Endotoxin TLR4 Signaling Regulates Lung Endothelial Paracellular Pathway

描述(申请人提供)：革兰氏阴性细菌血流感染及其伴随的内毒素血症与血管渗漏综合征有关，包括危及生命的急性呼吸窘迫综合征(ARDS)。内皮细胞S表达内毒素或细菌脂多糖的受体TLR4，但TLR4信号转导途径如何与肺微血管内皮细胞旁细胞通路开放有关尚不清楚。我们已经证明，内毒素增加了EC蛋白的酪氨酸磷酸化，丰富到EC-EC边界，并诱导肌动蛋白重组。先前的PTK抑制和F-肌动蛋白稳定都可以对抗内毒素诱导的屏障破坏。我们现在提供的数据表明，粘着带蛋白(ZA)、VE-钙粘附素、3-连环素和p120ctn是脂多糖诱导酪氨酸磷酸化的底物，激活一个或多个src家族蛋白酪氨酸激酶(Sfk)S是脂多糖诱导打开细胞旁通路的先决条件。1.鉴定人肺微血管内皮细胞TLR4应答、MYD88依赖的信号/接头分子(S)，该分子将内毒素刺激与sFK激活和旁细胞通路开放偶联。2.明确内毒素对ZA多蛋白复合体内蛋白质-蛋白质相互作用、ZA-肌动蛋白细胞骨架连接以及相对的VE-钙粘蛋白胞外结构域间相互作用的影响。3.鉴定内毒素通过增加连接蛋白和/或肌动蛋白骨架蛋白的酪氨酸磷酸化，诱导ZA重组，打开内皮细胞旁通路的SFK(S)。4.确定PTP是否对内毒素刺激反应中SFK的激活、ZA蛋白酪氨酸磷酸化的增加、ZA多蛋白复合体的重组和/或细胞旁通路的开放起到反调节作用。理解内毒素调节EC-EC亲和性黏附和细胞旁途径的机制(S)，不仅对脓毒症相关的急性肺损伤和血管内液体和大分子进入组织，而且对中性粒细胞外溢也有意义。与公共卫生相关：许多细菌会在人类体内引起危及生命的感染，它们会释放一种名为内毒素的物质，这种物质可以与血管细胞上的受体结合。当内毒素与这种受体结合时，它可以触发这些细胞之间的间隙打开，体液泄漏到组织中，包括肺的空气空间。了解导致这种高死亡率、血管渗漏综合征的生化事件应该为治疗干预提供潜在的靶点。

DESCRIPTION (provided by applicant): Gram-negative bacterial bloodstream infections and their attendant endotoxemia are associated with vascular leak syndromes, including the life-threatening, Acute Respiratory Distress Syndrome (ARDS). One receptor for endotoxin or bacterial lipopolysaccharide (LPS), Toll-like receptor (TLR)-4, is expressed by endothelial cell (EC)s. Although much is known about TLR4 signaling, how it is coupled to opening of the pulmonary microvascular endothelial paracellular pathway is unknown. We have demonstrated that LPS increases tyrosine phosphorylation of EC proteins enriched to EC-EC boundaries and induces actin reorganization. Prior PTK inhibition and F-actin stabilization each protects against LPS-induced barrier disruption. We now present data identifying the zonula adherens (ZA) proteins, VE-cadherin, 3-catenin, and p120ctn, as substrates for LPS- induced tyrosine phosphorylation, and activation of one or more src family PTK (SFK)s as a prerequisite for LPS-induced opening of the paracellular pathway. We propose the following SPECIFIC AIMS: 1. To identify the TLR4-responsive, MyD88-dependent signaling/adapter molecule(s) that couple the LPS stimulus to SFK activation and opening of the paracellular pathway in human lung microvascular endothelia (HMVEC-Ls). 2. To define LPS-induced changes in protein-protein interactions within the ZA multiprotein complex, the ZA-actin cytoskeletal linkage, and between opposing VE-cadherin ectodomains. 3. To identify the SFK(s) through which LPS increases tyrosine phosphorylation of junctional and/or actin cytoskeletal proteins, induces ZA reorganization, and opens the endothelial paracellular pathway. 4. To establish whether PTP¿ counter-regulates SFK activation, increased tyrosine phosphorylation of ZA proteins, reorganization of the ZA multiprotein complex, and/or opening of the paracellular pathway, in response to the LPS stimulus. Understanding the mechanism(s) through which LPS regulates EC-EC homophilic adhesion and the paracellular pathway has implications not only for sepsis-associated acute lung injury and the movement of intravascular fluid and macromolecules into tissues but for neutrophil extravasation as well. PUBLIC HEALTH RELEVANCE: Numerous bacteria that cause life-threatening infections in humans, release a substance called endotoxin, which can bind to a receptor on the cell that line blood vessels. When endotoxin engages this receptor, it can trigger opening of the spaces between these cells and leakiness of body fluids into the tissues, including the air-spaces of the lung. Understanding the biochemical events that lead to this high-mortality, vascular leak syndrome should offer potential targets for therapeutic interventions.