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

描述(申请人提供)：凝血酶原蛋白(TSP)-1是一种~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，引起多种EC反应。粘着带(ZA)是一种特殊的结构，它通过连接素将肌动蛋白细胞骨架连接到血管内皮细胞(VE)的细胞质结构域-钙粘附素，钙粘附素是一种表面受体，介导同嗜性EC-EC黏附。在人肺微血管内皮细胞中，我们已经证明TSP-1增加了ZA蛋白、VE-钙粘蛋白、y-连环蛋白和p120ctn的酪氨酸磷酸化，并打开了细胞旁通路。先前的蛋白酪氨酸激酶(PTK)抑制对TSP-1诱导的屏障破坏具有保护作用，而蛋白酪氨酸磷酸酶(PTP)抑制则增强了TSP-1诱导的屏障破坏。我们现在提出的证据表明，TSP-1激活了受体PTKs、ErbB1和ErbB2，并通过这种激活打开了内皮细胞旁通路。此外，PTP(一种调节VE-钙粘蛋白酪氨酸磷酸化和内皮细胞旁细胞途径的受体PTP)可以反向调节EGFR/ErbB2的激活。我们提出的具体目标如下：1)确定TSP-1中开启人肺微血管内皮细胞酪氨酸磷酸化反应性细胞旁通路所需的特异性序列(S)。2)阐明TSP-1激活EGFR/ErbB_2的机制(S)，包括a)将其EGF样重复序列与EGFR胞外区直接结合，b)刺激EC释放一种EGFR配体，和/或c)激活另一种TSP-1受体(S)，反式激活EGFR/erb B_2。3)探讨TSP-1活化EGFR/erb B2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的机制(S)。4)明确PTP在TSP-1诱导的EGFR/ErbB2激活中的反调节作用。了解TSP-1调节EC-EC亲和性黏附和细胞旁途径的机制(S)对于伤口愈合、组织重塑和肿瘤细胞存活以及细胞、大分子和液体从血流到血管外组织的运动中的血管生成具有重要意义。

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.