S-Nitrosation of β-Catenin and p120 Catenin

Abstract

Rationale Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of eNOS to cytosol and stimulation of eNOS-derived NO signaling cascade. The mechanisms by which NO signaling regulates permeability at adherens junctions are still incompletely understood. Objective We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. Methods and Results We measured PAF-stimulated S-nitrosation of β-catenin and p120-catenin (p120) in three cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein) and CVEC (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of β-catenin and p120 at the adherens junction and with hyperpermeability. TNF-α increased NO production and caused similar increase in S-nitrosation as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S-nitrosation of β-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of NO production and of S-nitrosation blocked PAF-induced S-nitrosation and hyperpermeability whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with VE-cadherin. Conclusions Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

“…This notion is supported by our findings that siRNA‐mediated knockdown of Rnd3 expression caused longer time courses of thrombin‐induced barrier dysfunction in HUVEC monolayers in association with extended periods of RhoA activation. It is worth noting that the general concept of signals localized in the perinuclear or centralized areas of the cytoplasm rapidly leading to elevated permeability of the endothelium has previously been shown in studies of endothelial nitric oxide synthase activation localized to the cytosol or Golgi apparatus,72, 73, 74 leading to the downstream S‐nitrosylation of junction‐associated proteins 75…”

Sphingosine-1-phosphate receptor 2 protects against anaphylactic shock through suppression of endothelial nitric oxide synthase in mice

“…12 Recent studies showed that VEGF induced Cui 19 S-nitrosylation of β-catenin, resulting in the dissociation of β-catenin from VE-cadherin and disassembly of AJ complexes. 31,34 In pulmonary EC, PAF and S1P induced S-nitrosylation of β-catenin with disassembly of VE-cadherin and β-catenin at cell-cell contacts, which was augmented by S1pr2 deficiency (Fig 5). Of note, pharmacological blockade of eNOS restored the aggravation of S-nitrosylation of β-catenin and AJ disassembly by S1pr2 deficiency.…”

“…22,23,25 Recent studies 31,34 demonstrated that VEGF stimulation of eNOS induced S-nitrosylation of β-catenin, leading to disassembly of AJ complexes. We examined how S1P 2 influenced PAF-induced disassembly of AJ and S-nitrosylation of β-catenin in a monolayer of MLEC.…”

eNOS derived nitric oxide regulates endothelial barrier function via VE cadherin and Rho GTPases

“…The regulation of AJ dynamics strongly supports the autocrine role of NO in endothelial cells, separate from the canonical, paracrine role of eNOS in controlling blood flow and hemodynamics. The idea that eNOS-derived NO regulates junctional dynamics is supported by recent results showing that NO posttranslationally nitrosylates b-catenin and p120-catenin promoting its dissociation from VE-cadherin (Marín et al, 2012;Thibeault et al, 2010), and tyrosine nitrates p190RhoGAP (Siddiqui et al, 2011) effects leading to enhanced permeability. Our data showing enhanced TIAM1-VE-cadherin-mediated stabilization of cortical actin explains why mice deficient in eNOS or mice harboring a S1176A allele exhibit reduced vascular leakage in vivo and sheds light on why anti-VEGF therapy reduces vascular permeability but promotes increased blood pressure in patients.…”