The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, beta-catenin, and alpha- catenin with vascular endothelial cadherin (VE-cadherin)

In this paper we report that the assembly of interendothelial junctions containing the cell type-specific vascular endothelial cadherin (VE- cadherin or cadherin-5) is a dynamic process which is affected by the functional state of the cells. Immunofluorescence double labeling of endothelial cells (EC) cultures indicated that VE-cadherin, alpha- catenin, and beta-catenin colocalized in areas of cell to cell contact both in sparse and confluent EC monolayers. In contrast, plakoglobin became associated with cell-cell junctions only in tightly confluent cells concomitantly with an increase in its protein and mRNA levels. Furthermore, the amount of plakoglobin coimmunoprecipitated with VE- cadherin, increased in closely packed monolayers. Artificial wounding of confluent EC monolayers resulted in a major reorganization of VE- cadherin, alpha-catenin, beta-catenin, and plakoglobin. All these proteins decreased in intensity at the boundaries of EC migrating into the lesion. In contrast, EC located immediately behind the migrating front retained junctional VE-cadherin, alpha-catenin, and beta-catenin while plakoglobin was absent from these sites. In line with this observation, the amount of plakoglobin coimmunoprecipitated with VE- cadherin decreased in migrating EC. These data suggest that VE- cadherin, alpha-catenin, and beta-catenin are already associated with each other at early stages of intercellular adhesion and become readily organized at nascant cell contacts. Plakoglobin, on the other hand, associates with junctions only when cells approach confluence. When cells migrate, this order is reversed, namely, plakoglobin dissociates first and, then, VE-cadherin, alpha-catenin, and beta-catenin disassemble from the junctions. The late association of plakoglobin with junctions suggests that while VE-cadherin/alpha-catenin/beta- catenin complex can function as an early recognition mechanism between EC, the formation of mature, cytoskeleton-bound junctions requires plakoglobin synthesis and organization.     

Monocytes induce reversible focal changes in vascular endothelial cadherin complex during transendothelial migration under flow

The vascular endothelial cell cadherin complex (VE-cadherin, alpha-, beta-, and gamma-catenin, and p120/p100) localizes to adherens junctions surrounding vascular endothelial cells and may play a critical role in the transendothelial migration of circulating blood leukocytes. Previously, we have reported that neutrophil adhesion to human umbilical vein endothelial cell (HUVEC) monolayers, under static conditions, results in a dramatic loss of the VE-cadherin complex. Subsequent studies by us and others (Moll, T., E. Dejana, and D. Vestweber. 1998. J. Cell Biol. 140:403-407) suggested that this phenomenon might reflect degradation by neutrophil proteases released during specimen preparation. We postulated that some form of disruption of the VE-cadherin complex might, nonetheless, be a physiological process during leukocyte transmigration. In the present study, the findings demonstrate a specific, localized effect of migrating leukocytes on the VE-cadherin complex in cytokine-activated HUVEC monolayers. Monocytes and in vitro differentiated U937 cells induce focal loss in the staining of VE-cadherin, alpha-catenin, beta-catenin, and plakoglobin during transendothelial migration under physiological flow conditions. These events are inhibited by antibodies that prevent transendothelial migration and are reversed following transmigration. Together, these data suggest that an endothelial-dependent step of transient and focal disruption of the VE-cadherin complex occurs during leukocyte transmigration.     

Regulation of VE-cadherin linkage to the cytoskeleton in endothelial cells exposed to fluid shear stress

Endothelial cells exposed to shear stress realigned and elongated in the direction of flow through the coordinated remodeling of their adherens junctions and actin cytoskeleton. The elaborate networks of VE-cadherin complexes in static cultures became more uniform and compact in response to shear. In contrast, the cortical actin present in static cultures was reorganized into numerous stress fiber bundles distributed parallel to the direction of flow. Exposure to shear did not significantly alter the expression of the junctional proteins VE-cadherin, beta-catenin, and alpha-catenin, but the composition of the junctional complexes did change. We detected a marked decrease in the alpha-catenin associated with VE-cadherin complexes in endothelial monolayers subjected to shear. This loss of alpha-catenin, the protein that links beta-catenin-bound cadherin to the actin cytoskeleton, was not due to decreased quantities of beta-catenin associated with VE-cadherin. Instead, the loss of alpha-catenin from the junctional complexes coincided with the increased tyrosine phosphorylation of beta-catenin associated with VE-cadherin. The change in beta-catenin phosphorylation closely correlated with the shear-induced loss of the protein tyrosine phosphatase SHP-2 from VE-cadherin complexes. Thus, the functional interaction of alpha-catenin with VE-cadherin-bound beta-catenin is regulated by the extent of tyrosine phosphorylation of beta-catenin. This, concomitantly, is regulated by SHP-2 associated with VE-cadherin complexes.     

ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration

Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.     

Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin-based cell-cell adhesion by regulating beta-catenin tyrosine phosphorylation

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 beta-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/CAKbeta-related non-kinase), an N-terminal deletion mutant that acts in a dominant negative fashion, not only abolishes the increase in beta-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 beta-catenin. This signaling is initiated upon loss of VE-cadherin function and is important for our insight in the modulation of endothelial integrity.     

Dynamics of vascular endothelial-cadherin and beta-catenin localization by vascular endothelial growth factor-induced angiogenesis in human umbilical vein cells

The adherens junctional molecule, vascular endothelial cadherin (VE-cadherin), functions to maintain adherens junction stability and to suppress apoptosis of endothelial cells by forming a complex with vascular endothelial growth factor (VEGF) receptor 2 and members of the armadillo family of cytoplasmic proteins. In order to investigate the dynamics of the association of VE-cadherin with adherens junctions during the initial stages of angiogenesis, human umbilical cord endothelial cells (HUVECs) were stimulated with VEGF to undergo angiogenesis in type-I collagen three-dimensional culture. In confluent monolayers of HUVECs, VE-cadherin and its signaling partner, beta-catenin, as well as the paracellular transmembrane adhesion molecule platelet-endothelial cell adhesion molecule (PECAM-1), were all present in regions of cell-cell contact. Within 3 h of stimulation of angiogenesis, VE-cadherin and beta-catenin were lost from these regions. In contrast, the distribution pattern of PECAM-1 did not alter. After 6 h the majority of endothelial cells had migrated to form a network of capillary cords with cell-cell contacts that contained all three molecules. By metabolic labeling of HUVECs it was found that de novo synthesis of VE-cadherin was not essential for the formation of new adherens junctions. Coimmunoprecipitation and immunoblotting experiments showed that the VE-cadherin and beta-catenin remained associated after they were lost from adherens junctions. Detergent extraction of cells with Triton X-100 indicted that the majority of VE-cadherin and beta-catenin was Triton soluble, indicating that they are only weakly associated with the actin-based cytoskeleton.     

Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions

Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE- cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE- cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen- reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.     

Structure of the dimerization and beta-catenin-binding region of alpha-catenin

In adherens junctions, alpha-catenin links the cadherin-beta-catenin complex to the actin-based cytoskeleton. alpha-catenin is a homodimer in solution, but forms a 1:1 heterodimer with beta-catenin. The crystal structure of the alpha-catenin dimerization domain, residues 82-279, shows that alpha-catenin dimerizes through formation of a four-helix bundle in which two antiparallel helices are contributed by each protomer. A slightly larger fragment, comprising residues 57-264, binds to beta-catenin. A chimera consisting of the alpha-catenin-binding region of beta-catenin linked to the amino terminus of alpha-catenin 57-264 behaves as a monomer in solution, as expected, since beta-catenin binding disrupts the alpha-catenin dimer. The crystal structure of this chimera reveals the interaction between alpha- and beta-catenin, and provides a basis for understanding adherens junction assembly.     

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.     

Histamine stimulates phosphorylation of adherens junction proteins and alters their link to vimentin

Histamine increases microvascular permeability by creating small transitory (100-400 nm) gaps between adjacent endothelial cells at sites of vascular endothelial (VE)-cadherin-based adhesion. We examined the effects of histamine on the proteins within the VE-cadherin-based adherens junction in primary human umbilical vein endothelial cells. VE-cadherin is linked not only by beta- and alpha-catenin to cortical actin but also by gamma-catenin to the intermediate filament vimentin. In mature human umbilical vein cultures, the VE-cadherin immunoprecipitate contained equivalent amounts of alpha- and beta-catenin, 130% as much beta- as gamma-catenin, and 50% as much actin as vimentin. Within 60 s, histamine decreased the fraction of VE-cadherin in the insoluble portion of the cell lysate by 35 +/- 1.5%. At the same time, histamine decreased the amount of vimentin that immunoprecipitated with VE-cadherin by 50 +/- 6%. Histamine did not affect the amount of actin or the amount of alpha-, beta-, or gamma-catenin that immunoprecipitated with VE-cadherin. Within 60 s, histamine simulated a doubling in the phosphorylation of VE-cadherin and beta- and gamma-catenin. The VE-cadherin immunoprecipitate contained kinase activity that phosphorylated VE-cadherin and gamma-catenin in vitro.     

Formation of E-cadherin/beta-catenin-based adherens junctions in hepatocytes requires serine-10 in p27(Kip1)

The adhesion between epithelial cells at adherens junctions is regulated by signaling pathways that mediate the intracellular trafficking and assembly of its core components. Insight into the molecular mechanisms of this is necessary to understand how adherens junctions contribute to the functional organization of epithelial tissues. Here, we demonstrate that in human hepatic HepG2 cells, oncostatin M-p42/44 mitogen-activated protein kinase signaling stimulates the phosphorylation of p27(Kip1) on Ser-10 and promotes cell-cell adhesion. The overexpression of wild-type p27 or a phospho-mimetic p27S10D mutant in HepG2 cells induces a hyper-adhesive phenotype. In contrast, the overexpression of a nonphosphorylatable p27S10A mutant prevents the mobilization of E-cadherin and beta-catenin at the cell surface, reduces basal cell-cell adhesion strength, and prevents the stimulatory effect of oncostatin M on cell-cell adhesion. As part of the underlying molecular mechanism, it is shown that in p27S10A-expressing cells beta-catenin interacts with p27 and is prevented from interacting with E-cadherin. The intracellular retention of E-cadherin and beta-catenin is also observed in hepatocytes from p27S10A knockin mice that express the p27S10A mutant instead of wild-type p27. Together, these data suggest that the formation of adherens junctions in hepatocytes requires Ser-10 in p27.     

Activated neutrophils induce hyperpermeability and phosphorylation of adherens junction proteins in coronary venular endothelial cells.

The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which beta-catenin is known to connect the junctional protein vascular endothelial (VE)-cadherin to the cytoskeleton and to play a signaling role in the regulation of junction-cytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on beta-catenin and VE-cadherin modification. Treatment of cultured bovine coronary endothelial monolayers with C5a-activated neutrophils resulted in an increase in permeability as measured by albumin clearance across the monolayer. Furthermore, large scale intercellular gap formation was observed in coincidence with the hyperpermeability response. Immunofluorescence analysis showed that beta-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of control cells to a diffuse pattern for both proteins and finger-like projections for beta-catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, beta-catenin and VE-cadherin from neutrophil-treated endothelial cells showed a significant increase in tyrosine phosphorylation. Our results are the first to link neutrophil-mediated changes in adherens junctions with intercellular gap formation and hyperpermeability in microvascular endothelial cells. These data suggest that neutrophils may regulate endothelial barrier function through a process conferring conformational changes to beta-catenin and VE-cadherin.     

Opposing effect of angiopoietin-1 on VEGF-mediated disruption of endothelial cell-cell interactions requires activation of PKC beta

Angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF) cooperate in migration and survival of endothelial cells by activation of phosphatidylinositol-3 (PI-3) kinase and mitogen activating protein (MAP) kinase pathways. However, Ang1 opposes the effect of VEGF on vascular permeability. We found that Ang1 also blocks VEGF-mediated diffusion of fluoresin isothiocyanate (FITC)-labeled albumin across an endothelial cell monolayer. VEGF-mediated vascular permeability has been attributed, in part, to activation of phospholipase A(2) and subsequent formation of platelet activating factor. However, Ang1 had no effect on VEGF-induced activation of phospholipase A(2) or the release of arachidonic acid. VEGF-mediated permeability was associated with disruption of endothelial cell junctional complexes, dissociation of beta-catenin from VE-cadherin, and accumulation of beta-catenin in the cytosol. In contrast, Ang1 enhanced the interaction of beta-catenin with VE-cadherin and impaired VEGF-mediated dissociation of this complex. Ang1 also blocked VEGF-induced translocation of protein kinase C (PKC) and beta2 to the membrane, but had no effect on activation of PKC alpha. In addition, staurosporine and a PKC beta inhibitor, LY379196, blocked VEGF-mediated dissociation of beta-catenin from VE-cadherin, diffusion of albumin across the endothelial cell monolayer, and translocation of PKC beta isoforms. These data indicate that VEGF-mediated disruption of endothelial cell-cell interactions requires activation of PKC beta isoforms and that this pathway is blocked by Ang1.     

Alpha-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly

Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.     

Activation of MMP-2, cleavage of matrix proteins,and adherens junctions during a snake venom metalloproteinase-induced endothelial cell apoptosis

Snake venom metalloproteinases (SVMPs) are structurally and functionally similar to matrix metalloproteinases (MMPs). We have previously demonstrated that a SVMP, named gaminelysin, can induce endothelial cell apoptosis [Biochem J. 357 (2001) 719]. In this study, the action mechanism of graminelysin in causing endothelial cell apoptosis was further investigated. We showed that the apoptosis was initiated with cell shape change and extracellular matrix degradation and occurred before cell detachment. Cleaved forms of MMP-2 might act in concert with graminelysin to cause apoptosis. During apoptosis, adherens junctions, including VE-cadherin and beta- and gamma-catenin were cleaved and alpha-catenin was decreased. VE-cadherin and beta-catenin at cell periphery were decreased and the discontinuity in alignment was found as observed with immunofluorescence microscopy. This was accompanied with a diffuse beta-catenin staining in the cytoplasm and a decreased F-actin stress fibers in some rounded cells. The decrease of VE-cadherin and beta-catenin in Triton-insoluble fractions confirmed that the association of adherens junctions with actin cytoskeleton was altered during apoptosis. Graminelysin-induced cleavage in adherens junctions was paralleled with the changes in paracellular permeability. We also detected the activation of caspase-3 and the decrease of Bcl-2/Bax ratio during apoptosis. However, caspase inhibitors showed differential effects in blocking the cleavage of PARP, adherens junctions, and DNA fragmentation. Taken together, the data presented suggest that metalloproteinase can control cell fates via the degradation of matrix proteins, the change of cell shape, and the cleavage of adherens junctions.     

A distinct PAR complex associates physically with VE-cadherin in vertebrate endothelial cells

A cell polarity complex consisting of partitioning defective 3 (PAR-3), atypical protein kinase C (aPKC) and PAR-6 has a central role in the development of cell polarity in epithelial cells. In vertebrate epithelial cells, this complex localizes to tight junctions. Here, we provide evidence for the existence of a distinct PAR protein complex in endothelial cells. Both PAR-3 and PAR-6 associate directly with the adherens junction protein vascular endothelial cadherin (VE-cadherin). This association is direct and mediated through non-overlapping domains in VE-cadherin. PAR-3 and PAR-6 are recruited independently to cell-cell contacts. Surprisingly, the VE-cadherin-associated PAR protein complex lacks aPKC. Ectopic expression of VE-cadherin in epithelial cells affects tight junction formation. Our findings suggest that in endothelial cells, another PAR protein complex exists that localizes to adherens junctions and does not promote cellular polarization through aPKC activity. They also point to a direct role of a cadherin in the regulation of cell polarity in vertebrates.     

VE-cadherin and beta-catenin binding dynamics during histamine-induced endothelial hyperpermeability

Beta-catenin plays an important role in the regulation of vascular endothelial cell-cell adhesions and barrier function by linking the VE-cadherin junction complex to the cytoskeleton. The purpose of this study was to evaluate the effect of beta-catenin and VE-cadherin interactions on endothelial permeability during inflammatory stimulation by histamine. We first assessed the ability of a beta-catenin binding polypeptide known as inhibitor of beta-catenin and T cell factor (ICAT) to compete beta-catenin binding to VE-cadherin in vitro. We then overexpressed recombinant FLAG-ICAT in human umbilical vein endothelial cells (HUVECs) to study its impact on endothelial barrier function controlled by cell-cell adhesions. The binding of beta-catenin to VE-cadherin was quantified before and after stimulation with histamine along with measurements of transendothelial electrical resistance (TER) and apparent permeability to albumin (P(a)) under the same conditions. The results showed that ICAT bound to beta-catenin and competitively inhibited binding of the VE-cadherin cytoplasmic domain to beta-catenin in a concentration-dependent manner. Overexpression of FLAG-ICAT in endothelial cell monolayers did not affect their basal permeability properties, as indicated by unaltered TER and P(a); however, the magnitude and duration of histamine-induced decreases in TER were significantly augmented. Likewise, the increase in P(a) in the presence of histamine was exacerbated. Overexpression of FLAG-ICAT also significantly decreased the level of beta-catenin-associated VE-cadherin following histamine stimulation. Taken together, these data suggest that inflammatory agents like histamine cause a transient and reversible disruption of binding between beta-catenin and VE-cadherin, during which endothelial permeability is elevated.