29Short Report

IntroductionIntercellular adhesion molecule 1 (ICAM1) on the luminal surfacemembrane of vascular endothelial cells (ECs) plays a crucial roleduring the egress of lymphocytes from the vascular compartment.It binds to �2 integrins, such as leucocyte-function-associatedantigen 1 (LFA-1), a heteromer of �L (ITGAL, also known asCD11a) and ITGB2 (CD18), on lymphocytes and promotes theirstable arrest (Butcher, 1991). However, endothelial ICAM1 is notonly a docking receptor for lymphocyte adhesion and migration butits engagement also triggers endothelial signaling cascades thatcontribute fundamentally to vascular compliance to lymphocytediapedesis and the endothelial inflammatory response (Turowski etal., 2005). ICAM1-induced signaling comprises changes in levelsof intracellular Ca2+ and dynamic actin as well as activation of thesmall GTPase Rho, and Src and C protein kinases. Despite theidentification of an increasing number of key players involved inICAM1-mediated endothelial signaling, downstream effectors andmolecular endpoints that ultimately modulate lymphocytemigration remain elusive.

Both tight and adherens junction (AJ) proteins form attachmentsbetween ECs, and are major sites of regulation in the transport ofmolecules and cells across vascular barriers (Bazzoni and Dejana,

2004). At least in the case of paracellular leukocyte extravasation,various junction molecules disengage and, in turn, form homo- andheterophilic interactions with leukocyte adhesion proteins (Imhofand Aurrand-Lions, 2004; Muller, 2003; Rao et al., 2007). In thisprocess the EC has been ascribed a rather passive role, wherejunctions are forced open by the migrating leukocyte (Imhof andAurrand-Lions, 2004; Shaw et al., 2001). We investigated whetherintercellular junction modulation is part of the endothelial responseto lymphocyte migration.

Results and DiscussionMigration of antigen-activated lymphocytes across brainmicrovascular ECs in vitro does not require stimulation ofinflammatory cytokines, occurs in the absence of chemokinegradients and is predominantly dependent on ICAM1 but notvascular cell adhesion molecule 1 (VCAM1) (Greenwood et al.,1995). ICAM1-mediated signaling pathways involve RhoGTPases and MAP kinase cascades and have generally beenstudied by antibody-mediated crosslinking of serum-starved cells(Turowski et al., 2005). In the rat brain microvascular EC cellline (GPNT) ICAM1 activation led to a rapid and transientincrease of tyrosine phosphorylation of a number of proteins

Lymphocytes emigrate from the circulation to target tissuesthrough the microvascular endothelial cell (EC) barrier.During paracellular transmigration cell-cell junctions havebeen proposed to disengage and provide homophilic andheterophilic interaction surfaces in a zip-like process. However,it is not known whether ECs modulate junction proteins duringthis process. Here we show that tyrosine phosphorylation ofadherens junction vascular endothelial cadherin (VEC) isrequired for successful transendothelial lymphocyte migration.We found that adhesion of lymphocytes or activation of theendothelial intercellular adhesion molecule 1 (ICAM1) led totyrosine phosphorylation of VEC. Substitution of tyrosine forphenylalanine in VEC at positions 645, 731 or 733 producedECs that were significantly less permissive to lymphocytemigration. We also found that these same tyrosine residuesare involved in ICAM1-dependent changes of VEC

phosphorylation. ICAM1 activation enhanced transendothelialpermeability, suggesting the occurrence of junctiondisassembly. In agreement, the expression of VEC mutated atY645F, Y731F or Y733F predominantly affected lymphocytetransmigration in paracellular areas. Taken together, theseresults demonstrate that phosphorylation of adherensjunctions constitutes a molecular endpoint of lymphocyte-induced vascular EC signaling and may be exploited as a newtarget of anti-inflammatory therapies.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/121/1/29/DC1

Key words: Lymphocyte migration, VE-cadherin, Tyrosinephosphorylation, Brain endothelium, ICAM1

Summary

Phosphorylation of vascular endothelial cadherincontrols lymphocyte emigrationPatric Turowski1,*, Roberta Martinelli1, Rebecca Crawford1,‡, David Wateridge1,§, Anna-Pia Papageorgiou1,Maria Grazia Lampugnani2, Alexander C. Gamp5, Dietmar Vestweber5, Peter Adamson1,¶,Elisabetta Dejana2,3,4 and John Greenwood1,*1Division of Cell Biology, Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK2Mario Negri Institute for Pharmacological Research, 3IFOM-IEO Campus, Via Adamello 16 and 4Department of Biomolecular Sciences andBiotechnologies, Faculty of Sciences, University of Milan, 20139 Milan, Italy5Max-Planck-Institute of Molecular Biomedicine, Röntgenstr. 20, 48149 Münster, Germany*Authors for correspondence (e-mails: p.turowski@ucl.ac.uk; j.greenwood@ucl.ac.uk)‡Present address: Kennedy Institute of Rheumatology, Imperial College, London, W6 8LH, UK§Present address: Faculty of Life Sciences, University of Manchester, Manchester, UK¶Present address: Ophthaltec Limited, London, SW1Y 4QU, UK

Accepted 25 September 2007Journal of Cell Science 121, 29-37 Published by The Company of Biologists 2008doi:10.1242/jcs.022681

30 Journal of Cell Science 121 (1)

Fig. 1. Tyrosine phosphorylation of VEC following ICAM1 crosslinking or adhesion of lymphocytes. (A) GPNT cells were grown to confluence, serum starvedand ICAM1 crosslinked (XL) for the indicated times. Total protein extracts (~50 �g) were analyzed by immunoblotting with antibodies against phosphorylatedtyrosine. Blots were subsequently stripped and probed for �-catenin as loading control. Four proteins with apparent molecular masses of 220 kDa, 140 kDa, 94 kDaand 83 kDa [previously identified as cortactin (Durieu-Trautmann et al., 1994)] displayed clearly enhanced tyrosine phosphorylation and are indicated by filledarrowheads. Open arrowhead indicates the position of the IgG heavy chains of the crosslinking antibody. (B) Confluent GPNT cells were serum starved and either(a,m) left untreated or ICAM1 crosslinked for (b) 15 minutes or (c-l) the different times indicated. Cells were fixed, extracted and stained for (a,b) surface ICAM1,(c-g) phosphorylated tyrosine, (h-l) F-actin or (m) VEC. Bar, 10 �m. (C-F) Confluent (C,D,F) GPNT cells or (E) mouse brain endothelioma EC, bEND5, wereserum starved and subjected to crosslinking of ICAM1 (XL) or unrelated surface molecules (MHC class I; endomucin, EMCN). At the indicated times cells werewashed and lysed. VEC immunoprecipitates were then analyzed by immunoblotting using either antibodies against phosphorylated tyrosine or VEC. (D) Theamount of tyrosine-phosphorylated VEC (see C) was quantified by densitometry from five independent experiments and expressed as fold-increase of untreatedcontrols (mean ± s.e.m.). (F) Prior to ICAM1 crosslinking (15 minutes), and where indicated, cells were pre-treated using PP2 (10 �M, 30 minutes), C3 transferase(2 �g/ml, 16 hours), cytochalasin D (CD, 2 �M, 30 minutes) or BAPTA (BA, 20 �M, 30 minutes). (G,H) Confluent GPNT cells were co-cultured with ratperipheral lymph node (PLN) lymphocytes (approximately five lymphocytes per EC). At the indicated times cells were lysed and VEC immunoprecipitatesprepared and analyzed as described above. (H) Data from four independent experiments were quantified by densitometry, normalized and expressed as fold-increase of untreated controls (mean ± s.e.m.). Significant differences were determined by Student’s t-test (*P<0.003, **P<0.002). In all blots the position of sizemarkers (in kDa) is indicated on the left.

31VE-cadherin in ICAM1-mediated T-cell migration

which was maximal within 10-15 minutes of ICAM1 stimulation(Fig. 1A). When analyzed by immunocytochemistry (Fig. 1B) theincrease in phosphorylated tyrosine was most marked at theintercellular junction area and preceded major actin re-arrangements typically seen following ICAM1 crosslinking(Adamson et al., 1999). Immunoprecipitation and subsequentanalysis of phosphorylated tyrosine of junction proteins revealedthat VEC (Fig. 1C,E), but not the tight junction protein 1 (ZO-1), occludin, or any of the catenins (Fig. 2), displayed alteredtyrosine phosphorylation following ICAM1 crosslinking.Phosphorylation of VEC was rapid and transient, reaching amaximum after 15 minutes of ICAM1 crosslinking (Fig. 1D). Itdepended on upstream signaling by Rho GTPase, dynamic actinand also intracellular Ca2+ (Fig. 1F), all of which are essential forsuccessful lymphocyte migration (Turowski et al., 2005). Noinvolvement of Src family protein kinases was observed in ourexperimental set up. This is in contrast to a recent report byAllingham et al. (Allingham et al., 2007) and may reflect thatdifferent signaling modules are activated and used by differentsubsets of ECs and/or leukocytes. Indeed, T-cell adhesion toGPNT cells also induced a significant rise in VEC tyrosinephosphorylation (Fig. 1G,H), albeit with a slightly delayed timecourse (presumably reflecting settling and adhesion times). Takentogether these data suggest that interendothelial junctions can bemolecular endpoints of ICAM1-mediated signaling duringleukocyte adhesion and migration.

To study the functional significance of ICAM1-mediated VECphosphorylation, the structure of the cytoplasmic domain of VECwas further analyzed in silico and by mutagenesis. The cytoplasmictail of VEC contains eight conserved tyrosine residues (Fig. 3A),three of which are predicted to constitute phospho-acceptor sites

for insulin receptor family (Y645, Y731) or Src family (Y685)protein kinases (http://kinasephos.mbc.nctu.edu.tw, data notshown). A structural computer model of the VEC intracellulardomain bound to �-catenin suggested that Y725, Y731 and Y733are exposed and accessible to phosphorylation, whereas Y685,Y757 and Y774 are oriented towards the bound �-catenin and,possibly, inaccessible (Fig. 3B). Y645 and Y658 are locatedoutside of this area in a domain predicted to mediate binding top120 (Lampugnani et al., 2002).

Recently, Y658, Y685 and Y731 have been described as targetphosphorylation sites (Potter et al., 2005; Wallez et al., 2006), andwe therefore asked whether phosphorylation of any of these threetyrosines residues is important for lymphocyte migration. For thiswe derived EC lines from VEC-null mouse endotheliomas that re-expressed wild-type (wt) VEC, Y658F-VEC, Y685F-VEC orY731F-VEC. The VEC expression levels and localization weresimilar in all four cell lines (Fig. 4A,B), suggesting that Y to Fmutations do not interfere with junctional targeting. Migration ofactivated T cells across VEC-null ECs was very low butsignificantly enhanced when wt VEC was re-expressed (Fig. 4C).A similar, twofold increase was observed when either a clonal cellline that stably re-expressed wt VEC or a pool of cells transientlyexpressing EGFP-VEC was analyzed. Absolute migration ratesmeasured after 4 hours of lymphocyte co-culture increased from~10% to 20-25% when VEC was reconstituted in VEC-nullendothelioma cells and were then in the same range as thoseusually observed for GPNT cells (ca. 30%) indicating that theexpression of VEC is vital to the functional integrity of the vascularendothelium. This was somewhat unexpected because VECdisruption using antibodies has been reported to enhance neutrophilmigration in vivo (Gotsch et al., 1997). However, VEC elimination

Fig. 2. Analysis of tyrosine phosphorylation of other junction proteins in ICAM1 stimulated ECs. (A-E) Confluent GPNT cells were serum starved and ICAM1crosslinked (XL). At the indicated times cells were washed, lysed and subjected to immunoprecipitation of (A) ZO-1 and (B-E) catenins as indicated.Immunoprecipitates were then analyzed by immunoblotting using antibodies against phosphorylated tyrosine, ZO-1, VEC or catenin. Black and white arrowheadsin A-E indicate the position of migration of VEC and relevant catenins, respectively, as determined by stripping and re-probing of the immunoblots.Immunoprecipitates of p120 did not contain detectable VEC, whether phosphorylated (black arrowhead) or not (data not shown). (F) The chicken occludin-expressing GPNT cell line (see supplementary material Fig. S1) was grown to confluence, serum starved and ICAM1 crosslinked (XL). At the indicated timeschicken occludin was immunoprecipitated and analyzed by immunoblotting for phosphorylated tyrosine or occludin. In all blots the position of size markers (inkDa) is indicated on the left.

32

leads to significant changes in the composition of AJs (Zanetta etal., 2005) and might, in this way, inhibit leukocyte migrationindirectly. Alternatively, VEC might have opposing effects on themigration of lymphocytes and neutrophils. When cells that expressVEC mutants were analyzed we found that the re-expression ofVECs carrying single mutations in Y658 or Y685 did not have anyeffect on lymphocyte transmigration (Fig. 4D). However, mutationof Y731 led to an approximately twofold reduction in lymphocytemigration. This indicated that VEC phosphorylation may beimportant for leukocyte migration.

To test the role of intracellular tyrosines of VEC moresystematically and in an EC system more relevant to inflammation,VEC mutants containing single Y to F substitutions weretransiently expressed as N-terminal fusion proteins of EGFP inGPNT cells. Expression levels and overall localization of theindividual VEC mutants were similar and mostly restricted to theperiphery of the cell (Fig. 5A). Some cells expressed the protein atvery high levels throughout the cell but these appeared non-viableand were never integrated into endothelial monolayers. In all otherexpressing cells, EGFP was almost exclusively found at cell-celljunctions once EC monolayers were formed (Fig. 5B). Again weobserved that the introduction of Y731F-VEC significantlyreduced lymphocyte migration. In addition we also observedimpaired lymphocyte migration across GPNT cells that expressed

Y645F-VEC or Y733F-VEC (Fig. 5C). By contrast, theintroduction of wt VEC or any other Y to F mutant did not affectmigration. The inhibitory effect of Y645F-VEC and Y733F-VECwas also corroborated in a VEC-null background. Transientexpression of Y645F-VEC–GFP or Y733F-VEC–GFP but not anyother VEC-GFP mutants, produced endothelioma cells with asimilar propensity to reduced lymphocyte migration (Fig. 5D). Inall cases the mutant VEC clearly interfered on the level oflymphocyte diapedesis because adhesion rates were comparable.

Commercially available antibodies against VEC phosphorylatedat Y658 or Y731 (Potter et al., 2005) were not reactive with VECimmunoprecipitates from ICAM1-crosslinked cells (data notshown). They also appeared to recognize a protein of the same sizewhether full-length or truncated VEC (Lampugnani et al., 2002)was expressed (data not shown). To examine whether ICAM1-induced tyrosine phosphorylation involved the tyrosine at positions645, 731 or 733 the phosphorylation of wt and mutant GFP-VECwas analyzed in CHO expressing ICAM1 cells (CHO-ICAM1).Crosslinking of ectopic human ICAM1 on CHO cells inducedtyrosine phosphorylation of co-expressed GFP-VEC in a timedependent manner (Fig. 6A), similar to that observed in GPNTcells. When the phosphorylation status of the Y to F mutants ofGPF-VEC was tested, only mutant Y733F-VEC displayedsignificantly reduced tyrosine phosphorylation in response to

Journal of Cell Science 121 (1)

Fig. 3. Conserved tyrosines in the cytoplasmic domain of VEC. (A) Sequence alignment of cytoplasmic domains of VEC from mouse, rat, human and chicken, andmouse E-cadherin. Tyrosines conserved in VEC and their relative position (mouse versus human) are indicated in green. Identical residues, conserved and semi-conserved substitutions are indicated by asterisks, colons and dots, respectively. Grey boxes correspond to those parts of E-cadherin that interact with �-cateninwhen crystallized together (Huber and Weis, 2001). (B) Mouse VEC was computer-modeled on the crystal structure of mouse E-cadherin in the E-cadherin–�-catenin complex (i.e. boxed in A) (pdb: 1i7x, 1i7w). Shown in the left panel is a ribbon representation of this model with the position and orientation of sixtyrosines highlighted in green. The right panels represent enlarged views of areas surrounding these tyrosines. The �-catenin chain is in a space fillingrepresentation. Significantly, in this model, Y685, the residue predominantly phosphorylated following VEGF stimulation (Wallez et al., 2006), is not accessiblewhen �-catenin is bound.

33VE-cadherin in ICAM1-mediated T-cell migration

Fig. 4. Y731 within the intracellular domain of VEC isimportant for lymphocyte migration. Mouse endothelioma celllines, null for VEC and stably re-expressing wt or Y to Fmutants of VEC were grown to confluence. (A) Equal amountsof proteins were analyzed by immunoblotting using anti-VECand anti-ERK antibodies. The position of size markers (in kDa)is indicated on the left. (B) Immunocytochemical analysis of theVEC distribution. Bar, 10 �m. (C) Mouse endothelioma celllines, null for VEC, stably re-expressing wt VEC or transientlynucleofected with the VEC-GFP-expressing plasmid pEGFP-N�-VEC were grown to confluence. They were then incubated withantigen-specific T cells, which were allowed to adhere andmigrate for 4 hours. Adhesion (white) and migration (black)across these EC populations were then determined as describedin the Materials and Methods section. Results are expressed asthe percent increase of VEC-null EC (mean ± s.e.m. of sixreplicates from five independent experiments). (D) Lymphocytemigration across the indicated stable mouse endothelioma celllines. Adhesion (white) and migration (black) across individualtransfected EC populations were then measured as above.Results are expressed as % of control cells re-expressing wtVEC (mean ± s.e.m. of six replicates from at least threeindependent experiments). Significant differences weredetermined by Student’s t-test (*P<0.005, **P<0.0001).

Fig. 5. Y to F substitutions in theintracellular domain of VEC atpositions 645, 731 or 733 affectlymphocyte migration in a dominantmanner. GPNT cells werenucleofected with wt or Y to Fmutants of pEGFP-N�-VEC. Onaverage 80% of cells expressed VEC-EGFP over a period of 3-4 days.(A) Transfected GPNT cells werefixed after 2 days and VEC-GFPdistribution was analyzed byfluorescent microscopy. Bar, 50 �m.(B) Three days after transfection,GPNT cells were fixed and VEC-GFP was expression analyzed usingconfocal microscopy. Bar, 10 �m.(C) Nucleofected GPNT cells weregrown to confluence for 24-48 hoursat which point equal expression wasverified by fluorescent microscopy(see A). Lymphocyte adhesion(white) and migration (black) werethen measured as described in Fig. 4.(D) Mouse VEC-null endotheliomacells (see Fig. 4) were nucleofectedwith wt or the indicated Y to Fmutants of pEGFP-N�-VEC before Tcell adhesion and migration wasassessed. Significant differences weredetermined by Student’s t-test(*P<0.005; **P<0.0001).

34

ICAM1 ligation (Fig. 6B), suggesting that this mutation interferedwith tyrosine phosphorylation of VEC. Next, GFP-VEC was alsoisolated and analyzed from 32P-labelled cells. Significantly, GFP-VEC was immunoprecipitated as a strongly phosphorylatedprotein, with the majority of phosphate present as phosphorylatedserine and only very little phosphorylated tyrosine (data notshown). This observation is also in agreement with the recentlyreported phosphorylation of S665 by PAK (Gavard and Gutkind,2006). Tryptic phosphopeptide mapping revealed at least fivegroups of peptides with apparently variable phosphorylation levels(Fig. 6C,D; supplementary material Fig. S2). When ICAM1 wascrosslinked most phosphopeptides remained unchanged but twopeptides displayed significantly reduced chromatographic mobility,indicative of additional phosphorylation (arrows in Fig. 6D). TheY645F mutation suppressed phosphorylation of one peptide butenhanced that of the other, indicating that this mutation interfereswith the recognition of the phosphorylation site. Mutation Y731Finduced a perfect reversal to VEC from non-crosslinked cellssuggesting that this site was directly phosphorylated. Overallphosphorylation was low in Y733F-VEC and the migration of mostphosphopeptides was altered, which suggests that this mutationrenders most sites unphosphorylatable. More refined analysis ofphosphopeptides by mass spectroscopy will be required to

delineate the exact phosphorylation changes that occur on tyrosineand serine residues when ECs are stimulated. In summary, VECmutations of Y645, Y731 or Y733, which dominantly inhibitlymphocyte migration, also affect ICAM1-mediated VECphosphorylation, suggesting that these events are mechanisticallylinked.

VEC has been reported to be phosphorylated on tyrosine undervarious conditions (Andriopoulou et al., 1999; Esser et al., 1998)and, indeed, we found that tyrosine phosphorylation was alsoincreased when GPNT cells were treated with a variety ofvasoactive compounds (Fig. 7A). Generally it is thought that suchphosphorylation induces the disruption of AJs, which can bemeasured by concomitant rises in transendothelial permeability.We found that ICAM1 crosslinking is likely to affect AJorganization because it also induced an increase in paracellularpermeability (Fig. 7B) and monolayer impedance (data not shown).However, in contrast to growth factor stimulation, where this isfrequently associated with altered catenin binding (Lampugnani etal., 1997; Potter et al., 2005; Weis et al., 2004), ICAM1 ligationdid not affect the levels of VEC-associated �-, �- or �- catenin(Fig. 7C,D), suggesting that the molecular mechanism may bedifferent. Intuitively, ICAM1-mediated VEC phosphorylationmight lead to the disengagement of AJs and thus facilitate the path

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Fig. 6. ICAM1 induced VEC phosphorylation in wt and mutant VEC. (A) CHO-ICAM1 cells were transfected with wt pEGFP-N�-VEC or not, grown toconfluence and then starved. Cells were then subjected to ICAM1 crosslinking and VEC-GFP immunoprecipitated and analyzed by immunoblotting forphosphorylated tyrosine and VEC. C, untransfected controls; PV, sample from cells pretreated with pervandate (100 �M). (B) As described for A, except that theCHO-ICAM1 cells were transfected with wt or Y to F mutants of VEC as indicated. ICAM1 crosslinking was 10 minutes. (C) The sequence of the cytoplasmicdomain of mouse VEC (as shown in Fig. 3A) has been used to predict tryptic peptides. Amino acids in small letters in peptide 11 are from the linker sequence toEGFP (which is not shown). Five out of the eleven peptides (bold) contain many phosphorylatable serine and tyrosine residues in line with our observation thatVEC is strongly phosphorylated on serine and less so on tyrosine (data not shown). Note, in contrast to the report by Wallez et al. (Wallez et al., 2006) we haveassumed that trypsin digestion does not occur when a proline is found at the carboxylic side of lysine or arginine. (D) CHO-ICAM1 cells were transfected withpEGFP-N�-VEC as described above. Cells were labeled with 32P and then subjected to ICAM1 crosslinking or not. VEC-GFP was immunoprecipitated andprocessed for tryptic peptide mapping. Arrows denote the position of crosslinking-specific phosphopeptides. The three maps displayed in a single row werechromatographed in the same tank and Rf values were directly comparable. Enlarged sections of the phosphopeptide maps showing ICAM1 crosslinking specificphosphopeptides are shown in supplementary material Fig. S2.

35VE-cadherin in ICAM1-mediated T-cell migration

during paracellular migration. In support of such a mechanism wefound that the expression of Y645F-VEC, Y731F-VEC or Y733F-VEC led to a significant reduction of migration in paracellularareas of the EC monolayer (Fig. 7E) and this correlated well withthe general rate of inhibition (see Fig. 5C). Therefore, paracellularbut not transcellular migration might be affected by dysfunctionalVEC. However, more careful analyses using dynamic confocalmicroscopy of stable transfectants are required to draw a definiteconclusion, in particular because transcellular migration also relieson juxta-junctional positioning of the lymphocyte and,subsequently, on a transcytotic pathway (Carman et al., 2007;Millan et al., 2006) – in which VEC may also be involved (Gavardand Gutkind, 2006; Xiao et al., 2005).

Leukocyte adhesion and subsequent ICAM1 engagement on ECinduces an endothelial response and a complex network ofsignaling pathways regulating the inflammatory response of theendothelium (Turowski et al., 2005). From the results presentedhere, tyrosine phosphorylation of VEC meets all the criteria for amolecular endpoint of an endothelial signaling cascade thatregulates lymphocyte migration: it is induced by surfaceengagement of ICAM1, it can be placed downstream of pathwaysthat have previously been identified to affect lymphocyte migrationand it appears to regulate paracellular migration directly. Theimportance of this mechanism and its general validity is

underscored by recent findings by Allingham et al. (Allingham etal., 2007) who reported similar VEC phosphorylation to operatewhen neutrophils migrate across human umbilical vein endothelialcells. Collectively, these results demonstrate that VEC plays a moreactive role during leukocyte migration than previously anticipated.Single amino acid mutations, namely at Y645, Y731 or Y733,suppressed this function and, surprisingly, did so in a dominantfashion. Since dominant effects are rarely observed with mutationsintroducing non-phosphorylatable amino acids (as opposed tophospho-mimetic substitutions), it will be interesting to elucidatehow these mutations interfere with the function of endogenousVEC during leukocyte migration. Currently, anti-adhesiontherapies are amongst the most specific and advanced strategies totreat inflammation. The identification of a dominant molecularendpoint of adhesion-induced signaling may constitute a validtarget for novel anti-inflammatory therapies.

Materials and MethodsCell culture and treatmentThe rat brain microvascular EC line GPNT (Romero et al., 2003) and mouse brainendothelioma cells bEND5 (Lyck et al., 2003) were grown as previously described.Mouse ECs with a homozygous null mutation of the VEC gene and the cell line re-expressing wild-type VEC derived by retroviral gene transfer have been describedin detail (Lampugnani et al., 2002). Additionally, cell lines that stably expressY658F-VEC, Y685F-VEC, or Y731F-VEC have been generated in the same manner.

Fig. 7. ICAM1-mediated VEC phosphorylation affects paracellular migration and coincides with increased EC permeability. (A) GPNT cells were grown toconfluence, serum starved and then either left untreated (NT) or subjected to ICAM1 crosslinking (XL), 50 ng/ml VEGF, 10 �M lysophosphatidic acid (LPA), 10�M bradykinin (BK), 100 �M histamine (HST) or 1 U/ml thrombin (TBN) for 15 minutes. Subsequently, cells were lysed and VEC immunoprecipitates analyzedby immunoblots using anti-phosphorylated tyrosine or anti-VEC antibodies. (B) The flux of 4 kDa or 140 kDa FITC-dextran across confluent GPNT cellmonolayers was measured when ICAM1 was crosslinked (XL) or not (NT). In each case, the FITC-dextran flux was linear over 120 minutes. The values shown aremean permeability changes that occurred over the initial linear 50-minute period following crosslinking in three independent experiments. (C,D) Confluent GPNTcells were serum starved and ICAM1 crosslinked (XL). At the indicated times cells were lysed and subjected to immunoprecipitation of VEC (C) or �-catenin (D).Immunoprecipitates were then analyzed by immunoblotting using antibodies against phosphorylated tyrosine, �-, �-, �-catenins or VEC. Similar results wereachieved when the order of the proteins for immunoprecipitates and immunoblots was inverted (data not shown). In all blots the position of size markers (in kDa) isindicated on the left. (E) GPNT cells were nucleofected with wt or Y to F mutants of pEGFP-N�-VEC as described in Fig. 3. They were then incubated withantigen-specific T cells, which were allowed to adhere and migrate for 1-4 h. Subsequently time-lapsed microscopy was performed over a period of 5-10 minutes todetermine the fraction of T cells migrating in the paracellular area of the EC. Results are the mean ± s.e.m. of six replicates from at least three independentexperiments. Significant differences were determined by Student’s t-test (*P<0.05, **P=0.005, ***P<0.0001).

36

Wild-type Chinese hamster ovary (CHO) cells and stable transfectants expressinghuman ICAM1 (CHO-ICAM1; a kind gift from Jeremy Pearson, King’s CollegeLondon, UK) were cultured in DMEM/F12 supplemented with 10% FCS.

Antibody-mediated ICAM1 crosslinking was performed as previously describedin serum-starved cells (Adamson et al., 1999) using monoclonal antibodies againstrat (clone 1A29), mouse (clones YNI-1 and 25ZC7) or human (clone 15.2) ICAM1.

Lymphocyte-EC co-cultures were carried out with peripheral lymph node cells(PLNCs) isolated from Lewis rats (Harlan Olac) as previously described (Adamsonet al., 1999). For biochemical analysis of the ECs, PLNCs were washed and fixedusing 3.7% formaldehyde before being added to the ECs, thus avoiding analysis oflymphocyte proteins after cell lysis. For adhesion assays, PLNCs were fluorescentlylabeled with 1 �M calcein-AM (Molecular Probes) before addition to EC monolayers(approximately ten labeled PLNCs per EC). Co-cultures were left at 37°C for 60minutes and adherent T cells quantified in a fluorescent plate reader. Migration assayswere performed using a myelin basic protein (MBP) Ag-specific T-cell line and time-lapse video microscopy as previously reported (Adamson et al., 1999). Migrationwas considered paracellular when it clearly occurred in defined areas of the peripheryof the EC that appeared bright by phase-contrast microscopy. Only T cells that hadcompleted transmigration were counted as migrated cells.

Permeability assays on GPNT cells were conducted in transwell inserts usingFITC-dextrans (Sigma) of varying molecular weight (1 mg/ml) (Esser et al., 1998).

Cell lysis, immunoprecipitations and western blotsTotal cell extracts were prepared in 50 mM Tris-Cl pH 7.5, 1% SDS, 1 mMorthovanadate. For immunoprecipitations cells were lysed in a buffer containing 10mM HEPES-NaOH pH 7.4, 100 mM NaCl, 50 mM �-glycerophosphate, 2 mMMgCl2, 5 mM EGTA, 5 mM EDTA, 1% Igepal (Sigma), 1 mM orthovanadate, 1mM NaF and protease inhibitors (Roche). Clarified cell extracts were incubatedwith 1 �g of antibodies against VEC (Santa Cruz, 6458), �- or �-catenin (Sigma),�-catenin or p120 catenin (BD Biosciences) or 5 �g of anti-GFP antibody (Abcam290). Antigen-antibody complexes were collected using protein A- or protein G-sepharose beads (GE Healthcare). SDS-PAGE and immunoblotting was performedas previously described (Turowski et al., 1999) using antibodies againstphosphorylated tyrosine (4G10, PY20 or RC20H), VEC or catenin. For quantitativeanalyses immunoblots were scanned and quantified using the NIH imaging softwareImageJ. The signal intensity of the phosphorylated tyrosine residues was normalizedto the corresponding amount of VEC, and expressed as fold-increase of untreatedcontrols.

ImmunofluorescenceGPNT cells were fixed in 3.7% formaldehyde and extracted with acetone (–20°C).For the detection of phosphorylated tyrosine the monoclonal antibody 4G10(Upstate) was biotinylated and used at 2 �g/ml. Cells were then stained usingstreptavidin-coupled Texas Red (GE Healthcare) and phalloidin conjugated toOregon Green (Molecular Probes; 1:50). VEC was revealed using a polyclonal goatantibody (sc-6458, Santa Cruz). Cells were mounted using Moviol 4-88 and analyzedby standard epifluorescent or confocal microscopy (Turowski et al., 1999).

Plasmids, mutagenesis and transfectionsMouse VEC was mutagenized using the Quickchange mutagenesis (Stratagene).Subsequently, wt and mutant VEC were cloned into pEGFP-N1 (BD Biosciences)to result in expression of the open reading frame of mouse VEC (aa 1- 784), followedby the linker sequence LVDPPVAT and the entire sequence of EGFP. Resultingplasmids (pEGFP-N�-VEC) were verified by DNA sequencing and purified usingendotoxin-free preparation methods (Qiagen). Subsequently, GPNT cells or mouseVEC-null endothelioma cells were nucleofected using 20 �g plasmid per 2�106 cellsaccording to the manufacturer’s instructions (Amaxa). CHO cells were transfectedusing Lipofectamine 2000 (Invitrogen).

32P labeling and phosphoamino acid and phosphopeptide analysisTransfected CHO-ICAM1 cells were labeled with 32P (PBS 13; GE Healthcare; 20MBq/ml) in phosphate-free DMEM for 3 hours, ICAM1 crosslinked, lysed and VEC-GFP was precipitated using 5 �g of a polyclonal anti-GFP antibody (Abcam 290).Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocelluloseor PVDF. Radiolabelled VEC was excised and subjected to acid hydrolysis forphosphoamino acid analysis or to digestion with trypsin using standard procedures(Boyle et al., 1991). Amino acids or phosphopeptides were subsequently resolvedon cellulose TLC plates by electrophoresis and chromatography and detected byautoradiography.

Computer model of the VEC cytoplasmic domainA homology-based model was obtained using the coordinates of E-cadherin-�-catenin complex (pdb: 1i7x, 1i7w). Cytoplamic domains of VEC from variousspecies and E-cadherin were aligned using ClustalW. The optimal alignment wasused by PyMol to build the VEC model.

We thank David Barford for help with deriving the structure modelof the VEC–�-catenin complex. This work was supported by a

Programme Grant from the Wellcome Trust (062403, awarded to J.G.and P.A.) and in part by the European Community (NoE MAIN502935, to E.D.).

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