Thrombospondin-1 Opens Endothelial Paracellular Pathway via EGFR/ErbB2 Activation

描述（由申请方提供）：血小板反应蛋白（TSP）-1是一种约420 KDa的三聚体、多结构域糖蛋白，可与多种内皮细胞（EC）受体结合并激发多种EC应答。粘附小带（ZA）是一种特化结构，其通过连环蛋白将肌动蛋白细胞骨架偶联至血管内皮（VE）-钙粘蛋白的胞质结构域，所述钙粘蛋白是介导嗜同性EC-EC粘附的表面受体。在人肺微血管内皮细胞中，我们已经证明TSP-1增加ZA蛋白、VE-钙粘蛋白、γ-连环蛋白和p120 ctn的酪氨酸磷酸化，并打开细胞旁通路。先前的蛋白酪氨酸激酶（PTK）抑制剂保护TSP-1诱导的屏障破坏，而蛋白酪氨酸磷酸酶（PTP）抑制剂增强了它。我们现在提出的证据表明，TSP-1激活受体PTKs，ErbB 1和ErbB 2，并通过这种激活打开内皮细胞旁通路。此外，PTP（调节VE-钙粘蛋白酪氨酸磷酸化和内皮细胞旁途径的受体PTP）反调节EGFR/ErbB 2活化。我们提出了以下具体目的：1）确定TSP-1中打开人肺微血管内皮细胞中酪氨酸磷酸化反应性细胞旁途径所需的特异性序列。2)阐明TSP-1激活EGFR/erbB 2的机制，包括a）其EGF样重复序列与EGFR胞外域的直接结合，B）刺激EC释放EGFR配体，和/或c）激活另一种反式激活EGFR/erbB 2的TSP-1受体。3)确定EGFR/erbB 2的TSP-1激活与ZA和/或其他连接蛋白酪氨酸磷酸化增加、ZA重组和/或内皮细胞旁通路开放偶联的机制。4)确定PTP的反调节作用（在TSP-1诱导的EGFR/ErbB 2活化中）。了解TSP-1调节EC-EC嗜同性粘附和细胞旁途径的机制对伤口愈合、组织重塑和肿瘤细胞存活背景下的血管生成以及细胞、大分子和流体从血流进入血管外组织的运动具有意义。

DESCRIPTION (provided by applicant): Thrombospondin (TSP)-1 is an ~420KDa trimeric, multidomain glycoprotein that engages multiple endothelial cell (EC) receptors and elicits multiple EC responses. The zonula adherens (ZA) is a specialized structure that, through the catenins, couples the actin cytoskeleton to the cytoplasmic domain of vascular endothelial (VE)-cadherin, a surface receptor that mediates homophilic EC-EC adhesion. In human pulmonary microvascular endothelia, we have demonstrated that TSP-1 increases tyrosine phosphorylation of the ZA proteins, VE-cadherin, y-catenin and p120ctn, and opens the paracellular pathway. Prior protein tyrosine kinase (PTK) inhibition protects against TSP-1-induced barrier disruption whereas protein tyrosine phosphatase (PTP) inhibition enhances it. We now present evidence that TSP-1 activates the receptor PTKs, ErbB1 and ErbB2, and opens the endothelial paracellular pathway through this activation. Further, PTP(, a receptor PTP that regulates VE-cadherin tyrosine phosphorylation and the endothelial paracellular pathway, counter-regulates EGFR/ErbB2 activation. We propose the following Specific Aims: 1) To define the specific sequence(s) within TSP-1 required to open the tyrosine phosphorylation - responsive paracellular pathway in human pulmonary microvascular endothelia. 2) To elucidate the mechanism(s) through which TSP-1 activates EGFR/erbB2, including a) direct binding of its EGF-like repeats to the ectodomain of EGFR, b) stimulation of EC release of an EGFR ligand, and/or c) activation of another TSP-1 receptor(s) that transactivates EGFR/erbB2. 3) To determine the mechanism(s) through which TSP-1 activation of EGFR/erbB2 is coupled to increased tyrosine phosphorylation of ZA and/or other junctional proteins, ZA reorganization, and/or opening of the endothelial paracellular pathway. 4) To define the counter-regulatory role for PTP( in TSP-1-induced EGFR/ErbB2 activation. Understanding the mechanism(s) through which TSP-1 regulates EC-EC homophilic adhesion and the paracellular pathway has implications for angiogenesis within the context of wound healing, tissue remodeling, and tumor cell survival, as well as the movement of cells, macromolecules, and fluids from the bloodstream into extravascular tissues.