Endothelial TAK1 Signaling and Resolution of Pulmonary Edema in Sepsis

 描述(由申请人提供)：与急性肺损伤(ALI)相关的脓毒症是住院患者的常见死亡原因。ALI在很大程度上是肺血管渗漏和富含蛋白质的水肿所致，目前尚缺乏有效的治疗方法。在这里，我们提出了一种新的策略，通过刺激通常在肺损伤后激活的内源性恢复过程来逆转ALI。凝血酶是脓毒症时产生的一种水肿性因子，通过激活内皮细胞表面的蛋白水解酶激活受体-1(PAR-1)介导肺血管渗漏。PAR-1通过钙离子通道(SOCs)诱导钙离子内流，分解内皮细胞黏附连接(AJs)，导致肺血管渗漏增加。内质网(ER)定位的钙离子感受器蛋白基质相互作用分子-1(STIM1)在激活SOC诱导内皮细胞(ECs)钙离子内流(SOCE)中起着至关重要的作用。现在，我们在三苯氧胺诱导的内皮细胞限制性TAK1缺失(Map3k7i∆EC)的小鼠模型中发现，TAK1通过调节内皮细胞中STIM1、糖原合成酶β-3β和β-连环蛋白的功能，在解决PAR-1介导的肺水肿形成中发挥关键作用。我们做了以下观察(支持数据)：1)TAK1缺失的内皮细胞在PAR-1激活后表现为SOCE和通透性增加；II)PAR-1诱导的∆EC小鼠体内肺血管通透性不可逆；III)β-Catenin在Map3k7i∆EC小鼠体内的表达显著降低；4)糖原合成酶β-3β在Map3k7i∆EC小鼠的内皮细胞中持续活跃，这可能是导致Map3k7iβ-Catenin表达显著降低的原因之一；Vi)令人惊讶的是，我们观察到在EC限制性的∆基因敲除小鼠(GSK-3βIβEC)中，SOCE通过TAK1的激活而失活GSK-3β；vii)重要的是，PAR-1介导的肺血管渗漏在他莫昔芬诱导的EC限制性GSK-3∆敲除小鼠中显著减少。基于这些新的观察结果，在目标1中，我们将检验这样一个假设，即STIM1介导的SOCE继而激活TAK1导致STIM1磷酸化，进而抑制SOCE并抑制肺血管通透性。在目标2中，我们将验证一种假设，即STIM1介导的SOCE继而激活Tak1使GSK-3β磷酸化，使GSK-3β失活，进而促进内皮AJ上β-Catenin表达的增加，以恢复内皮屏障的完整性，从而解决肺水肿。更好地了解SOCE下游TAK1功能的信号机制将导致解决脓毒症肺水肿的新的治疗方法。

 DESCRIPTION (provided by applicant): Sepsis associated with acute lung injury (ALI) is a common cause of death in hospitalized patients. ALI is in large part the result of lung vascular leakage and protein rich edema and there is a lack of effective therapy. Here, we have proposed a novel strategy to reverse ALI by stimulating an endogenous recovery process that is usually activated after lung injury. Thrombin, an edema-genic factor generated during sepsis, mediates pulmonary vascular leakage by activating protease-activated receptor-1 (PAR-1) on the endothelial cell surface. PAR-1-induced Ca2+ entry via store-operated Ca2+-entry channels (SOCs), disassembles endothelial adherens junctions (AJs) to cause increased lung vascular leak. An endoplasmic reticulum (ER) localized Ca2+ sensor protein stromal interacting molecule-1 (STIM1), is crucial for activating SOC to induce store- operated Ca2+-entry (SOCE) in endothelial cells (ECs). Now, we have identified in a murine model of tamoxifen-inducible endothelial cell (EC)-restricted TAK1 (Map3k7) deletion (Map3k7i∆EC), a key role for TAK1 in resolving PAR-1-mediated pulmonary edema formation through regulation of the functions of STIM1, glycogen synthase kinase-3β (GSK-3β) and β-catenin in ECs. We made the following observations (Supporting Data): i) TAK1 null ECs exhibited augmented SOCE and permeability in response to PAR-1 activation; ii) PAR-1-induced lung vascular permeability in vivo was not reversible in Map3k7i∆EC mice; iii) β- catenin expression was markedly reduced in ECs of Map3k7i∆EC mice; iv) glycogen synthase kinase-3β (GSK- 3β) was persistently active in ECs of Map3k7i∆EC mice, which may account for the markedly reduced expression of β-catenin in ECs of Map3k7i∆EC mice; v) PAR-1-medatied TAK1 activation was prevented in ECs of EC-restricted STIM1 knockout (Stim1∆EC) mice; vi) surprisingly, we observed that SOCE signals the inactivation of GSK-3β via TAK1 activation in ECs; vii) importantly, PAR-1-mediated lung vascular leak was markedly reduced in tamoxifen-inducible EC-restricted GSK-3β knockout (GSK-3βi∆EC) mice. Based on these novel observations, in Aim 1, we will test the hypothesis that TAK1 activation secondary to STIM1-mediated SOCE induces STIM1 phosphorylation which in turn inhibits SOCE and dampens lung vascular permeability. In Aim 2, we will test the hypothesis that TAK1 activation secondary to STIM1-mediated SOCE phosphorylates GSK-3β to inactivate GSK-3β, which in turn promotes increased β-catenin expression at endothelial AJs to restore endothelial barrier integrity and thereby resolves pulmonary edema. A better understanding of the signaling mechanisms of TAK1 functions downstream of SOCE will lead to novel therapeutic approaches that will resolve pulmonary edema in sepsis.