Rho-mediated Signaling in Lung Endothelial Cells Induced by Neutrophil Adhesion

描述（由申请方提供）：从血液中募集中性粒细胞并进入周围肺组织是肺部炎症的关键事件。要做到这一点，嗜中性粒细胞必须首先粘附在血管内皮细胞表面表达的细胞粘附分子（特别是E-选择素和ICAM-1）上。这些粘附相互作用提供附着并允许中性粒细胞在内皮表面上产生牵引力，使得它们在探测内皮连接或它们可以穿过内皮屏障的其他位点时可以在内皮表面上迁移。这些粘附分子的参与也触发了内皮细胞中促进迁移的信号通路。在已经鉴定的E-选择素和ICAM-1下游的许多信号传导途径中，几种Rho家族GTP酶已经涉及介导允许嗜中性粒细胞通过的细胞骨架和细胞连接的变化。关于不同Rho蛋白的协调以及它们如何响应于E-选择素和ICAM-1连接而被激活的知之甚少。此外，尚不清楚中性粒细胞对这些粘附分子施加的牵引力是否会影响其促进中性粒细胞穿过内皮的信号通路。然而，这些过程是高度协调和严格调节的，以使宿主防御的益处最大化并使由内皮细胞损伤引起的损伤最小化，特别是在水肿干扰气体交换的肺中。为了解决这些问题，我们提出了以下具体目标。目的1将研究关键Rho蛋白（RhoA，Rac 1，RhoG和Cdc 42）在肺微血管内皮细胞上响应E-选择素和ICAM- 1的接合和交联的活化动力学。每个Rho GTdR的基于FRET的生物传感器将用于跟踪其激活的时间和位置。新的光操纵技术将用于在精确的时间和地点激活或抑制特定的GTP酶，以检查GTP酶的相互作用如何影响中性粒细胞的迁移。目的2鉴定和操纵E-选择素和ICAM-1下游的鸟嘌呤核苷酸交换因子（GEFs），它们调节Rho蛋白的活性。目的3将确定E-选择素和ICAM-1上的张力是否启动Rho蛋白的激活。使用三维力显微镜，我们将研究是否模仿中性粒细胞对E-选择素和ICAM-1的张力启动或调节信号转导Rho GTPases。目的4将确定中性粒细胞在内皮细胞表面的迁移如何通过E-选择素和ICAM-1诱导内皮细胞表面的张力沿着和跨内皮细胞的张力，以及它们的连接是否调节VE-钙粘蛋白复合物的分解。总而言之，拟议的研究解决了炎症期间调节中性粒细胞通过内皮的重要信号通路。他们将有助于内皮细胞粘附分子信号转导的综合模型，纳入空间和时间控制，这是新的和重要的全面了解炎症。这些研究可能会确定治疗炎症性疾病的新靶点。 公共卫生相关性：该基金旨在了解中性粒细胞（一种白色血细胞）如何粘附并穿过肺血管的内皮细胞衬里，以便在炎症期间进入组织。虽然炎症和中性粒细胞募集是宿主防御病原体和其他损伤的正常方面，但中性粒细胞的不适当募集是许多炎症和免疫疾病的基础。了解中性粒细胞募集过程中跨内皮细胞屏障的步骤的机制可能会导致新的抑制剂和改进的治疗方法的发展。

DESCRIPTION (provided by applicant): The recruitment of neutrophils out of the blood and into surrounding lung tissues is a critical event in pulmonary inflammation. For this to occur, neutrophils must first adhere to cell adhesion molecules (particularly E-selectin and ICAM-1) expressed on the surface of endothelial cells lining blood vessels. These adhesive interactions provide attachment and allow neutrophils to generate traction on the endothelial surface, so that they can migrate over it as they probe for endothelial junctions or other sites where they can cross the endothelial barrier. Engagement of these adhesion molecules also triggers signaling pathways in the endothelial cells that promote transmigration. Of the many signaling pathways that have been identified downstream from E-selectin and ICAM-1, several Rho family GTPases have been implicated in mediating the changes in the cytoskeleton and cell junctions that allow neutrophil passage. Little is known about the co-ordination of the different Rho proteins and how they become activated in response to E-selectin and ICAM-1 ligation. Additionally, it is not known whether tractional forces exerted by neutrophils on these adhesion molecules affect their signaling pathways to promote neutrophil transit across the endothelium. However, these processes are highly co- ordinated and tightly regulated to maximize the benefits of host defense and minimize the injury resulting from endothelial cell damage, particularly in the lungs where edema interferes with gas exchange. To tackle these questions, we propose the following specific aims. Aim 1 will examine the dynamics of activation of key Rho proteins (RhoA, Rac1, RhoG and Cdc42) in response to engagement and crosslinking of E-selectin and ICAM- 1 on lung microvascular endothelial cells. FRET-based biosensors for each Rho GTPase will be used to follow the time and location of their activation. Novel photomanipulation techniques will be used to activate or inhibit specific GTPases at precise times and places to examine how interactions of the GTPases affect neutrophil transmigration. Aim 2 will identify and manipulate guanine nucleotide exchange factors (GEFs) that are downstream of E-selectin and ICAM-1 and that regulate Rho protein activity. Aim 3 will determine whether tension on E-selectin and ICAM-1 initiates activation of Rho proteins. Using 3D force microscopy, we will examine whether mimicking the tension applied by neutrophils on E-selectin and ICAM-1 initiates or modulates signaling to Rho GTPases. Aim 4 will determine how neutrophil migration over endothelial cell surfaces induces tension along and across endothelial cells through E-selectin and ICAM-1, and whether their ligation modulates disassembly of VE-cadherin complexes. Taken together, the proposed studies address important signaling pathways that regulate neutrophil passage across the endothelium during inflammation. They will contribute to an integrated model of endothelial adhesion molecule signaling, incorporating spatial and temporal control that is novel and important to a comprehensive understanding of inflammation. These studies may identify new targets for therapies in the treatment of inflammatory diseases. PUBLIC HEALTH RELEVANCE: This grant seeks to understand how neutrophils (one type of white blood cell) adhere and pass through the endothelial cell lining of lung blood vessels in order to enter tissues during inflammation. Although inflammation and neutrophil recruitment is a normal aspect of host defense against pathogens and other injuries, the inappropriate recruitment of neutrophils underlies many inflammatory and immune diseases. Understanding the mechanisms underlying the steps in the process of neutrophil recruitment across an endothelial cell barrier may lead to the development of novel inhibitors and improved therapies.