Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to Decrease Barrier Function of Endothelial Monolayers
ABSTRACT Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4-6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and beta-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.
- SourceAvailable from: Haixia Gong
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- "Src activation in turn mediated phosphorylation of VE-cadherin at Tyr 658, the phosphorylation site known to promote the dissociation of p120-catenin from VE-cadherin and the subsequent internalization of VE-cadherin (Xiao et al., 2003; Potter et al., 2005; Hatanaka et al., 2011). Although the role of Src and resulting VE-cadherin phosphorylation at Tyr658 in mediating disassembly of AJs has been previously proposed (Adam et al., 2010), our results are the first to show that in the context of Src activation induced by G13–VE-cadherin interaction, Src phosphorylation of VE-cadherin at Tyr656 is an important mechanism of AJ disassembly. This contention is reinforced by the finding that endothelial cell expression of phosphorylation-resistant Y658F VE-cadherin not only restored endothelial AJ integrity but also protected AJs from disruption induced by multiple inflammatory mediators. "
ABSTRACT: The heterotrimeric G protein Gα13 transduces signals from G protein-coupled receptors (GPCRs) to induce cell spreading, differentiation, migration, and cell polarity. Here, we describe a novel GPCR-independent function of Gα13 in regulating the stability of endothelial cell adherens junctions (AJs). We observed that the oxidant H2O2, which is released in response to multiple proinflammatory mediators, induced the interaction of Gα13 with VE-cadherin. Gα13 binding to VE-cadherin in turn induced Src activation and VE-cadherin phosphorylation at Tyr 658, the p120-catenin binding site thought to be responsible for VE-cadherin internalization. Inhibition of Gα13-VE-cadherin interaction using an interfering peptide derived from the Gα13 binding motif on VE-cadherin abrogated the disruption of AJs in response to inflammatory mediators. These studies identify a unique role of Gα13 binding to VE-cadherin in mediating VE-cadherin internalization and endothelial barrier disruption and inflammation.Journal of Experimental Medicine 03/2014; 211(3). DOI:10.1084/jem.20131190 · 13.91 Impact Factor
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- "However , the precise mechanisms through which Src exerts its action are far from being completely clear. In vitro studies have shown that phosphorylation of VE-cadherin by Src is not enough to increase endothelial permeability (Adam et al., 2010). Accordingly , a recent study by our group (Orsenigo et al., 2012) showed that in vivo, under resting conditions, VE-cadherin is phosphorylated on tyrosine in the veins, but not in the arteries. "
ABSTRACT: VE-cadherin is a component of endothelial cell-to-cell adherens junctions, and it has a key role in the maintenance of vascular integrity. During embryo development, VE-cadherin is required for the organization of a stable vascular system, and in the adult it controls vascular permeability and inhibits unrestrained vascular growth. The mechanisms of action of VE-cadherin are complex and include reshaping and organization of the endothelial cell cytoskeleton and modulation of gene transcription. Here we review some of the most important pathways through which VE-cadherin modulates vascular homeostasis and discuss the emerging concepts in the overall biological role of this protein.Developmental Cell 09/2013; 26(5):441-54. DOI:10.1016/j.devcel.2013.08.020 · 10.37 Impact Factor
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- "In the present study we did not dissect the steps coupling PKC, a serine/ threonine kinase, with phosphorylation of tyrosine 731 of VE-cadherin. For example, PKC can activate Src (a tyrosine kinase) and Src is known to phosphorylate VE-cadherin at Y731 (Adam et al., 2010). Because PKC activity was required for the response to each treatment (Hcy, CHPG, NMDA; Fig. 5), we do not know whether PKC is also involved in the potentiation of the NMDAr channel by mGluR5. "
ABSTRACT: Elevated plasma homocysteine (Hcy) is an independent risk factor for vascular disease and stroke in part by causing generalized endothelial dysfunction. A receptor that is sensitive to Hcy and its intracellular signaling systems has not been identified. β-catenin is a pleiotropic regulator of transcription and cell function. Using a brain microvascular endothelial cell line (bEnd.3), we tested the hypothesis that Hcy causes receptor-dependent nuclear translocation of β-catenin. Hcy increased phosphorylation of Y731 on vascular endothelial cadherin (VE-cadherin), a site involved in coupling β-catenin to VE-cadherin. This was blocked by inhibition of either metabotropic glutamate receptor 5 (mGluR5) or ionotropic glutamate receptor (NMDAr) and by shRNA knockdown of mGluR5. Expression of these receptors was confirmed by flow cytometry, immunohistochemistry, and western blotting. Directed pharmacology with specific agonists elucidated a signaling cascade where Hcy activates mGluR5 which activates NMDAr with subsequent PKC activation and uncoupling of the VE-cadherin/β-catenin complex. Moreover, Hcy caused a shift in localization of β-catenin from membrane-bound VE-cadherin to the cell nucleus, where it bound DNA, including a regulatory region of the gene for claudin-5, leading to reduced expression of claudin-5. Nuclear localization, DNA binding of β-catenin, and reduced claudin-5 expression were blocked by inhibition of mGluR5. Knockdown of mGluR5 expression with shRNA also rescued claudin-5 expression from the effects of Hcy treatment. These data uniquely identify mGluR5 as a master switch that drives β-catenin nuclear localization in vascular endothelium and regulates cell-cell coupling in response to elevated Hcy levels. These studies dissect a pharmacological opportunity for developing new therapeutic strategies in HHcy.Vascular Pharmacology 01/2012; 56(3-4):159-67. DOI:10.1016/j.vph.2012.01.004 · 4.62 Impact Factor