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1.
Vascular endothelial cadherin (VE-cadherin), which belongs to the classical cadherin family, is localized at adherens junctions exclusively in vascular endothelial cells. Biochemical and biomechanical cues regulate the VE-cadherin adhesive potential by triggering the intracellular signals. VE-cadherin-mediated cell adhesion is required for cell survival and endothelial cell deadhesion is required for vascular development. It is therefore crucial to understand how VE-cadherin-based cell adhesion is controlled. This review summarizes the inter-endothelial cell adhesions and introduces our recent advance in Rap1-regulated VE-cadherin adhesion. A further analysis of the VE-cadherin recycling system will aid the understanding of cell adhesion/deadhesion mechanisms mediated by VE-cadherin in response to extracellular stimuli during development and angiogenesis.  相似文献   

2.
T Ohmori  Y Yatomi  Y Wu  M Osada  K Satoh  Y Ozaki 《Biochemistry》2001,40(43):12992-13001
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130K transmembrane glycoprotein that belongs to the immunoglobulin gene superfamily and is expressed on the surface of hematological or vascular cells, including platelets and endothelial cells. Although the importance of this adhesion molecule in various cell-cell interactions is established, its function in platelets remains ill-defined. In the process of clarifying the mechanism by which the lectin wheat germ agglutinin (WGA) activates platelets, we unexpectedly discovered that PECAM-1 is involved in signal transduction pathways elicited by this N-acetyl-D-glucosamine (NAGlu)-reactive lectin. WGA, which is a very potent platelet stimulator, elicited a rapid surge in Syk and phospholipase C (PLC)-gamma 2 tyrosine phosphorylation and the resultant intracellular Ca(2+) mobilization; collagen, as reported, induced these responses, but in a much slower and weaker manner. WGA strongly induced tyrosine phosphorylation of a 130-140K protein, which was confirmed to be PECAM-1 by immunoprecipitation and immunodepletion studies. WGA-induced PECAM-1 tyrosine phosphorylation occurred rapidly, strongly and in a manner independent of platelet aggregation or cell-cell contact; these characteristics of PECAM-1 phosphorylation were not mimicked at all by receptor-mediated platelet agonists. In addition, WGA was found to associate with PECAM-1 itself, and anti-PECAM-1 antibody, as well as NAGlu, specifically inhibited WGA-induced platelet aggregation. In PECAM-1 immunoprecipitates, Src family tyrosine kinases existed, and a kinase activity was detected, which increased upon WGA stimulation. Furthermore, the Src family kinase inhibitor PP2 inhibited WGA-induced platelet aggregation, Ca(2+) mobilization, and PLC-gamma 2 tyrosine phosphorylation. Finally, WGA induced PECAM-1 tyrosine phosphorylation and cytoskeletal reorganization in vascular endothelial cells. Our results suggest that (i) PECAM-1 is involved in WGA-induced platelet activation, (ii) PECAM-1 clustering by WGA activates unique and strong platelet signaling pathways, leading to a rapid PLC activation via Src family kinases, and (iii) WGA is a useful tool for elucidating PECAM-1-mediated signaling with wide implications not confined to platelets.  相似文献   

3.
Rap1 is a small GTPase that regulates adherens junction maturation. It remains elusive how Rap1 is activated upon cell-cell contact. We demonstrate for the first time that Rap1 is activated upon homophilic engagement of vascular endothelial cadherin (VE-cadherin) at the cell-cell contacts in living cells and that MAGI-1 is required for VE-cadherin-dependent Rap1 activation. We found that MAGI-1 localized to cell-cell contacts presumably by associating with beta-catenin and that MAGI-1 bound to a guanine nucleotide exchange factor for Rap1, PDZ-GEF1. Depletion of MAGI-1 suppressed the cell-cell contact-induced Rap1 activation and the VE-cadherin-mediated cell-cell adhesion after Ca2+ switch. In addition, relocation of vinculin from cell-extracellular matrix contacts to cell-cell contacts after the Ca2+ switch was inhibited in MAGI-1-depleted cells. Furthermore, inactivation of Rap1 by overexpression of Rap1GAPII impaired the VE-cadherin-dependent cell adhesion. Collectively, MAGI-1 is important for VE-cadherin-dependent Rap1 activation upon cell-cell contact. In addition, once activated, Rap1 upon cell-cell contacts positively regulate the adherens junction formation by relocating vinculin that supports VE-cadherin-based cell adhesion.  相似文献   

4.
Huang YT  Chen SU  Chou CH  Lee H 《Cellular signalling》2008,20(8):1521-1527
Sphingosine 1-phosphate (S1P) is a multifunctional phospholipid which acts through a specific family of G protein-coupled receptors. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) form trans-homophilic binding at lateral cell border. Upon stimulation, its cytoplasmic tyrosine residues could be phosphorylated and interact with various downstream signaling molecules. In this study, we demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in human umbilical cord vein cells (HUVECs). By pharmacological inhibitors, it was suggested that G(i) and Src family kinases were involved in PECAM-1 phosphorylation. Moreover, cSrc and Fyn siRNA significantly suppressed S1P-induced PECAM-1 phosphorylation. These results suggested that S1P-induced PECAM-1 phosphorylation through G(i) and subsequent cSrc and Fyn. Our findings provide further understanding of S1P and PECAM-1 signaling as well as their functions in endothelial cells.  相似文献   

5.
Homophilic engagement of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) induces 'outside-in' signal transduction that results in phosphorylation events and recruitment and activation of signalling molecules. The formation of signalling scaffolds with PECAM-1 are important signalling events that modulate platelet secretion, aggregation and platelet thrombus formation. In this study, we describe a novel interaction between PECAM-1 and cytosolic calmodulin (CaM) in platelets. Reciprocal co-immunoprecipitation studies revealed that cytosolic CaM is constitutively associated with PECAM-1 in resting, thrombin activated and aggregated human platelets. Our studies demonstrate that CaM directly interacts with a PECAM-1 peptide (594-604) C595A containing the sequences (594)KAFYLRKAKAK(604). This CaM:PECAM-1 interaction has a threefold higher affinity than CaM:GPVI interaction. It is potentiated by the addition of calcium ions, and dissociated by the CaM inhibitor, trifluoperazine. Treatment of platelets with CaM inhibitors triggers cleavage of PECAM-1 in a time- and dose-dependent manner. Furthermore, this membrane proximal portion of PECAM-1 is conserved across mammalian species and the helical representation of basic/hydrophobic residues reveals a charge distribution analogous to other CaM-binding motifs in other proteins. Taken together, these results suggest that this highly charged cluster of amino acids in the PECAM-1 cytoplasmic domain directly interacts with CaM and this novel interaction appears to regulate cleavage of PECAM-1.  相似文献   

6.
Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, or platelets result in increased cell adhesion to immobilized integrin ligands. However, the mechanism by which this occurs is not clear, and it is possible that anti-PECAM-1 mAbs elicit this effect by simply sequestering PECAM-1, via antibody-induced patching and capping, away from stimulatory receptors that it normally regulates. To determine whether dimerization or oligomerization of PECAM-1 directly initiates signal transduction pathways that affect integrin function in an antibody-independent manner, stable human embryonic kidney-293 cell lines were produced that expressed chimeric PECAM-1 cDNAs containing one or two FK506-binding protein (FKBP) domains at their COOH terminus. Controlled dimerization initiated by addition of the bivalent, membrane-permeable FKBP dimerizer, AP1510, nearly doubled homophilic binding capacity, whereas AP1510-induced oligomers favored cis PECAM-1/PECAM-1 associations within the plane of the plasma membrane at the expense of trans homophilic adhesion. Importantly, AP1510-induced oligomerization resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2-transfected cells on immobilized fibronectin, a reaction that was mediated by the integrin alpha(5)beta(1). These data demonstrate that signals required for integrin activation can be elicited by clustering of PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosine-based inhibitory motif family of regulatory receptors.  相似文献   

7.
Reports in the past two years have shown that Cdc42, Rac1, and Rho - belonging to the Rho small GTPase family - participate in the regulation of cadherin-mediated cell-cell adhesion. IQGAP1, an effector of Cdc42 and Rac1, interacts with cadherin and beta-catenin and induces the dissociation of alpha-catenin from the cadherin-catenins complex leading to disruption of cell-cell adhesion: activated Cdc42 and Rac1 counteract the effect of IQGAP1. Thus, Cdc42 and Rac1 appear to regulate cadherin-mediated cell-cell adhesion acting through IQGAP1.  相似文献   

8.
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a newly assigned member of the Ig-immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. In this study, we hypothesized that PECAM-1 plays an essential in vivo role as a counterregulator of immediate hypersensitivity reactions. We found that PECAM-1 was highly expressed on the surface of immature bone marrow mast cells and at a lower density on mature peritoneal mast cells. Examination of skin biopsies from PECAM-1(+/+) and PECAM-1(-/-) mice revealed that absence of PECAM-1 did not affect mast cell development or the capacity of mast cells to populate tissues. To examine whether the absence of PECAM-1 would influence immediate hypersensitivity reactions, PECAM-1(+/+) and PECAM-1(-/-) mice were presensitized with anti-DNP mouse IgE and then challenged 20 h later with DNP-BSA or PBS. PECAM-1(-/-) mice exhibited elevated serum histamine concentrations after Ag stimulation compared with PECAM-1(+/+) mice, indicating an increased severity of systemic IgE-mediated anaphylaxis. PECAM-1(-/-) mice have increased sensitivity to local cutaneous IgE-dependent anaphylaxis compared with PECAM-1(+/+) mice, as assessed by greater tissue swelling of their ears and mast cell degranulation in situ. PECAM-1(-/-) bone marrow mast cells showed enhanced dense granule serotonin release after Fc epsilon RI cross-linking in vitro. These results suggest that PECAM-1 acts as a counterregulator in allergic disease susceptibility and severity and negatively modulates mast cell activation.  相似文献   

9.
10.
Coordinated migration of endothelial cells models the remodeling of existing endothelia as well as angiogenesis and vasculogenesis. Platelet-endothelial cell adhesion molecule-1, PECAM-1, a transmembrane endothelial adhesion protein, binds and activates the tyrosine phosphatase SHP-2 via phosphotyrosines 663 and 686. PECAM-1 phosphorylation and recruitment of SHP-2 are regulated by cell-cell and cell-substrate adhesion. We found that PECAM-1 is dephosphorylated on tyrosine 686 during endothelial migration, resulting in diffuse dispersal of PECAM-1 and SHP-2. Overexpression of native PECAM-1 slowed, and nonphosphorylatable PECAM-1 increased, endothelial migration, implying that the SHP-2-regulatory phosphotyrosines negatively regulate migration. Using differentially phosphorylated recombinant proteins we found that phosphotyrosine 686 preferentially mediates binding and 663 mediates activation of SHP-2 by PECAM-1. In PECAM-1-null endothelial cells, SHP-2 bound and dephosphorylated an alternative set of phosphoproteins and its distribution to the cytoskeletal fraction was significantly decreased. Tyrosine phosphorylation of beta-catenin and focal adhesion kinase was increased in endothelial cells overexpressing nonphosphorylatable PECAM-1. Thus homophilically engaged, tyrosine-phosphorylated PECAM-1 locally activates SHP-2 at cell-cell junctions; with disruption of the endothelial monolayer, selective dephosphorylation of PECAM-1 leads to redistribution of SHP-2 and pro-migratory changes in phosphorylation of cytoskeletal and focal contact components.  相似文献   

11.
Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.  相似文献   

12.
13.
14.
Gräbner R  Till U  Heller R 《Cytometry》2000,40(3):238-244
BACKGROUND: Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). METHODS: TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells. RESULTS: HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules. CONCLUSIONS: The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.  相似文献   

15.
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is widely used as a marker during vasculogenesis and angiogenesis from embryonic stem (ES) cells. However, the expression of PECAM-1 isoforms in ES cells has not been determined. The present study was designed to determine the role of PECAM-1 isoforms during in vitro endothelial differentiation of ES cells. It was found that undifferentiated ES cells expressed high level of PECAM-1, which primarily located at cell-cell junction, but the expression of PECAM-1 was sharply down-regulated during early ES cell differentiation. In addition, undifferentiated ES cells were found the expressed all eight known alternatively spliced PECAM-1 isoforms, among them the expression of PECAM-1 isoforms lacking exon 15 or 14&15 was predominant. Quantitative analysis revealed a significant increase in the expression of PECAM-1 isoform lacking exon 12&14&15 as vascular development of ES cells. These results indicate a constitutive expression of PECAM-1 in undifferentiated murine ES cells and suggest a developmental role of PECAM-1 isoform changes during vasculogenesis and angiogenesis.  相似文献   

16.
Coordinated regulation of endothelial cell migration is an integral process during angiogenesis. However, molecular mechanisms regulating endothelial cell migration remain largely unknown. Increased expression of cell adhesion molecules has been implicated during angiogenesis, yet the precise role of these molecules is unclear. Here, we examined the hypothesis that intercellular adhesion molecule-1 (ICAM-1) is important for endothelial cell migration. Total cell displacement and directional migration were significantly attenuated in ICAM-1-deficient endothelium. Closer examination of ICAM-1-deficient cells revealed decreased Akt Thr(308) and endothelial nitric-oxide synthase Ser(1177) phosphorylation and NO bioavailability, increased actin stress fiber formation, and a lack of distinct cell polarity compared with wild-type endothelium. Supplementation of ICAM-1 mutant cells with the NO donor DETA NONOate (0.1 microM) corrected the migration defect, diminished stress fiber formation, and enhanced pseudopod and uropod formation. These data demonstrate that ICAM-1 facilitates the development of cell polarity and modulates endothelial cell migration through a pathway regulating endothelial nitric-oxide synthase activation and organization of the actin cytoskeleton.  相似文献   

17.
Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.  相似文献   

18.
Folate deficiency increases stroke risk. We examined whether folate deficiency affects platelet endothelial cell adhesion molecule-1 (PECAM-1), which is an immunoglobulin-associated cell adhesion molecule and mediates the final common pathway of neutrophil transendothelial migration, in blood vessels in the gerbil dentate gyrus after transient forebrain ischemia. Gerbils were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to common carotid artery occlusion for 5 min. In the control diet (CD)- and FAD-treated sham-operated groups, weak PECAM-1 immunoreactivity was detected in the blood vessels located in the dentate gyrus. PECAM-1 immunoreactivity in both groups was increased by 4 days after ischemic insult. PECAM-1 immunoreactivity in the FAD-treated group was twice as high that in the CD-treated-sham-operated group 4 days after ischemic insult. Western blot analyses showed that the change patterns in PECAM-1 protein levels in the dentate gyrus in both groups after ischemic insult were similar to changes in PECAM-1 immunohistochemistry in the ischemic dentate gyrus. Our results suggest that folate deficiency enhances PECAM-1 in the dentate gyrus induced by transient ischemia.  相似文献   

19.
Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration.  相似文献   

20.
Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.  相似文献   

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