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* INSERM EMI 02-19, Laboratoire de Développement et Vieillissement de L’Endothélium, Département Réponse Dynamique Cellulaire, Commissariat à l’Energie Atomique, Grenoble Cedex 9, France; and
INSERM U525-IFR14 C
ur Muscle et Vaisseaux, Université P.M. Curie, Faculté de Médecine Pitié-Salpêtrière, Paris, Cedex 13, France
1Correspondence: INSERM EMI 0219, Département Réponse et Dynamique Cellulaire, Laboratoire de Développement et Vieillissement de l’Endothélium, CEA Grenoble, 17, rue des Martyrs, 38054 Grenoble Cedex 9, France. E-mail: ivilgrain{at}cea.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adherens junction • VE cadherin • Akt • angiogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. These cells are all targets of PAF bioactions as they bear PAF receptors (PAF-R) localized on their cell surface (3)
. The gene encoding human PAF-R possesses two 5' noncoding exons terminated by a tissue specific promoter; each exon is spliced to a common acceptor site on a third exon, which encodes a unique functional PAF-R protein of 39 kDa (4)
. Upon binding to its receptor, PAF stimulates signal transduction pathways, involving phospholipids turnover, through activation of phospholipase C (PLC) in platelets, macrophages, B cell lines, endothelial cells, and Kupffer cells (5
, 6)
. PAF equally activates PLA2, phospholipase D, and phosphatidylinositol 3-kinase (PtdIns3'-kinase) in various cells and tissues (6)
; it is involved in activation of mitogen-activated protein kinase (MAPK) (7
, 8)
and induces an early tyrosine phosphorylation of numerous signaling proteins such as focal adhesion kinase (p125FAK) in human endothelial cells (9)
, pp60c-src (10)
in platelets, and PLC, Fyn, Syk, Lyn, and p85 regulatory subunit of PtdIns3'-kinase in human B cell lines (11)
. It was recently shown that PAF enhances the angiogenic activity of certain polypeptide mediators such as tumor necrosis factor and hepatocyte growth factor by promoting endothelial cell motility, suggesting a role for PAF in angiogenesis (12)
.
Endothelial adherens junctions regulate the transendothelial flux of liquid and plasma proteins (13)
. The endothelial cell-specific VE-cadherin is a component of endothelial adherens junctions involved in mediating cell-cell interactions (14)
. Endothelial cell adherens junctions disassemble in response to proinflammatory mediators such as thrombin (15)
and histamine (16)
, resulting in increased transendothelial permeability. The endothelial junctional barrier is disrupted within 5 to 10 min, and VE-cadherin complex is redistributed to the membrane in association with increased endothelial permeability. Endothelial adherens junctions disappear, then reform within 2 h to restore endothelial junctional integrity and normal vasopermeability (15)
. Tyrosine and serine/threonine kinases and phosphatases acting on catenins, the proteins linking VE-cadherin to the actin cytoskeleton, seem to play an important role in the disassembly of endothelial adherens junctions (17)
.
The cytoplasmic tail of the classical cadherins, including VE-cadherin, comprises two well-characterized domains. The juxtamembrane domain binds to the catenin p120, an armadillo family protein thought to regulate cadherin adhesive interactions by modulating the activity of Rho family GTPases (18)
. At the carboxyl-terminal region of the cadherin cytoplasmic tail, a domain termed the catenin binding domain interacts with ß-catenin or plakoglobin (19)
. Accordingly, VE-cadherin cytoplasmic domain was shown to regulate endothelial protrusive activity in vitro, suggesting that VE-cadherin may be essential for the invasive process (20)
. In addition, gene ablation experiments strongly suggested that VE-cadherin might be involved in a VEGF-induced survival pathway (21)
.
The present study focused on the signaling triggered by PAF through PAF-R, leading to activation of tyrosine kinase phosphorylation pathways, in endothelial cell adherens junctions. Our data demonstrate that PAF induces activation of MAPK p44/42 and PtdIns3'-kinase signaling pathways, and finally triggers VE-cadherin tyrosine phosphorylation and the dissociation of adherens junction. We show for the first time a link between PAF-R signaling, tyrosine kinase phosphorylations, and adherens junctions in the regulation of endothelial cell barrier integrity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
PAF, phosphatidylinositol, phosphatidylinositol 3-kinase (PtdIns3'-kinase) inhibitor (wortmanin), tyrosine protein kinase inhibitor (herbimycin, bis-tyrphostin), benzamidine, leupeptin, pepstatin A, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). [
32P]-ATP (3000 Ci/mmol) and the enhanced chemiluminescence detection reagents were purchased from Perkin-Elmer (Lifesciences, Belgium). Nitrocellulose was obtained from Schleicher and Schuell (Ecquevilly, France). The micro-bicinchoninic acid protein assay reagent kit was from Pierce (Oud Beijerland, The Netherlands). Protein A-Sepharose was from Pharmacia (Netherlands). Thin-layer chromatography plates were from Merck (Rahway, NJ, USA).
Buffers
Buffer B was 10 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, and 0.5% (v/v) Nonidet P-40.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total endothelial cell RNA was isolated using the RNAgents Total RNA Isolation System (22)
. Amplification parameters were as follows: 40 cycles (94°C for 1 min, 55°C for 1 min, 72°C for 1 min) for PAF-R, for VE-cadheringene 5 min at 94°C, n (n=24, 26, 28, 30) cycles: 94°C for 1 min, 57°C for 1 min, 72°C for 1 min, followed by 10 min at 72°C for final extension using a PCR apparatus (Biometra Trio-Thermoblock). To ensure semiquantitative results, the number of PCR cycles for each set of primers was selected to be in the linear range of amplification. Hybridized filters were visualized and signals quantified using a Fluorimager (Molecular Dynamics, Sunnyvale, CA, USA). Primers and probes used in these studies were for murine PAF receptor, sense: 5' CAG TGT GCC CAT CCT TGT TG, and antisense 5' CCT GAT GGA AGT TGG TCT GG, 1, for murine VE-cadherin gene, sense, 5' ACG GAC AAG ATC AGC TCC TC, antisense 5' TCT CTT CAT CGA TGT GCA TT. RT-PCR products were analyzed by fractionation of 10 µL aliquots on a 2.5% agarose/TAE gel. Control samples analyzed in the absence of reverse transcriptase were free of genomic DNA.
Cell culture, extraction, and immunoprecipitation
Mouse embryonic heart endothelial cells (H5V) were obtained from C. Garlanda (Marie Negri Institute, Milan, Italy) and cultured in Dulbecco’s modified Eagle’s medium (DMEM), 4500 mg/L glucose, sodium pyruvate (Invitrogen corporation, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 1% penicillin, 1% streptomycin, and 200 mM L-glutamine (all from Life Technologies, Inc.).
Before PAF stimulation, endothelial cells, were pretreated for 10 min at 37°C with 5 µL in 5 mL of a mix solution of Na3VO4, 250 mM, hydrogen peroxide (H2O2) 5M, H2O 4:4:92). PAF stimulation was performed at 37°C for the indicated concentrations and time. Reactions were stopped by addition of 200 µL of ice-cold lysis buffer (buffer A).
Lysing cells in Triton lysis buffer
To avoid potential dephosphorylation of tyrosine residues, cells were rinsed twice with cold phosphate-buffered saline (PBS) and immediately lysed in cold buffer A: 20 mM Tris/acetate (pH 7.0), 0.27 M sucrose, 1% (v/v) Triton X-100, 1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM benzamidine, 4 µg/mL leupeptin and 1 µg/mL pepstatin A. Cell lysates were subjected to SDS-PAGE or stored at –20°C.
Lysing cells in SDS lysis buffer
In some experiments cells were rinsed twice with ice-cold PBS and lysed in ice-cold SDS lysis buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) SDS] supplemented with phosphatase inhibitors (1 mM Na3VO4, 1 mM NaF) and protease inhibitors (4 µg/mL aprotinin, 4 µg/mL leupeptin). Cells were harvested and sonicated for 10 s at 4°C and lysates were cleared by centrifugation at 12,000 g for 10 min at 4°C. Protein concentration of each sample was determined using a bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Samples were boiled in a 1:5 ratio (v/v) with 5x Laemmli buffer containing 2.5% (v/v) ß-mercaptoethanol and subjected to SDS-PAGE or stored at –20°C.
Immunoprecipitation
Equal amounts of protein from each cell lysate were incubated with the appropriate antibody and 40 µL of a 50% (v/v) mixture of protein A-Sepharose beads (Amersham Biosciences) for 30 min at 4°C on a rotator. The supernatants were then incubated with the anti-phosphotyrosine antibody or anti-PtdIns3'-kinase antibody (2 µg/mL) overnight at 4°C. Immunoprecipitates were collected by incubation with 20 µL of protein A-Sepharose. Immunoprecipitates were washed four times in ice-cold NP-40 lysis buffer and collected by centrifugation for 10 min at 4°C. Samples were eluted from protein A-Sepharose beads by boiling in a 1:5 ratio (v/v) with 5x Laemmli buffer containing a final concentration of 2.5% (v/v) ß-mercaptoethanol and subjected to SDS-PAGE.
MAP kinase assays
Activation of p42/p44 MAP kinase was determined through immunoblotting of endothelial cell lysates using antibodies that specifically recognize the phosphorylated amino acid residues on activated phospho-MAPK. After treatment, H5V cells were washed twice with ice-cold PBS and lysed with buffer A containing 10 mM sodium orthovanadate for 20 min on ice. The cell lysates were then harvested from the culture dishes and the cellular debris was removed by centrifugation. Of the total cell lysates, 80 µg of proteins was separated on a 10% denaturing polyacrylamide gel; proteins were subsequently transferred to nitrocellulose membranes.
PtdIns3'-kinase activity
PtdIns3'-kinase activity was measured by adding 100 µg of sonicated phosphatidylinositol and 10 µCi of [-32P]ATP, 30 mM MgCl2, and 35 µM ATP in a total volume of 60 µL. Reactions were performed for 20 min at 30°C and stopped by addition of 100 µL of 1 N HCl and 200 µL of chloroform/methanol (1:1 by vol). After centrifugation and removal of the upper layer, 100 µL of methanol/HCl (1:1) was added. After further centrifugation, lipids were separated on thin-layer chromatography (TLC) plates with a solvent system of chloroform/methanol/NH4OH (45:35:10 by vol.). TLC plate-associated radioactivity was determined using PhosphorImager (Bio-Rad) quantification. Immunoprecipitation with mouse monoclonal anti-p85 antibody was used as positive control for PtdIns3'-kinase activity.
Immunofluorescence studies
Endothelial cells (H5V) were plated onto coverslips at a density of 45,000 cells/mL and grown to confluence. After stimulation with PAF for 30 min at 37°C, cells were fixed with 3.5% paraformaldehyde in PBS for 20 min at room temperature and washed three times with PBS containing MgCl2 0.5 mM, CaCl2 1 mM. Permeabilization was performed with Triton X-100 (0.5% in PBS) for 10 min. Cells were washed three times in PBS and nonspecific binding sites were saturated with PBS/BSA (1 mg/mL) for 30 min. Incubation with the primary antibody (phosphotyrosine and VE-cadherin 2 µg/mL) was performed in PBS for 1 h at room temperature. After three washes with PBS, cells were incubated for 1 h with the cyanine-labeled secondary antibody. After three washes in PBS, nuclei were labeled with Hoescht 1 µg/mL for 5 min, then washed three times with PBS. The coverslips were then rinsed, dried in ethanol, and mounted on glass slides with Aquamount.
SDS/PAGE and Western blot
Endothelial cell lysates were analyzed by SDS/PAGE (12% acrylamide, 1% bis-acrylamide). Proteins were transferred from the gel to nitrocellulose for 1 h and residual binding sites were blocked by incubating the filters for 1 h in PBS containing 0.05% (v/v) Tween 20 and 5% (v/v) nonfat milk. Blots were subsequently incubated with the primary antibody for 1 h. After being washed, the blots were incubated for 1 h with HRP-conjugated rabbit anti-mouse IgG diluted in PBS containing 0.05% (v/v) Tween 20. Immunoreactive proteins were visualized by chemiluminescence.
Data analysis
Each data point represents the mean ± SD of the measures of three different wells or dishes in the same experiment. Each experiment was reproduced at least three times in identical or similar configuration with similar results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, amplification of VE-cadherin transcripts by RT-PCR produced a single product (154 bp) that corresponded in size to the mRNA expected for the murine VE-cadherin.
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PAF receptor mRNA levels were determined by RT-PCR, using the sets of primers corresponding to the coding region of the mouse PAF-R sequence (268 bp). Figure 1B
shows a single band corresponding in size to the RT-PCR product expected for mouse PAF-R mRNA. Analysis of mRNA from rat brain displayed an identical amplification product and served as a positive control for PAF-R expression.
Functionality of PAF-R in H5V cells
To determine whether the PAF-R was functional in H5V cells, we measured activation of MAPK, tyrosine kinases, PtdIns3'-kinase, and Akt, which play a crucial role in angiogenesis and are known to be activated by PAF in other cell types (22)
. The dose-dependent phosphorylation of MAPK p44/p42 was used as a sensitive readout of PAF activity. The phospho-p44/42 MAPK antibody detects p42 and p44 MAPK only when catalytically activated by phosphorylation at Thr-202/Tyr-204 (23)
. p44/42 MAPK phosphorylation, induced by PAF (10 nM), was observed as early as 2 min after its addition to the cells, then reached maximum at 5 min and remained elevated up to 20 min (Fig. 2
A). Addition of PAF (1-100 nM) increased p44/42 MAPK phosphorylation in a dose-dependent manner (Fig. 2B
).
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Tyrosine phosphorylation of membrane-associated proteins was determined by stimulation of serum-starved H5V cells and Western blot of cell lysates with an antiphosphotyrosine antibody. In PAF-treated cells, a dramatic time-dependent increase in the phosphotyrosine signal was observed especially for bands of high molecular mass, as proteins of 75 and 50 kDa remained unchanged (Fig. 2C
).
To evaluate PtdIns3'-kinase activity, we measured phosphorylation of the phosphatidylinositol. Total cellular extracts from control cells or cells treated with 10 nM PAF were incubated in the presence of phosphatidylinositol (PI) and 32P-ATP, as described in Materials and Methods. After extraction of phosphorylated lipids, they were further separated by TLC. As shown in Fig. 2D
, addition of PAF (10 nM) induced a strong increase in cellular of PtdIns3'-kinase activity. Pretreatment of H5V cells with a specific inhibitor of PtdIns3'-kinase, wortmannin (30 nM) for 20 min abolished the PAF-induced PtdIns3'-kinase activity (Fig. 2D, E
). Figure 2E
illustrates quantification of the radioactive spots using PhosphorImager.
To evaluate Akt activity, we measured the phosphorylation of Akt (Ser 473). Cells were plated in the presence or absence of PAF and immunoblotted with an anti-phospho-Akt antibody. As shown in Fig. 2F
, PAF (10 nM) increased Akt phosphorylation as early as 2 min after addition and produced a maximal effect at 10 min. Collectively, these results indicated to us that the PAF signaling pathways were functional in mouse endothelial cells.
Characterization of tyrosine phosphorylation of VE-cadherin in mouse endothelial cells
In an attempt to identify the proteins phosphorylated after PAF treatment of the cells, we examined the phosphorylation of VE-cadherin, as it has been proposed to be a point of convergence of signaling by endothelial specific growth factors, including VEGF (21)
. Endothelial cells were exposed to 10 nM PAF for 20 min. Cell lysates were immunoprecipitated with the anti-phosphotyrosine antibody; immunoprecipitates were analyzed by SDS-PAGE and Western blotted with the anti-VE-cadherin antibody. PAF treatment of endothelial cells resulted in a time-dependent phosphorylation of VE-cadherin protein. As shown in Fig. 3
A, B, the phosphotyrosine content of the 125 kDa protein increased rapidly within 5 min and was sustained for up to 20 min of PAF treatment. The effect of PAF on VE-cadherin tyrosine phosphorylation was dose dependent, already detectable at 0.1 nM (Fig. 3C, D
). Upon PAF challenge, the amount of VE-cadherin was not significantly modified in control cells at any time of stimulation (data not shown). These data demonstrate that VE-cadherin is regulated by tyrosine phosphorylation in endothelial cells after PAF stimulation.
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Effect of tyrosine kinase inhibitors
To elucidate the role of protein tyrosine kinases (PTK) in PAF-induced activation of VE-cadherin tyrosine phosphorylation, we used herbimycin and bis-tyrphostin AG126 as potent inhibitors of PTK. As shown on Fig. 4
A, when cells were pretreated with PTK inhibitors, before the addition of PAF, the pattern of P-tyrosine was reduced to control levels by both inhibitors. Treatment of cells with wortmanin, the PtdIns3'-kinase inhibitor, before PAF treatment yielded a pattern of tyrosine phosphorylation similar to PAF alone. In these conditions, analysis of VE-cadherin tyrosine phosphorylation (Fig. 4B, C
) by immunoprecipitation showed that tyrosine phosphorylation of VE-cadherin was strongly inhibited by herbimycin and tyrphostin but not by wortmanin. Thus, it can be suggested that PAF stimulated tyrosine phosphorylation of VE-cadherin independent of the PtdIns3'-kinase pathway.
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PAF stimulates complex formation between PtdIns3'-kinase and VE-Cadherin in endothelial cells
As the cytoplasmic tail of VE-cadherin contains several highly conserved tyrosine residues, we hypothesized that some are phosphorylated and could serve as recognition sites for SH2 domain proteins involved in intracellular signal transduction. As the prototypic p85 regulatory subunit of the PtdIns3'-kinase has two SH2 domains (24)
and because PAF activated PtdIns3'-kinase (Fig. 1D
) in endothelial cells, we examined whether an association between p85 subunit of PtdIns3'-kinase and VE-cadherin was detectable. Serum-starved cells were incubated with or without PAF (10 nM) and cell lysates were immunoprecipitated with the antibody against VE-cadherin. Immune complexes were separated on SDS/PAGE, transferred to nitrocellulose membrane, and immunoblotted with the antibody against the p85 subunit of PtdIns3'-kinase. As shown in Fig. 5
A, the level of PtdIns3'-kinase associated with VE-cadherin was barely detectable in untreated cells, whereas stimulation with PAF strongly induced the association of PtdIns3'-kinase with VE-cadherin (Fig. 5A
). Immunoprecipitation with the anti-PtdIns3'-kinase antibody and immunoblotting with the anti-VE-cadherin led to a similar conclusion (data not shown).
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To confirm the association of PtdIns3'-kinase with VE-cadherin, we performed the PtdIns3'-kinase assay using VE-cadherin immunoprecipitates. As shown in Fig. 5B, C
, PtdIns3'-kinase activity was barely detectable in VE-cadherin immunoprecipitates from untreated cells. In contrast, in PAF-treated cells the PtdIns3'-kinase activity associated with VE-cadherin increased by 4-fold. When cells were treated with specific tyrosine kinase inhibitors, the PtdIns3'-kinase activity associated with VE-cadherin remained similar to control level. The levels of VE-cadherin as determined by Western blot were not affected under these conditions (Fig. 5D
). These data strongly suggest that the PtdIns3'-kinase was associated with VE-cadherin in PAF-treated cells and that this association was dependent on VE-cadherin tyrosine phosphorylation, as it was inhibited by the tyrosine kinase inhibitor.
PAF induced endothelial cell morphological changes and dissociates adherens junctions
The phase-contrast micrographs of intact endothelial cells grown for 4 days revealed their cobblestone aspect (Fig. 6
, left upper panel). After exposure to 10 nM PAF for 10 min, pronounced morphological changes were observed (Fig. 6
, right upper panel). To determine the effects of PAF exposure on endothelial adherens junctions, we performed immunofluorescent staining with the anti-VE-cadherin antibody. The immunostaining of VE-cadherin in control cells was typical for adherens junctions in confluent cells, distributed mainly at the periphery of the cells (Fig. 6
, middle panel). When cells were stimulated with 10 nM PAF, the majority of VE-cadherin staining was redistributed throughout the cytoplasm and only a few adherens junctions were detected. In a similar way, tyrosine-phosphorylated proteins were revealed with the anti-phosphotyrosine antibody in PAF-treated cells and showed a pattern similar to that of the VE-cadherin staining. Control, untreated cells exhibited similar staining with anti-VE-cadherin and anti-phoshotyrosine antibodies (Fig. 6
, lower panel).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We show here for the first time that upon activation of PAF-R by PAF in mouse endothelial cells, several signaling events lead to tyrosine phosphorylation of the junctional protein VE-cadherin. Only a few in situ studies have been reported on PAF-R expression in blood vessels; they were related mainly to PAF-induced increases in vascular permeability, showing a widespread localization of PAF-R in microvascular beds and especially its ubiquitous presence on endothelia and in pericytes, fibroblasts, and macrophages associated with microvessels (26)
. In the present study, we demonstrated that PAF-R is expressed and functionally active in mouse endothelial cells as demonstrated by the rapid activation of a set of protein kinases and where the dose-dependent phosphorylation of MAPK p44/p42 was used as a sensitive readout of PAF-R activity. Our results agree with previous studies showing that the PAF-R, associated with heterotrimeric guanine nucleotide binding proteins, is responsible for transducing PAF signals into various intracellular cascades (27)
. It is now accepted that in addition to a well-characterized pathway leading to MAPK activation after ligand-induced tyrosine kinase receptor autophosphorylation, many G-protein-coupled receptors (GPCRs), which lack intrinsic kinase activity, are able to effectively activate MAPK (28)
. These include the receptors for such diverse ligands as bombesin, endothelin-1, somatostatin, interleukin-8, oxytocin, and lysophosphatidic acid (29)
.
PAF induces tyrosine phosphorylation of numerous cellular proteins such as p125fak in human endothelial cells and brain (30
, 31)
, p85 regulatory subunit of phosphatidylinositol 3-kinase (32)
, pp60src (10)
, Fyn, Syk, and Lyn in a human B cell line (32
, 33)
. In the present study we provide evidence that PAF induced a detectable tyrosine phosphorylation in H5V cell membranes; however, the precise identity of tyrosine kinases activated in this model remains unclear. The Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway are a major mechanism by which cytokine receptors transduce intracellular signals. Four mammalian Jaks have been identified (Jak1, Jak2, Jak3, and Tyk2) and characterized (34)
. Recently, the Tyk2 kinase was shown to be activated in response to PAF in myeloid cells and to associate with PAF-R independent of agonist binding (35)
. Thus, it remains to be determined whether Tyk2 could be a potential mediator of PAF signaling in endothelial cells since it was shown to be present and activated by another GPCR agonist, bradykinin, in endothelial cells (36)
.
Using a traditional lipid kinase assay to measure PtdIns3'-kinase activity and a PtdIns3'-kinase inhibitor, we found that PAF was able to stimulate PtdIns3'-kinase activity and promote PKB/Akt phosphorylation in mouse endothelial cells. PtdIns3'-kinase activation by PAF may have several functional implications in endothelial cells: it 1) promotes proliferation, 2) induces angiogenesis, and 3) regulates endothelial nitric oxide synthase activity and ultimately increases Akt activity (see ref 2
for a review). PKB/Akt activity protects against apoptosis through its phosphorylation and inhibition of proapoptotic mediators (37)
; PKB/Akt-mediated control of cell cycle progression is well established (38)
. Taken together, these data suggest that PAF may be a key player in endothelial cell survival and/or cell proliferation.
Tyrosine phosphorylation of VE-cadherin has been described in cells stimulated by VEGF, thrombin, or histamine and has been correlated with a rapid dissociation of adherens junctions, a critical step in angiogenesis and inflammatory processes (13)
. In the present study, we demonstrated for the first time that VE-cadherin is tyrosine phosphorylated in a time- and a dose-dependent manner in PAF-treated cells. Further studies are needed to identify the specific tyrosine kinase activated through the PAF-R. Covalent modification of the specific tyrosyl residues in growth factor receptors and signaling molecules is involved in cellular communication (39)
. A conserved domain of 
100 amino acids, the SH2 domain, specifically recognizes and binds to phosphotyrosine, thereby promoting interactions of activated receptors and signaling molecules together (39)
. Our data demonstrated that the p85 subunit of PtdIns3'-kinase may bind to the phosphorylated form of VE-cadherin in PAF-treated endothelial cells, as this binding was impaired by specific tyrosine kinase inhibitors. It is possible that both direct and indirect interactions exist between PtdIns3'-kinase and VE-cadherin. For example, mutational studies in the cytoplasmic domain of the type 2 VEGF receptor have suggested that the p85 subunit of PtdIns3'-kinase may bind directly to Tyr-799 and Tyr-1173 (40)
. Phosphorylation sites of the VE-cadherin in vivo need to be determined to support this hypothesis.
It has been shown that loss or truncation of VE-cadherin did not impair endothelial proliferation or differentiation, but increases endothelial apoptosis due to an inability of VE-cadherin deficient cells to respond to the survival activity of VEGF-A. The latter is known to mediate endothelial survival via binding to VEGFR-2, thereby activating the PtdIns3'-kinase and Akt, and also via increasing levels of the antiapoptotic protein Bcl2 (21)
. Thus, VE-cadherin appears to be required for these VEGF-A-dependent survival signals through formation of a VE-cadherin/ß-catenin/PtdIns3'-kinase/VEGFR-2 complex. Further studies will be required to identify the role of PtdIns3'-kinase/VE-cadherin association in PAF actions on endothelial cells.
It was demonstrated that PAF signaling is involved in angiogenesis (2)
. PAF is present in breast cancer tissues (41)
and correlates with tumor microvessel density (2)
. The adherens junctional molecule VE-cadherin functions to maintain adherens junction stability and to suppress apoptosis of endothelial cells by forming a complex with VEGFR-2 and members of the armadillo family of cytoplasmic proteins. Our results demonstrate that PAF induced changes in cell shape of endothelial monolayers at nanomolar concentrations that correlate with data from in vitro studies in cultured umbilical vein endothelial cells (42
, 43)
. Disturbance of endothelial junctional protein distribution induced by PAF is not regulated at the transcriptional or translational level, as total cellular junctional protein expression and mRNA expression of VE-cadherin were not affected by PAF (44)
. Thus, the PAF-induced tyrosine phosphorylation of VE-cadherin might be a likely mechanism involved in PAF-induced endothelial cell shape changes, since it has been suggested that PAF rapidly activates a cascade of phosphorylation steps that mediate cytoskeletal rearrangement (45)
.
In conclusion, we have demonstrated that PAF induced tyrosine phosphorylation of VE-cadherin and its association with PtdIns3'-kinase, which led to the dissociation of adherens junctions. Physical association between PtdIns3'-kinase, serving as a docking protein, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of PAF-induced endothelial cells activation.
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ACKNOWLEDGMENTS |
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Received for publication June 1, 2004. Accepted for publication November 8, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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