PI3KC2? inactivation stabilizes VE-cadherin junctions and preserves vascular integrity
Ontology highlight
ABSTRACT:
SUBMITTER: Typhaine Anquetil
PROVIDER: S-SCDT-EMBOR-2020-51299-T | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:The transient receptor potential vanilloid 4 (TRPV4) channel is a mechanosensor in endothelial cells (EC) that regulates cyclic strain-induced reorientation and flow-mediated nitric oxide production. We have recently demonstrated that TRPV4 expression is reduced in tumor EC and tumors grown in TRPV4KO mice exhibited enhanced growth and immature leaky vessels. However, the mechanism by which TRPV4 regulates tumor vascular integrity and metastasis is not known. Here, we demonstrate that VE-cadherin expression at the cell-cell contacts is significantly reduced in TRPV4-deficient tumor EC and TRPV4KO EC. In vivo angiogenesis assays with Matrigel of varying stiffness (700-900 Pa) revealed a significant stiffness-dependent reduction in VE-cadherin-positive vessels in Matrigel plugs from TRPV4KO mice compared with WT mice, despite an increase in vessel growth. Further, syngeneic Lewis Lung Carcinomatumor experiments demonstrated a significant decrease in VE-cadherin positive vessels in TRPV4KO tumors compared with WT. Functionally, enhanced tumor cell metastasis to the lung was observed in TRPV4KO mice. Our findings demonstrate that TRPV4 channels regulate tumor vessel integrity by maintaining VE-cadherin expression at cell-cell contacts and identifies TRPV4 as a novel target for metastasis.
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Project description:RATIONALE:Adherens junctions (AJs) are the primary intercellular junctions in microvessels responsible for endothelial barrier function. Homophilic adhesion of vascular endothelial (VE) cadherin forms AJs, which are stabilized by binding of p120-catenin (p120). p120 dissociation from VE-cadherin results in loss of VE-cadherin homotypic interaction and AJ disassembly; however, the signaling mechanisms regulating p120 dissociation from VE-cadherin are not understood. OBJECTIVE:To address the mechanism of protein kinase C (PKC)-? function in increasing endothelial permeability, we determined the role of PKC? phosphorylation of p120 in mediating disruption of AJ integrity. METHODS AND RESULTS:We showed that PKC? phosphorylation of p120 at serine (S)879 in response to thrombin or lipopolysaccharide challenge reduced p120 binding affinity for VE-cadherin and mediated AJ disassembly secondary to VE-cadherin internalization. In studies in mouse lung vessels, expression of the phosphodeficient S879A-p120 mutant prevented the increase in vascular permeability induced by activation of the thrombin receptor PAR-1. CONCLUSIONS:PKC? phosphorylation of p120 at S879 is a critical phospho-switch mediating disassociation of p120 from VE-cadherin that results in AJ disassembly. Therefore, blocking PKC?-mediated p120 phosphorylation represents a novel targeted anti-inflammatory strategy to prevent disruption of vascular endothelial barrier function.
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Project description:Vascular endothelial (VE) protein tyrosine phosphatase (PTP) is an endothelial-specific phosphatase that stabilizes VE-cadherin junctions. Although studies have focused on the role of VE-PTP in dephosphorylating VE-cadherin in the activated endothelium, little is known of VE-PTP's role in the quiescent endothelial monolayer. Here, we used the photoconvertible fluorescent protein VE-cadherin-Dendra2 to monitor VE-cadherin dynamics at adherens junctions (AJs) in confluent endothelial monolayers. We discovered that VE-PTP stabilizes VE-cadherin junctions by reducing the rate of VE-cadherin internalization independently of its phosphatase activity. VE-PTP serves as an adaptor protein that through binding and inhibiting the RhoGEF GEF-H1 modulates RhoA activity and tension across VE-cadherin junctions. Overexpression of the VE-PTP cytosolic domain mutant interacting with GEF-H1 in VE-PTP-depleted endothelial cells reduced GEF-H1 activity and restored VE-cadherin dynamics at AJs. Thus, VE-PTP stabilizes VE-cadherin junctions and restricts endothelial permeability by inhibiting GEF-H1, thereby limiting RhoA signaling at AJs and reducing the VE-cadherin internalization rate.
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Project description:Vascular endothelial (VE)-protein tyrosine phosphatase (PTP) associates with VE-cadherin, thereby supporting its adhesive activity and endothelial junction integrity. VE-PTP also associates with Tie-2, dampening the tyrosine kinase activity of this receptor that can support stabilization of endothelial junctions. Here, we have analyzed how interference with VE-PTP affects the stability of endothelial junctions in vivo. Blocking VE-PTP by antibodies, a specific pharmacological inhibitor (AKB-9778), and gene ablation counteracted vascular leak induction by inflammatory mediators. In addition, leukocyte transmigration through the endothelial barrier was attenuated. Interference with Tie-2 expression in vivo reversed junction-stabilizing effects of AKB-9778 into junction-destabilizing effects. Furthermore, lack of Tie-2 was sufficient to weaken the vessel barrier. Mechanistically, inhibition of VE-PTP stabilized endothelial junctions via Tie-2, which triggered activation of Rap1, which then caused the dissolution of radial stress fibers via Rac1 and suppression of nonmuscle myosin II. Remarkably, VE-cadherin gene ablation did not abolish the junction-stabilizing effect of the VE-PTP inhibitor. Collectively, we conclude that inhibition of VE-PTP stabilizes challenged endothelial junctions in vivo via Tie-2 by a VE-cadherin-independent mechanism. In the absence of Tie-2, however, VE-PTP inhibition destabilizes endothelial barrier integrity in agreement with the VE-cadherin-supportive effect of VE-PTP.
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Project description:BackgroundCross-talk between integrins and cadherins regulates cell function. We tested the hypothesis that vitronectin (VN), a multi-functional adhesion molecule present in the extracellular matrix and plasma, regulates vascular permeability via effects on VE-cadherin, a critical regulator of endothelial cell (EC) adhesion.Methodology/principal findingsAddition of multimeric VN (mult VN) significantly increased VE-cadherin internalization in human umbilical vein EC (HUVEC) monolayers. This effect was blocked by the anti-?(V)?(3) antibody, pharmacological inhibition and knockdown of Src kinase. In contrast to mult VN, monomeric VN did not trigger VE-cadherin internalization. In a modified Miles assay, VN deficiency impaired vascular endothelial growth factor-induced permeability. Furthermore, ischemia-induced enhancement of vascular permeability, expressed as the ratio of FITC-dextran leakage from the circulation into the ischemic and non-ischemic hindlimb muscle, was significantly greater in the WT mice than in the Vn(-/-) mice. Similarly, ischemia-mediated macrophage infiltration was significantly reduced in the Vn(-/-) mice vs. the WT controls. We evaluated changes in the multimerization of VN in ischemic tissue in a mouse hindlimb ischemia model. VN plays a previously unrecognized role in regulating endothelial permeability via conformational- and integrin-dependent effects on VE-cadherin trafficking.Conclusion/significanceThese results have important implications for the regulation of endothelial function and angiogenesis by VN under normal and pathological conditions.
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Project description:BACKGROUND:Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact. METHODOLOGY/PRINCIPAL FINDINGS:We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus. CONCLUSIONS/SIGNIFICANCE:Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.
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Project description:Inhibition of VE-PTP, an endothelial receptor-type tyrosine phosphatase, triggers phosphorylation of the tyrosine kinase receptor Tie-2, which leads to the suppression of inflammation-induced vascular permeability. Analyzing the underlying mechanism, we show here that inhibition of VE-PTP and activation of Tie-2 induce tyrosine phosphorylation of FGD5, a GTPase exchange factor (GEF) for Cdc42, and stimulate its translocation to cell contacts. Interfering with the expression of FGD5 blocks the junction-stabilizing effect of VE-PTP inhibition in vitro and in vivo. Likewise, FGD5 is required for strengthening cortical actin bundles and inhibiting radial stress fiber formation, which are each stimulated by VE-PTP inhibition. We identify Y820 of FGD5 as the direct substrate for VE-PTP. The phosphorylation of FGD5-Y820 is required for the stabilization of endothelial junctions and for the activation of Cdc42 by VE-PTP inhibition but is dispensable for the recruitment of FGD5 to endothelial cell contacts. Thus, activation of FGD5 is a two-step process that comprises membrane recruitment and phosphorylation of Y820. These steps are necessary for the junction-stabilizing effect stimulated by VE-PTP inhibition and Tie-2 activation.
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Project description:Endothelial junctions provide blood and lymph vessel integrity and are essential for the formation of a vascular system. They control the extravasation of solutes, leukocytes and metastatic cells from blood vessels and the uptake of fluid and leukocytes into the lymphatic vascular system. A multitude of adhesion molecules mediate and control the integrity and permeability of endothelial junctions. VE-cadherin is arguably the most important adhesion molecule for the formation of vascular structures, and the stability of their junctions. Interestingly, despite this prominence, its elimination from junctions in the adult organism has different consequences in the vasculature of different organs, both for blood and lymph vessels. In addition, even in tissues where the lack of VE-cadherin leads to strong plasma leaks from venules, the physical integrity of endothelial junctions is preserved. Obviously, other adhesion molecules can compensate for a loss of VE-cadherin and this review will discuss which other adhesive mechanisms contribute to the stability and regulation of endothelial junctions and cooperate with VE-cadherin in intact vessels. In addition to adhesion molecules, endothelial receptors will be discussed, which stimulate signaling processes that provide junction stability by modulating the actomyosin system, which reinforces tension of circumferential actin and dampens pulling forces of radial stress fibers. Finally, we will highlight most recent reports about the formation and control of the specialized button-like junctions of initial lymphatics, which represent the entry sites for fluid and cells into the lymphatic vascular system.
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Project description:Cadherin-mediated cell-cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell-cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell-cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell-cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell-cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.
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Project description:In order to identify genes regulated by VE-cadherin expression, we compared a mouse VE-cadherin null cell line (VEC null) with the same line reconstituted with VE-cadherin wild type cDNA (VEC positive). The morphological and functional properties of these cell lines were described previously [Lampugnani,M.G. et al. Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148. J. Cell Biol. 161, 793-804 (2003)]. By Affymetrix gene expression analysis we found several genes up-regulated by VE-cadherin, among which claudin-5 reached remarkably high levels. The up-regulation of these genes required not only VE-cadherin expression but also cell confluence suggesting that VE-cadherin clustering at junctions was needed.
2008-07-06 | GSE11674 | GEO