stud-

ies were performed by seeding 80,000 cells on Biocoat 8-wellglass culture slides (BD Falcon) precoated with 0.1% gelatin.Three days after seeding, cells were infected with adenovirus toexpress �-galactosidase, GFP, DN-Csk, or caSrc or treated asindicated. Cells were then fixed with 4% p-formaldehyde (USBCorp.) in PBS for 30 min at 4 °C, washed twice with PBS, andprocessed for immunofluorescence at room temperature.Briefly, cells were permeabilized with 0.1% Triton X-100(Sigma) in Tris-buffered saline for 15 min, treated withImage-iT FX signal enhancer (Invitrogen) for 30 min and thenblocked with 5.5% bovine serum in Tris-buffered saline con-taining 0.1% Triton X-100. Antibodies (described above) wereincubated for 2 h at room temperature. Secondary anti-goat,anti-rabbit, or anti-mouse antibodies conjugated with AlexaFluor 488,Alexa Fluor 594,Alexa Fluor 647 (Molecular Probes),or Cy2 (Jackson ImmunoResearch Laboratories) were incu-bated for 1 h at room temperature in the presence or absence ofAlexa Fluor 488- or Alexa Fluor 594-conjugated phalloidin and4�,6-diamidino-2-phenylindole (Molecular Probes). Slideswere mounted using Gel Mount aqueous mounting medium(Biomeda Corp.). Images were obtained using a Zeiss AxioObserver Z1 inverted microscope with the Apotome moduleand Zeiss AxioVision software.Statistical Analyses—ECIS data were analyzed by two-way

analysis of variance of repeated measurements and post hocBonferroni tests against GFP-infected cells. Error bars showS.D. (n� 3) in Figs. 1, 2, 4, 5, and 7. An � � 0.05 was consideredstatistically significant.

RESULTS

There is controversy in the literature with respect towhich tyrosine residues of VE-cadherin are phosphorylatedby VEGF treatment (14, 15). Therefore, we first wanted to definewhich tyrosine residues of VE-cadherin were phosphorylatedby VEGF in cultures of HDMECs and to determine whether thetyrosine phosphorylation was temporally associated withVEGF-induced changes in the integrity of the endothelialmonolayer. As shown in Fig. 1, the addition ofVEGF to amono-

FIGURE 1. VEGF-induced VE-cadherin tyrosine phosphorylation andincreased permeability depend on SFK activity in HDMEC monolayers.A, confluent, mature monolayers of HDMECs were serum-starved for 16 h(0.3% fetal bovine serum in EBM-2 medium, see “Experimental Procedures”),incubated with 10 �M PP1 Src kinase inhibitor or 0.1% DMSO (control) for 1 h,and then treated with 50 ng/ml VEGF or vehicle (0.1% bovine serum albuminin PBS) for different times prior to lysis. Western blot analysis using phospho-specific antibodies shows a fast increase in VE-cadherin phosphorylation thatquickly reverses. Src activity inhibition by PP1 pretreatment blocks VEGF-in-duced phosphorylation. pY658, phospho-Tyr-658; pY685, phospho-Tyr-685;pY731, phospho-Tyr-731; pY416, phospho-Tyr-416; pY118, phospho-Tyr-118.B, HDMEC monolayers were treated as above, and electric resistance wasmeasured by ECIS at 4000 Hz (n � 3). Results are representative of at leastthree independent experiments.




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layer of HDMECs resulted in a rapidincrease in VE-cadherin phosphor-ylation on three different sites,namely, Tyr-658, Tyr-685, and Tyr-731. Furthermore, the increase inVE-cadherin tyrosine phosphoryla-tion was temporally associated withthe VEGF-induced decrease ofresistance across the HDMECmonolayer. As expected, the VEGF-induced loss of monolayer barrierfunction and VE-cadherin phos-phorylation was dependent on Srcor other SFK activity because it wascompletely blocked by pretreat-ment with 2 �M PP1 (Fig. 1).

Together, these results suggestthat SFK-dependent tyrosine phos-phorylation of VE-cadherin may berequired for VEGF-induced loss ofbarrier function in HDMECs. Nev-ertheless, VEGF may induce theactivation of other signaling path-ways that may also lead to increasedpermeability. To better understandthe regulation of endothelial mono-layer permeability in the absence ofother confounding signals inducedby inflammatory mediators orgrowth factors, we initiated Src acti-vation by expressing DN-Csk.Adenoviral delivery of the DN-Cskplasmid produced a dose- and time-dependent increase in DN-Cskexpression and an increase in thetotal phosphotyrosine content, con-sistent with activation of Src byDN-Csk (Fig. 2, A and B). DN-Csk-in-duced tyrosine phosphorylationwas severalfold stronger than thatinduced by VEGF treatments (seesupplemental Fig. 1). To confirmSrc activation, we established thatthere was a decrease in the phos-phorylation of Tyr-527 (Fig. 2B) andan increase in the phosphorylationof Tyr-416 in the endogenous Src(see Figs. 3A and 4A). In addition,phosphorylation of paxillin at Tyr-118 and of FAK at Tyr-861 wasalso increased, consistent with anincrease in the activation of Src fam-ily kinases (supplemental Fig. 6).

Surprisingly, DN-Csk expressiondid not induce a decrease in barrierfunction, as assessed by changes intrans-endothelial electric resistance(TEER). As shown in Fig. 2C, doses

FIGURE 2. Overexpression of DN-Csk induces tyrosine kinase signaling but does not increase monolayerpermeability. A, confluent, mature monolayers of HDMECs were infected with adenovirus to express eitherGFP (1.7 � 109 pfu/ml) or DN-Csk (1.8 � 108 to 7.2 � 108 pfu/ml), and 24 h later, cells were lysed and probed forCsk expression and total phosphotyrosine content by Western blot. �-actin blots served as loading control.B, confluent monolayers of HDMECs were infected with 7.2 � 108 pfu/ml DN-Csk virus at time 0 and lysed 3–12h after infection. Cell lysates were then probed for Csk expression, total phosphotyrosine content, and phos-phorylated Tyr-527 (inactive) Src. C, confluent monolayers were infected with adenovirus to express either GFPor DN-Csk (pfu/ml as indicated). Monolayer resistance was measured by ECIS at 4000 Hz. A two-way repeatedmeasures analysis of variance was performed with Bonferroni post tests versus GFP-infected wells values (n �2). No significant changes were detected upon DN-Csk overexpression. Results are representative of at leastthree independent experiments.




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of adenovirus shown to induce Src activation in panels A and Bdid not decrease the resistance across the endothelial mono-layer, as would be expected with a loss of barrier function.Importantly, DN-Csk expression failed to increase endothelialmonolayer permeability, although it promoted SFK activationat the sites of cell-cell contact, as shown by immunofluores-cence to phosphorylated Tyr-416 Src (Fig. 3A). Consistent withthe ECIS data, cell-cell junctions can still be observed at cell-cellcontacts, as assessed by VE-cadherin labeling (Fig. 3B). How-ever, we did observe a change in the morphology of the VE-cadherin-mediated junction, as characterized by an increase incell overlap with a net-like structure of labeled VE-cadherin atthese overlapping regions (Fig. 3B and supplemental Fig. 5).

We next tested whether the overexpression of caSrc resultedin a loss of barrier function. We detected caSrc expression in

HDMECs starting at 6–8 h after adenoviral infection (Fig. 4A).Expression of caSrc promoted total tyrosine phosphorylation tolevels that were comparable with those achieved with DN-Cskexpression. As opposed to DN-Csk expression, caSrc induced amarked decrease in barrier function (Fig. 4B). Interestingly, thedecrease in TEER occurred between 12 and 16 h, whereas theincrease in active Src was observed as early as 8 h after infectionwith the caSrc construct, a time frame similar to that ofDN-Csk(Fig. 4A, pY416 Src). Consistent with the decrease in TEER,expression of caSrc produced large gaps between cells (supple-mental Fig. 2).The difference in altering barrier response followingDN-Csk

and caSrc expression led us to test whether there was a differ-ence in the localization of active Src following expression ofthese two constructs. As shown in Fig. 3A, DN-Csk expressioninduced SFK activation that was localized at the cell-cell bor-ders, whereas caSrc expression induced phospho-Tyr-416staining that was more widely distributed and not restricted tocell periphery (supplemental Fig. 2). This difference was notdue to total Csk or Src localization because we observed thatDN-Csk and caSrc virus induced Csk and Src expression,respectively, that localized throughout the cell (supplemen-tal Fig. 3). The appearance of gaps between caSrc-infectedcells led us to test whether caSrc induced changes in the actincytoskeleton in these cells. Consistent with the relocaliza-tion of active Src, caSrc induced a drastic rearrangement ofactin stress fibers (supplemental Fig. 4). These fibers wereshorter and thicker and appeared to be bundled together.Contrary to caSrc expression, DN-Csk-expressing cells didnot show any overt actin reorganization when comparedwith control cells (supplemental Fig. 4).Because VE-cadherin phosphorylation may participate in

changes in endothelial cell barrier function, we tested whetherdifferences in tyrosine phosphorylation of VE-cadherin in cellsexpressing either DN-Csk or caSrc could explain differences inthe regulation of barrier function by these two constructs. Asshown in Fig. 5, both caSrc and DN-Csk expression inducedVE-cadherin phosphorylation on Tyr-658 and Tyr-731,whereas only DN-Csk expression promoted Tyr-685 phosphor-ylation. Time course experiments showed a close relationshipbetween SFK activation and VE-cadherin tyrosine phosphory-lation (compare Fig. 4A with Fig. 5A). We detected VE-cad-herin phosphorylation as early as 6 h after adenoviral infectionof either DN-Csk-containing or caSrc-containing virus. Tyro-sine phosphorylation of VE-cadherin was increased at both 8and 12 h after infection with caSrc adenovirus; however, therewas no change in TEER until 15 h after caSrc infection. Thus,the increase in VE-cadherin tyrosine phosphorylation was nottemporally associated with decrease in TEER.We also observed that in most cases, caSrc expression

induced a stronger Tyr-658 and Tyr-731 VE-cadherin phos-phorylation level than DN-Csk, which suggested that maybeDN-Csk infection was not inducing a level of phosphorylationsufficient to promote the loss of barrier function in these cells.To determine whether increased levels of tyrosine phosphory-lation of VE-cadherin would induce changes in permeability,we infected endothelial monolayers with higher levels of ade-novirus to increase the amount of expressed DN-Csk. In addi-

FIGURE 3. DN-Csk overexpression induces changes in the cell-cell bor-ders. A, confluent, mature monolayers of HDMECs were infected with adeno-virus to express either GFP or DN-Csk and fixed 24 h later. The levels andlocalization of active (pTyr-416) Src (pY416 Src) were determined by indirectimmunofluorescence. Ctrl, control. B, HDMECs were infected with adenovirusto overexpress GFP or DN-Csk and fixed 6, 18, or 30 h after infection. Cells werestained for VE-cadherin (red) or nuclei (4�,6-diamidino-2-phenylindole, blue).White arrows point to areas of cytoplasmic overlay in DN-Csk-overexpressingcells. Results are representative of at least three independent experiments.




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tion, we compared changes in tyrosine phosphorylation ofVE-cadherin at 24 and 48 h after infection. As shown in Fig. 5B,a 4-fold increase in adenovirus infection produced a greaterexpression of DN-Csk and induced a higher level of VE-cad-herin phosphorylation on tyrosines 658, 685, and 731. Theincrease in DN-Csk expression (Fig. 5B, lane 4X DN-Csk)resulted in a level of VE-cadherin tyrosine phosphorylationcomparable with that of caSrc. Of note is that the level of VE-cadherin phosphorylationwas greater at 48 h than it was at 24 hfor both levels of DN-Csk expression. Nevertheless, the higherlevels ofDN-Csk expression did not produce a change in barrierfunction as TEER was not altered during the 48 h of increasedDN-Csk expression (Fig. 5C). Conversely, we infectedHDMECs with lower amounts of caSrc virus, resulting inreduced levels of VE-cadherin phosphorylation but only a slightdelay (�2 h) in the increase in monolayer permeability (Fig.5D). Taken together, these results demonstrate that VE-cad-herin phosphorylation is not sufficient to produce a decrease inendothelial barrier function.It has been suggested that VE-cadherin phosphorylationmay

reduce its ability to bind to p120 and �-catenin at the adherensjunction complex. Thus, we determinedwhether the changes inVE-cadherin phosphorylation induced by DN-Csk expressionaltered VE-cadherin attachment to catenins. Co-localization ofVE-cadherinwith p120 in areas of cell-cell overlap suggests thatthere is an intact adherens junction complex in DN-Csk-ex-pressing cells (Fig. 6A), consistent with the lack of induction ofpermeability by this construct. Confocal microscopy imagesshowed that VE-cadherin staining at the cell-cell overlapregions is in the same plane as the VE-cadherin located at thecell-cell borders, suggesting that this overlapmay be due to thin

cell extensions (supplemental Fig.5A). We also observed that VE-cad-herin additionally co-localized atthe cell borders with �-catenin and�-catenin (supplemental Fig. 5B).Furthermore, by immunoprecipita-tion of VE-cadherin followed byimmunoblotting for either p120 or�-catenin, we found that both p120and �-catenin are associated withVE-cadherin in cells expressingDN-Csk despite VE-cadherin phos-phorylation (Fig. 6B). Althoughgaps were found between ECmono-layers expressing caSrc, �-cateninand p120 could also be co-immuno-precipitated with VE-cadherin inlysates from these cells (Fig. 6B), andp120,�-catenin, and �-cateninwereall co-localized at the cell-cell junc-tion (supplemental Fig. 5).We then tested the possibility

that DN-Csk overexpression maysequester important proteins re-quired to promote an increase inmonolayer permeability, thus pre-venting SFK-mediated loss in bar-

rier function. If this was the case, then Csk knockdown wouldnot only induce SFK activation but also a permeability increase.Electroporation of siRNAs specific to Csk promoted VE-cad-herin phosphorylation at tyrosines Tyr-658 and Tyr-731 butnot at Tyr-685 (Fig. 7A). Nevertheless, the same siRNAs did notprevent themonolayer from forming a restrictivemonolayer, asassessed by TEER (Fig. 7B), demonstrating that SFK activationalone is not sufficient to alter barrier function.Baumeister et al. (23) reported thatCsk binds toVE-cadherin

in a Tyr-685-dependent fashion. Furthermore, the same groupproposed that Csk binding to VE-cadherin may prevent theaccess of phosphatases to tyrosine 685. Consistent with thisfinding, overexpression of DN-Csk, but not Csk knockdown,induced VE-cadherin Tyr-685 phosphorylation (Fig. 7A).Regardless of the method employed to activate Src, the subse-quent increase of VE-cadherin tyrosine phosphorylation wasdependent on continuous SFK activity because the addition ofPP1 Src inhibitor for 6 h reversed DN-Csk- or caSrc-inducedVE-cadherin phosphorylation in cells overexpressing eitherDN-Csk or caSrc for 24 h (Fig. 8). PP2, another pharmacologicalinhibitor, also prevented caSrc-induced TEER and the phos-phorylation of VE-cadherin (supplemental Fig. 6).A VE-cadherin-Csk complex has been postulated to serve as

a scaffold for other signaling molecules. In fact, VE-cadherinand Csk are required for VEGF-mediated Akt phosphorylation(5). Supporting this notion, we observed that DN-Csk, but notcaSrc, induced the phosphorylation of Akt (Fig. 8). Interest-ingly, this phosphorylation was not completely dependent onSFK activity, a finding consistent with a role for Csk as a scaf-folding protein. On the other hand, caSrc, but not DN-Csk,promoted a mild increase in Erk1/2 phosphorylation that was

FIGURE 4. Overexpression of caSrc induces a robust decrease in monolayer barrier function. A, confluent,mature monolayers of HDMECs were infected with adenovirus to express GFP (1.7 � 109 pfu/ml), DN-Csk (7.2 �108 pfu/ml), or caSrc (3.2 � 106 pfu/ml), and 6 –12 h later, cells were lysed and probed for Csk and Src expres-sion, total phosphotyrosine content, and Src activation (pY416 Src) by Western blot. �-actin blots served asloading control. B, confluent monolayers were infected as in A, and monolayer resistance was measured byECIS at 4000 Hz. A two-way repeated measures analysis of variance was performed with Bonferroni post testsversus GFP-infected wells values (n � 4). The arrow marks the first time point in which caSrc induced a signifi-cant decrease in cell permeability (p � 0.001). No significant changes were detected upon DN-Csk overexpres-sion. Results are representative of five independent experiments. Error bars show S.D. (n � 3).




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dependent on SFK activity. Inhibi-tion of either Akt or Erk activationby pretreatment with the pharma-cological inhibitors LY294002 orU0126 did not have any effect onTEER nor showed any modulationof TEER in the presence of DN-Cskor caSrc (supplemental Fig. 7).

DISCUSSION

Previous studies have demon-strated that Src signaling is requiredfor receptor-induced increase invascular permeability (6, 24, 25).Weconfirmed this requirement inHDMECs as the Src inhibitor PP1was able to prevent VEGF-inducedchanges in permeability. In addi-tion, the decrease in permeabilitywas temporally associated with anincrease in phosphorylation of VE-cadherin on three tyrosine residuesthat have been implicated in theregulation of endothelial barrierfunction. Nevertheless, receptoractivation by VEGF, as well asother inflammatory mediators andgrowth factors, is known to stimu-late many different signaling path-ways in parallel (26), raising thequestion of whether Src signalingwas not only required but also suffi-cient to regulate endothelial mono-layer permeability. To answer thisquestion, we used three systems todirectly increase activity of SFKsindependent of receptor activation.Src is the prototypical member of

a family of tyrosine kinases with ahigh degree of conservation (27).Inhibition of Csk activity by eithergene knockdown or expression of adominant negative construct drasti-cally promotes SFK signaling (28).Interestingly, expression of Csk inendothelial cells increases VE-cad-herin phosphorylation (23), andCskinteraction with VE-cadherin hasbeen implicated in VEGF signalingand endothelial cell function (5).Surprisingly, we found that Src acti-vation and the subsequent tyrosinephosphorylation of VE-cadherinfollowing expression of dominantnegative Csk or by decreasing thelevels of Csk did not result in anincrease in endothelial permeabilityas assessed by changes in TEER.

FIGURE 5. DN-Csk expression and downstream SFK signaling induce VE-cadherin phosphorylation butdo not induce change in cell permeability. A, HDMECs were infected with adenovirus to overexpress GFP,DN-Csk, or caSrc and lysed 6 –12 h later. Cell lysates were assayed by Western blot for VE-cadherin phosphor-ylation at tyrosines 658 (pY658), 685 (pY685), and 731 (pY731). Total VE-cadherin blots served as loading control.pY118, phospho-Tyr-118. B, confluent HDMEC monolayers were infected with 6.8 � 109 pfu/ml GFP virus, 7.2 � 108

pfu/ml DN-Csk virus (labeled DN-Csk), 2.88 � 109 pfu/ml DN-Csk virus (labeled 4X DN-Csk), or 3.2 � 106 pfu/ml caSrcvirus and lysed 24 or 48 h later. Lysates were immunoblotted for VE-cadherin phosphorylation, phospho-Tyr-188paxillin, or Csk to document infection levels. Total VE-cadherin and �-actin blots served as loading controls; NI,non-infected. C, HDMEC cells were infected with adenovirus to express GFP or increasing amounts of DN-Csk at time0. Monolayer resistance was measured by ECIS at 4000 Hz. D, HDMEC cells were infected with adenovirus to expressGFP or different amounts of caSrc at time 0. Monolayer resistance was measured as above. A two-way repeatedmeasures analysis of variance was performed with Bonferroni post tests versus GFP-infected wells values (n � 3). Nosignificant differences were observed at any DN-Csk infection amount at any time point. Results are representativeof three independent experiments. Error bars show S.D. (n � 3).




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This finding suggests that Src activity may be required formediator-induced increases in permeability but that anincrease in Src-induced VE-cadherin tyrosine phosphorylationis not sufficient to decrease endothelial barrier function.In contrast to activation of Src through a loss of Csk activity,

forced expression of caSrc resulted in a decrease in barrier func-tion. The discrepancy between caSrc and DN-Csk expressioneffectsmay lie in themode of Src activation by these constructs.Although DN-Csk expression induces activation of endoge-nous SFKs, expression of caSrc does not depend on the endog-enous SFKs but instead provides active Src throughout the cell.DN-Csk, however, would promote the activation of SFK at their

normal subcellular localization.Thus, the induction of endothelialcell permeability in caSrc-express-ing cells may be due to the activa-tion of Src signaling at sites whereactivated Src would not typically befound in the cell. This is supportedby immunofluorescence data show-ing that in DN-Csk-expressing cells,active (pTyr-416) Src is localized atthe plasma membrane at the sitesof cell-cell contact, whereas incaSrc-expressing cells, active Src islocalized mainly at intracellularcompartments, rather than just atcell-cell borders.Alternatively, the observed

changes in TEER with forced ex-pression of caSrc may not be adirect effect of protein phosphoryla-tion but may involve changes ingene expression that are not foundwith DN-Csk expression. This issupported by the data showing a4–6-h delay between increases inprotein tyrosine phosphorylation,including VE-cadherin, and thedecrease in TEER induced byexpression of caSrc. Althoughchanges in gene expression mayparticipate in the loss of TEER, thisis not mediated by activation ofErk1/2 as inhibition of Erk1/2 phos-phorylation by the inhibitor U0126did not prevent the decrease inTEER following caSrc expression.Using synthetic peptides of the

VE-cadherin cytoplasmic domain,Wallez and co-workers (29) demon-strated that Tyr-685 of VE-cadherincan be phosphorylated by Src. Fur-thermore, these investigators dem-onstrated that VEGF increasedphosphorylation of Tyr-685 inhuman umbilical vein endothelialcells. We observed that the phos-

phorylation of Tyr-685 in HDMECs treated with VEGF wastemporally associated with a decrease in TEER. We alsoobserved an increase in Tyr-685 phosphorylation when Src wasactivated by expression of DN-Csk. This is consistent with thefindings of Baumeister et al. (23), who found increases in VE-cadherin phosphorylation in CHO cells coexpressing VE-cad-herin and either DN-Csk or wild type Csk. Our findings thatTyr-685 is not phosphorylated upon siRNA-mediated Cskknockdown supports the concept that Csk presence is requiredfor VE-cadherin phosphorylation at Tyr-685. These results alsoshow that Src activation in the absence ofCsk is not sufficient tophosphorylate Tyr-685, suggesting that Tyr-685 phosphoryla-

FIGURE 6. DN-Csk and caSrc overexpression does not prevent VE-cadherin binding to p120 and �-cate-nin or co-localization to the adherens junctions. A, HDMECs were infected with adenovirus to overexpressGFP, DN-Csk, or caSrc and fixed 24 h later. Cells were then stained with antibodies against p120 (green) andVE-cadherin (red). Nuclei were stained with 4�,6-diamidino-2-phenylindole (DAPI) (blue). The white arrows pointto the sites of cell-cell overlapping, which still showed p120 and VE-cadherin co-localization. B, HDMECs wereinfected with adenovirus to overexpress GFP, DN-Csk, or caSrc and lysed 16 h after infection. Immunoprecipi-tates (IP) of p120, �-catenin (�-cat), or VE-cadherin (VE-cad) were run on SDS-PAGE together with beads onlyprecipitates (IP Ctrl) and total cell lysates (TCL) and immunoblotted as described in the figure. �-actin was usedas loading control in the total cell lysates, whereas Src and Csk immunoblots served as infection controls.Results are representative of three independent experiments.




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tion requires events other than Src activation. This is furthersupported by our data showing that Tyr-685 is not phosphory-lated with forced expression of caSrc. However, the increase inTyr-685 phosphorylation by expression of DN-Csk was inhib-ited by PP1 or PP2, showing that Src activation is indeedrequired for VE-cadherin phosphorylation on this tyrosine res-idue. This finding supports the model that SFK can phosphor-ylate Tyr-685 but that Csk may serve to inhibit dephosphoryla-tion of Tyr-685 by phosphatases. This is consistent with thehypothesis that VEGF-induced phosphorylation of Tyr-685requires Csk-VE-cadherin interaction (5). By comparing theeffect of DN-Csk and caSrc constructs on the phosphorylationof Tyr-685 and on TEER, it is possible to hypothesize that Tyr-685 phosphorylation may in fact prevent, rather than promote,

SFK-mediatedTEER induction.Nevertheless, this hypothesis isnot compatible with two other observations: 1) that the siRNA-mediated knockdown of Csk does not induce the phosphoryla-tion of Tyr-685 nor promote TEER and 2) that VEGF-mediatedincrease inTEER is concurrentwith an increase inVE-cadherinphosphorylation at Tyr-685.Our findings that caSrc did not increase Tyr-685 phosphor-

ylation are not consistent with those of Wallez et al. (15), whofound increased Tyr-685 phosphorylation with expression ofcaSrc inCHOcells expressingVE-cadherin.However, our find-ings that overexpression of caSrc in endothelial cell monolayersincreases both Tyr-658 and Tyr-731 phosphorylation are con-sistent with the findings of Potter et al. (14), who demonstratedthat these are the two primary tyrosine residues in VE-cadherinthat are phosphorylated when caSrc is expressed in CHO cells

FIGURE 7. siRNA-mediated Csk knockdown promotes Tyr-658 (pY658)and Tyr-731 (pY731) but not Tyr-685 (pY685) VE-cadherin phosphoryla-tion and does not increase monolayer permeability. A, HDMEC cells wereelectroporated with 2 �g of luciferase (Luc)- or Csk-specific siRNAs, and 72 hlater, Csk expression and VE-cadherin phosphorylation were assessed byWestern blot. Total VE-cadherin blots served as loading control. B, after siRNAelectroporation, HDMEC cells were seeded onto ECIS chambers for mono-layer resistance monitoring. Notice the increase in resistance after seeding,due to cell spreading, proliferation, and cell-cell contact maturation, thatreaches a plateau after �96 h after seeding. Csk-siRNA-carrying cells show thesame increase in resistance as control cells. Error bars show S.D. (n � 3).

FIGURE 8. DN-Csk- and caSrc-induced signaling depends on continuousSFK activity. HDMEC cells were infected with adenovirus to overexpress GFP,DN-Csk, or caSrc for 18 h and maintained in EBM-2 medium with 0.3% fetalbovine serum and no other added factors. Cells were then incubated for 6 h inthe presence of 10 �M PP1 or 0.1% DMSO prior to lysis and Western blotanalysis to assess VE-cadherin phosphorylation (A) and Erk and Akt activation(B). Total VE-cadherin, total Erk, and total Akt blots served as loading control.Results are representative of at least three independent experiments. pY658,phospho-Tyr-658; pY685, phospho-Tyr-685; pY731, phospho-Tyr-731; pErk1/2,phospho-Erk1/2; pAkt, phospho-Akt.




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also expressing VE-cadherin. We show that Tyr-658 and Tyr-731 of VE-cadherin are also phosphorylated when Src is acti-vated by expression of DN-Csk or knockdown of Csk. We alsofound increases in Tyr-658 and Tyr-731 phosphorylation fol-lowing VEGF treatment of HDMEC monolayers. These twosites have also been shown to be phosphorylated in response tointercellular adhesion molecule-1 (ICAM-1) ligation (16) andto require Src activation.A number of recent studies have begun to use the phenylala-

nine substitutions at the different tyrosine residues in VE-cad-herin as a method to investigate the role of VE-cadherin phos-phorylation in the regulation of cell function. Studies usingVE-cadherin-expressing CHO cells have shown that substitu-tion of Y685F prevented contact inhibition of growth (23). Pot-ter et al. (14) showed that substitution of Y658F and Y731Fprevented vanadate-induced decreases in the barrier functionof CHO cells expressing mutant forms of VE-cadherin. Theseinvestigators also demonstrated that CHO cells expressingmutant VE-cadherin with glutamic acid substitutions of Tyr-658 or Tyr-731 did not form a restrictive barrier when com-pared with CHO cells expressing wild type VE-cadherin. Thestudy of Potter et al. (14) supports the hypothesis that tyrosinephosphorylation of VE-cadherin following treatment withinflammatorymediators results in a loss of barrier function, andmany of these studies have implicated Src as a major signalingmolecule in the pathway leading to VE-cadherin phosphoryla-tion and a loss of barrier function (reviewed in Refs. 30 and 31).In contrast, our data demonstrate that VE-cadherin tyrosine

phosphorylation is not sufficient to decrease the barrier func-tion of endothelial cell monolayers. Indeed, Src activation byexpression of DN-Csk or knockdown of Csk resulted in thephosphorylation of VE-cadherin on Tyr-658 and Tyr-731 butdid not change TEER across the monolayers. Although caSrcresulted in a decrease in resistance, this change occurred wellafter the phosphorylation of Tyr-658 andTyr-731was observed(6–8 h for phosphorylation versus 12–15 h for decreasedTEER). Thus, the tyrosine phosphorylation of VE-cadherin fol-lowing caSrc expression is not temporally associated with thedecrease in barrier function. The discrepancy betweenour find-ings and those of Potter et al. (14) may be the result of thedifferent cell types as previous investigations have demon-strated that cell context is important between CHO cells andendothelial cells with respect to VE-cadherin and cell signaling(32). It is also possible that in HDMECs, the pool of active Srcinduced byDN-Csk expression or siRNA-mediated Csk knock-down is not capable of activating a VAV2-Rac-p21 activatedkinase pathway thatmay result in the phosphorylation of serine665 of VE-cadherin, binding to �-arrestin and VE-cadherinendocytosis (33). However, we did not see an increase in VE-cadherin endocytosis in our fluorescence experiments, evenafter caSrc overexpression, suggesting that this pathway maynot be essential in this model. Also, it is possible that otherGTPases regulating permeability may be differentially regu-lated (see Ref. 34 and references therein).Surprisingly, we also did not find a change in the association

of VE-cadherin to either �-catenin or p120. Activation of SFKsignaling resulted in phosphorylation of VE-cadherin and p120,although these phosphorylations did not prevent the co-local-

ization of adherens junction proteins at the cell-cell junction orthe ability to co-immunoprecipitate VE-cadherin, p120, and�-catenin. Moreover, although caSrc expression induced amarked increase in permeability, co-localization, and co-im-munoprecipitation of VE-cadherin, p120 and �-catenin werenot impaired, suggesting that even in extreme circumstances,the adherens junction protein complex may be intact.Both Akt and Erk were activated by VEGF treatments in

HDMECs. DN-Csk expression inducedAkt activation, whereascaSrc expression promoted Erk activation. Nevertheless, nei-ther LY294002 nor U0126 treatments were able to modulateTEER in these conditions. This suggests that phosphatidylino-sitol 3-kinase-Akt and mitogen-activated protein kinase/Erkkinase (MEK)-Erk may be parallel pathways not involved inTEER or that the activation of the pathways upstream of thesekinases, rather than the endpoints tested, are responsible forthe changes in barrier function.Our data show that Src-induced VE-cadherin phosphoryla-

tion at Tyr-658, Tyr-685, and Tyr-731 is not sufficient toincrease barrier function. Furthermore, our results suggest thatVE-cadherin phosphorylation is not always followed by junc-tion disassembly. This does not mean that VE-cadherin phos-phorylation is not required for mediator-induced decreases inendothelial barrier function. For example, VE-cadherin phos-phorylation at Tyr-658 and Tyr-731 has been shown to berequired for trans-endothelial migration of leukocytes as Tyr toPhe mutation of either Tyr-658 or Tyr-731 decreases trans-endothelial cell migration (35, 36). The phosphorylation of VE-cadherin on these residues is the result of ligation of ICAM-1and the activation of SFK and Pyk2. These tyrosine residuesmay not be the only important residues phosphorylated in VE-cadherin as attachment of antigen-activated lymphocytes toICAM-1 induced the phosphorylation of tyrosines 645, 731,and 733, a process mediated by Rho GTPase, Ca2�, and actinrearrangement but not by Src activation (17). In addition, it hasalso been shown thatmutation of Tyr-658 or Tyr-731 attenuateVEGF-induced decreases in barrier function (37). These studiescoupled with our results would suggest that activation of mul-tiple signaling pathways by inflammatory mediators, includingthose resulting in VE-cadherin phosphorylation, may all berequired to produce a change in permeability. In this model,VE-cadherin would serve a permissive role in which phosphor-ylation would be required for the change in permeability tooccur but would not be sufficient to change permeability byitself. This is supported by our data that demonstrate that acti-vation of a single pathway leading to VE-cadherin tyrosinephosphorylation may not be sufficient to promote disassemblyof the adherens junction and suggest that further research isneeded to elucidate the role of VE-cadherin phosphorylation inthe regulation of endothelial barrier function.

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Alejandro P. Adam, Amy L. Sharenko, Kevin Pumiglia and Peter A. VincentDecrease Barrier Function of Endothelial Monolayers

Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to

doi: 10.1074/jbc.M109.079277 originally published online January 4, 20102010, 285:7045-7055.J. Biol. Chem.

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src-induced tyrosine phosphorylation of ve-cadherin is not

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