PTP-mu Regulates Lung Endothelial Paracellular Pathway

描述(申请人提供)：内皮细胞(EC)-EC黏附 连接或粘连带(ZA)和肺血管内皮细胞 细胞旁路部分是通过蛋白质酪氨酸调节的 磷酸化。几种蛋白酪氨酸磷酸酶(PTPs)与 与和/或去磷酸化的ZA多蛋白复合体的成分似乎 调节ZA蛋白酪氨酸的磷酸化和组装状态。一 这种名为PTP-u的蛋白在肺组织中高度表达，几乎只在肺组织中表达。 在EC中表达，与血管内皮细胞胞浆结构域结合 (Ve)-钙粘附素和/或一个或多个链状物，并通过其自身 胞外结构域参与同嗜性黏附。我们将用一个不朽的 人微血管内皮细胞系与四环素反应， 逆转录病毒介导的基因转移系统稳定高效表达野生型 PTP-MU，显性负性催化失活PTP-MU和反义PTP-MU 结合免疫共沉淀，体外GST-融合蛋白结合，以及 凝胶覆盖分析，荧光显微镜，磷酸酪氨酸免疫印迹， 和肌动蛋白池测量，以满足以下具体目标：1) 确定PTP-MU催化功能是否调节微血管 内皮细胞旁路；2)确定PTP-MU结构 对其与VE-钙粘附素和/或其他连接蛋白结合的要求 人肺微血管内皮细胞中的蛋白质；3)鉴定连接和/或 人微血管内皮细胞PTP-MU肌动蛋白结合蛋白底物。4)至 确定PTP-MU催化功能障碍是否干扰ZA-肌动蛋白 细胞骨架连接和/或诱导肌动蛋白重组；以及5)确定 PTP-MU过表达是否对激动剂驱动的内皮细胞有保护作用 障碍物破坏和/或增强障碍物恢复。这些研究将检验 PTP-MU在维持基础状态中是否起到反调节作用 肺微血管内皮细胞屏障功能和/或参与 开放细胞旁通路以响应已建立的介质 渗透性。

DESCRIPTION (provided by applicant): The endothelial cell (EC)-EC adherens junction or the zonula adherens (ZA) and the pulmonary vascular endothelial paracellular pathway are regulated, in part, through protein tyrosine phosphorylation. Several protein tyrosine phosphatases (PTPs) that associate with and/or dephosphorylate components of the ZA multiprotein complex appear to regulate the state of ZA protein tyrosine phosphorylation and assembly. One such PTP, PTP-mu, is highly expressed in lung tissue, almost exclusively expressed in EC, binds to the cytoplasmic domain of vascular endothelial (VE)-cadherin and/or to one or more of the catenins, and through its own ectodomain participates in homophilic adhesion. We will use an immortalized human microvascular EC line together with a tetracycline-responsive, retrovirus-mediated gene transfer system to stably overexpress wild-type PTP-mu, a dominant-negative catalytically-inactive PTP-mu and antisense PTP-mu together with coimmunoprecipitation, in vitro GST-fusion protein binding, and gel-overlay assays, fluorescence microscopy, phosphotyrosine immunoblotting, and actin pool measurements to address the following Specific Aims: 1) To determine whether PTP-mu catalytic function regulates the microvascular endothelial paracellular pathway; 2) To define the PTP-mu structural requirements for its association with VE-cadherin and/or other junctional proteins in human lung microvascular EC; 3) To identify the junctional and/or actin-binding protein substrates for PTP-mu in human microvascular EC. 4) To determine whether PTP-mu catalytic dysfunction disrupts the ZA-actin cytoskeletal linkage and/or induces actin reorganization; and 5) To determine whether PTP-mu overexpression protects against agonist-driven endothelial barrier disruption and/or enhances barrier recovery. These studies will test whether PTP-mu serves a counter-regulatory role in the maintenance of basal pulmonary microvascular endothelial barrier function and/or participates in the opening of the paracellular pathway in response to established mediators of permeability.