PTP-mu Regulates Lung Endothelial Paracellular Pathway

性状（由申请方提供）：内皮细胞（EC）-粘附EC 连接或粘附小带（ZA）和肺血管内皮细胞 细胞旁途径的调节，部分通过蛋白酪氨酸， 磷酸化几种蛋白酪氨酸磷酸酶（PTP）， 和/或去磷酸化ZA多蛋白复合物的组分似乎 调节ZA蛋白酪氨酸磷酸化和组装的状态。一 这种PTP，PTP-mu，几乎只在肺组织中高度表达 在EC中表达，结合血管内皮细胞的胞质结构域 （VE）-钙粘蛋白和/或一种或多种连环蛋白，并通过其自身的 胞外域参与嗜同性粘附。我们将用一个不朽的 人微血管EC与四环素反应性、 逆转录病毒介导的基因转移系统，以稳定过表达野生型 PTP-mu，一种显性负性无催化活性的PTP-mu和反义PTP-mu 连同免疫共沉淀，体外GST融合蛋白结合，和 凝胶覆盖试验，荧光显微术，磷酸酪氨酸免疫印迹， 和肌动蛋白池测量，以解决以下具体目的：1） 确定PTP-mu催化功能是否调节微血管 内皮细胞旁途径; 2）为了定义PTP-mu结构， 要求其与VE-钙粘蛋白和/或其他连接蛋白结合， 人肺微血管EC中的蛋白质; 3）鉴定人肺微血管EC中的连接和/或 人微血管内皮细胞中PTP-mu的肌动蛋白结合蛋白底物。4)到 确定PTP-mu催化功能障碍是否破坏了ZA-肌动蛋白 细胞骨架连接和/或诱导肌动蛋白重组;和5）确定 PTP-mu过表达是否能保护受激动剂驱动的内皮细胞 阻挡层破坏和/或增强阻挡层恢复。这些研究将测试 PTP-mu是否在维持基础胰岛素抵抗中起反调节作用， 肺微血管内皮屏障功能和/或参与 开放的旁细胞途径，以响应建立的介质， 磁导率

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.