iNOS Activation Regulates β-catenin Association with Its Partners in Endothelial Cells

BackgroundSignals that disrupt β-catenin association to cadherins may influence the translocation of β-catenin to the nucleus to regulate transcription. Post-translational modification of proteins is a signalling event that may lead to changes in structural conformation, association or function of the target proteins. NO and its derivatives induce nitration of proteins during inflammation. It has been described that animals treated with NO donors showed increased permeability due to modulation of VE-cadherin/catenin complex. We, therefore, aim to evaluate the effect of iNOS activation on the expression, nuclear localisation and function of β-catenin in endothelial cells. Methodology/Principal FindingsExpression, nuclear localisation, post-translational modifications and function of β-catenin was analysed by cell fractionation, immunoprecipitation, immunoblots, QRT-PCR and permeability assays in murine endothelial cells (H5V). Influence of macrophage activation on expression of VE-cadherin/p120-catenin/β-catenin complex in co-cultured H5V cells was also assessed. Activation of macrophages to produce NO provoked a decrease in VE-cadherin/p120-catenin/β-catenin expression in H5V cells. Phosphorylation of β-catenin, p120-catenin and VE-cadherin, and reduction in the barrier properties of the cell monolayer was associated with iNOS induction. Moreover, high NO levels provoked nitration of β-catenin, and induced its translocation to the nucleus. In the nucleus of NOS activated cells, nitration levels of β-catenin influenced its association with TCF4 and p65 proteins. High levels of NO altered β-catenin mediated gene expression of NFκB and Wnt target genes without affecting cell viability. ConclusionsNOS activity modulates β-catenin post-translational modifications, function and its association with different partners to promote endothelial cell survival. Therapeutic manipulation of iNOS levels may remove a critical cytoprotective mechanism of importance in tumour angiogenesis.背景：干扰β-连环蛋白（β-catenin）与钙粘蛋白结合的信号通路，可能影响β-连环蛋白向细胞核的转位过程，进而调控基因转录。蛋白质的翻译后修饰是一类信号事件，可导致靶蛋白的结构构象、相互结合状态或功能发生改变。一氧化氮（NO）及其衍生物在炎症过程中可诱导蛋白质发生硝基化修饰。已有研究证实，经一氧化氮供体处理的动物，其血管内皮钙粘蛋白/连环蛋白复合物的调控异常会引发血管通透性升高。为此，本研究旨在评估诱导型一氧化氮合酶（iNOS）活化对内皮细胞中β-连环蛋白的表达、核定位及功能的影响。 方法与主要结果：本研究通过细胞分级分离、免疫沉淀、免疫印迹、实时定量聚合酶链反应（QRT-PCR）以及通透性实验，对小鼠内皮细胞（H5V）中β-连环蛋白的表达、核定位、翻译后修饰及功能进行了分析。同时，本研究还评估了巨噬细胞活化对共培养H5V细胞中血管内皮钙粘蛋白/p120-连环蛋白/β-连环蛋白复合物表达的影响。实验结果显示，巨噬细胞活化产生一氧化氮后，H5V细胞内的血管内皮钙粘蛋白/p120-连环蛋白/β-连环蛋白表达水平显著下降。β-连环蛋白、p120-连环蛋白与血管内皮钙粘蛋白的磷酸化水平升高，以及细胞单层屏障功能的降低，均与诱导型一氧化氮合酶的诱导表达密切相关。此外，高浓度一氧化氮可诱导β-连环蛋白发生硝基化修饰，并促进其向细胞核转位。在诱导型一氧化氮合酶活化的细胞核中，β-连环蛋白的硝基化水平会影响其与TCF4及p65蛋白的结合能力。高浓度一氧化氮会改变β-连环蛋白介导的核因子κB（NFκB）与Wnt通路靶基因的表达，但不会对细胞活力产生影响。 结论：诱导型一氧化氮合酶的活性可通过调控β-连环蛋白的翻译后修饰、功能及其与不同伴侣蛋白的相互结合，促进内皮细胞存活。对诱导型一氧化氮合酶水平进行治疗性干预，可能会消除肿瘤血管生成中至关重要的一种关键细胞保护机制。