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

描述（由申请人提供）：中性粒细胞从血液中募集到周围肺组织是肺部炎症的关键事件。为此，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。这些粘附的相互作用提供了附着，并允许中性粒细胞在内皮表面产生牵引力，这样它们就可以在探测内皮连接或其他可以穿过内皮屏障的部位时迁移到内皮表面。这些粘附分子的参与也会触发内皮细胞中促进转运的信号通路。在e -选择素和ICAM-1下游已确定的许多信号通路中，几个Rho家族gtpase参与介导细胞骨架和细胞连接的变化，这些变化允许中性粒细胞通过。对于不同Rho蛋白的协调以及它们如何响应e -选择素和ICAM-1连接而被激活，人们知之甚少。此外，目前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它们的信号通路，从而促进中性粒细胞穿越内皮。然而，这些过程是高度协调和严格调节的，以最大限度地发挥宿主防御的好处，并最大限度地减少内皮细胞损伤造成的损伤，特别是在水肿干扰气体交换的肺部。为解决这些问题，我们提出以下具体目标。目的1将研究关键Rho蛋白（RhoA, Rac1， RhoG和Cdc42）的激活动力学，以响应e -选择素和ICAM- 1在肺微血管内皮细胞上的接合和交联。每个Rho GTPase将使用基于fret的生物传感器来跟踪其激活的时间和位置。新的图像处理技术将用于在精确的时间和地点激活或抑制特定的gtpase，以研究gtpase的相互作用如何影响中性粒细胞的转运。目的2将鉴定和操纵鸟嘌呤核苷酸交换因子（gef），这些因子位于e -选择素和ICAM-1的下游，并调节Rho蛋白的活性。Aim 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3D力显微镜，我们将检查中性粒细胞对e -选择素和ICAM-1施加的张力是否启动或调节Rho GTPases的信号传导。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诱导沿内皮细胞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。综上所述，提出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。他们将有助于建立内皮粘附分子信号的综合模型，将空间和时间控制结合起来，这对全面理解炎症是新颖而重要的。这些研究可能为炎症性疾病的治疗找到新的靶点。

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.