Endothelial TAK1 Signaling and Resolution of Pulmonary Edema in Sepsis

 描述（由申请方提供）：急性肺损伤（ALI）相关败血症是住院患者死亡的常见原因。ALI在很大程度上是肺血管渗漏和富含蛋白质的水肿的结果，并且缺乏有效的治疗。在这里，我们提出了一种新的策略，通过刺激肺损伤后通常被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致水肿因子，通过激活内皮细胞表面的蛋白酶激活受体1（PAR-1）介导肺血管渗漏。PAR-1通过钙池操纵的钙通道（SOC）诱导钙内流，分解内皮粘附连接（AJs），导致肺血管渗漏增加。基质相互作用分子-1（stromal interacting molecule-1，STIM 1）是一种位于内质网（endoplasmic reticulum，ER）的钙传感蛋白，在激活SOC诱导内皮细胞钙池操纵性内流（store- operated Ca ~（2+）-entry，SOCE）过程中起重要作用。现在，我们已经在他莫昔芬诱导的内皮细胞（EC）限制性TAK 1（Map 3 k7）缺失（Map 3 k7 i EKEC）的小鼠模型中确定了TAK 1通过调节STIM 1、糖原合成酶激酶-3 β（GSK-3β）和EC中β-连环蛋白的功能在解决PAR-1介导的肺水肿形成中的关键作用。我们提出了以下意见（支持数据）：i）TAK 1无效EC表现出响应PAR-1激活的SOCE和渗透性增加; ii）在Map 3 k7 i AEC小鼠中，PAR-1诱导的肺血管渗透性在体内是不可逆的; iii）在Map 3 k7 i AEC小鼠的EC中，β-连环蛋白表达显著降低;（4）糖原合成酶激酶-3 β（glycogen synthase kinase-3β，GSK- 3β）在Map 3 k7 i大肠癌小鼠的内皮细胞中具有持续活性，这可能是β-catenin在Map 3 k7 i大肠癌小鼠的内皮细胞中表达显著降低的原因; v）PAR-1介导的TAK 1活化在EC限制性STIM 1敲除的EC中被阻止vi）令人惊讶的是，我们观察到SOCE通过EC中的TAK 1活化而发出GSK-3β失活的信号; 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-3β敲除（GSK-3β iEC）小鼠中显著减少。基于这些新的观察结果，在目标1中，我们将检验以下假设：继发于STIM 1介导的SOCE的TAK 1激活诱导STIM 1磷酸化，STIM 1磷酸化反过来抑制SOCE并抑制肺血管通透性。在目标2中，我们将检验以下假设：STIM 1介导的SOCE继发的TAK 1激活将GSK-3β磷酸化为GSK-3β，这反过来促进内皮AJs的β-连环蛋白表达增加，以恢复内皮屏障完整性，从而解决肺水肿。更好地理解SOCE下游TAK 1功能的信号传导机制将导致解决脓毒症肺水肿的新治疗方法。

 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.