S-Nitrosation of β-catenin and p120 catenin: a novel regulatory mechanism in endothelial hyperpermeability.
S-Nitrosation of β-catenin and p120 catenin: a novel regulatory mechanism in endothelial hyperpermeability.
复制标题
DOI：
10.1161/circresaha.112.274548
复制
发表时间：
2012-08-17
影响因子：
20.1
通讯作者：
Sánchez FA
中科院分区：
文献类型：
--
作者：
Marín N;Zamorano P;Carrasco R;Mujica P;González FG;Quezada C;Meininger CJ;Boric MP;Durán WN;Sánchez FA
内皮粘附连接蛋白是控制微血管通透性的重要元件。血小板激活因子 (PAF) 通过 eNOS 易位至细胞质并刺激 eNOS 衍生的 NO 信号级联反应来增加大分子的通透性。 NO 信号传导调节粘附连接通透性的机制仍不完全清楚。我们探讨了 PAF 通过粘附连接蛋白的 S-亚硝化 (SNO) 刺激通透性过高的假设。我们在三种细胞系中测量了 PAF 刺激的 β-连环蛋白和 p120-连环蛋白 (p120) 的 S-亚硝化：ECV-eNOSGFP、EAhy926（源自人脐静脉）和 CVEC（源自牛心脏内皮）以及体内小鼠提睾肌。 SNO 与粘附连接处 β-连环蛋白和 p120 丰度的减少以及通透性过高相关。 TNF-α 增加 NO 的产生，并引起与 PAF 类似的 S-亚硝化增加。为了确定 eNOS 亚细胞定位在此过程中的重要性，我们使用了转染胞质 eNOS (GFPeNOSG2A) 和质膜 eNOS (GFPeNOSCAAX) 的 ECV-304 细胞。 PAF 诱导 β-连环蛋白和 p120 的 S-亚硝化，并显着减少细胞中这些蛋白质与胞质 eNOS 之间的关联，但在 eNOS 锚定于细胞膜的细胞中则不然。 NO 产生和 S-亚硝化抑制剂可阻断 PAF 诱导的 S-亚硝化和通透性过高，而 cGMP 途径的抑制则没有效果。对纯化 p120 的质谱分析鉴定出半胱氨酸 579 是该区域中的主要 S-亚硝化残基，推测与 VE-钙粘蛋白相互作用。我们的结果表明，激动剂诱导的 SNO 有助于连接膜蛋白的变化，从而增强内皮细胞的通透性。
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. We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. 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. Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.