Project 1- Role of Kindlins in Blood and Vascular Cell Biology

项目 1 - Kindlins 在血液和血管细胞生物学中的作用

• 批准号: 10661631
• 负责人: EDWARD Franklin PLOW
• 金额: $ 56.3万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10661631
• 关键词: Actins; Adaptor Signaling Protein; Adhesions; Affect; Affinity; Angiogenesis Pathway; Animal Model; Animals; Arterial Fatty Streak; Atherosclerosis; Avidity; Binding; Binding Sites; Blood; Blood Vessels; C-terminal; CRISPR/Cas technology; Cardiac Myocytes; Categories; Cell Adhesion; Cells; Cellular biology; Chondrocytes; Classification; Collaborations; Coronary; Cytoplasmic Tail; Cytoskeleton; Data; Distal; Embryo; Endothelial Cells; Enterobacteria phage P1 Cre recombinase; Fibroblasts; Gene Expression; Genes; Human; Integrin beta Chains; Integrins; Kidney; Knock-out; Knowledge; Ligands; Location; Macrophage-1 Antigen; Mediating; Membrane; Mus; Mutation; Mutation Analysis; Patients; Pattern; Pericytes; Permeability; Publishing; Renal function; Role; Signal Transduction; Site; Smooth Muscle Myocytes; Specificity; Structure of beta Cell of islet; Structure-Activity Relationship; Systemic Lupus Erythematosus; Talin; Tamoxifen; Testing; Tube; Vascular Permeabilities; alpha catenin; angiogenesis; animal tissue; atherogenesis; cancer cell; cell fixing; cell type; extracellular; human disease; in vivo; insight; kidney biopsy; knock-down; migration; mutant; nephrogenesis; novel; postnatal; response; small hairpin RNA; therapeutic target; tissue fixing; transcriptome sequencing; wound healing

Project Summary Kindlin-2 (K2) is the most broadly distributed of the three mammalian kindlins. It is present in fibrobasts, chondrocytes, and cardiomocytes, and its global knockout is embryonically lethal. As shown herein, K2 is highly expressed in the three key cells of blood vessels, endothelial cells, smooth muscle cells and pericytes, where it contributes to the functional responses of these cells. Yet, our information on the role of K2 in these cells is rudimentary with many gaps in knowledge that we will seek to fill. The most well-accepted function of K2 relates to its essential role in integrin activation, but with more than 20 identified binding partners, K2 may very well connect to multiple signaling and cytoskeletal nodes. Accordingly, the hypothesis that K2's functions may be subdivided as being integrin-dependent and integrin-independent will be critically assessed. In Aim 1, this proposition will be tested by expressing either wild-type or K2Q614W615/AA (K2QW/AA) mutant, which disables its high affinity binding to integrin β subunits, and assessing specific functional responses in the three major categories of blood vessel cells. In Aim 2, the role of K2 in EC, SMC and PC will be evaluated in vivo using cell-type specific, conditional K2KO. Vascular responses to be assessed are permeability, angiogenesis, atherosclerosis and wound healing. These studies should clearly resolve the functions of K2 in the vasculature. In studies relevant to human disease, K2 expression patterns in kidneys of Systemic Lupus Erythematosus (SLE) patients and in coronary blood vessels and will be examined. In Aim 3, structure-function relationships and mechanisms by which K2 mediates cellular responses will be investigated. These studies include an effort to precisely locate two integrin-independent functions (actin and catenin binding sites in K2). Surprisingly, our structural analyses indicate that a second K2 binding site exists in the membrane proximal region (as distinguished from its well-characterized membrane distal site) in integrin β cytoplasmic tails. The function and specificity of this site in eliciting K2-dependent signaling across integrins will be investigated, and the existence and function of a homologous site for kindlin-3 in activation and signaling across αMβ2 will also be determined. Overall, these studies should establish that K2 is a master regulator of vascular cell biology and define the mechanisms underlying this role in vivo.

项目摘要 Kindlin-2（K2）是三种哺乳动物Kindlins中最广泛的分布。它存在于纤维上， 软骨细胞和心脏细胞及其全球敲除具有胚胎致死。如本文所示，K2是 在血管，内皮细胞，平滑肌细胞和周细胞的三个关键细胞中高度表达， 它有助于这些细胞的功能响应。但是，我们有关K2在这些角色中的作用的信息 细胞是基本的，我们将寻求填补的知识中的许多空白。最受接受的功能 K2与其在整联蛋白激活中的重要作用的关系，但是有20多个确定的约束伙伴，K2可以 很好地连接到多个信号传导和细胞骨架节点。彼此之间，关于K2功能的假设 可能会细分为整联蛋白依赖性，与整合素独立的依赖性将进行严格评估。在AIM 1中， 该命题将通过表达野生型或K2Q614W615/AA（K2QW/AA）突变体来测试，该突变体 禁用其高亲和力与整联蛋白β亚基的结合，并评估这三个响应 血管细胞的主要类别。在AIM 2中，K2在EC，SMC和PC中的作用将在体内评估 使用细胞类型特异性的条件K2KO。要评估的血管反应是渗透性，血管生成， 动脉粥样硬化和伤口愈合。这些研究应清楚地解决K2在脉管系统中的功能。 在与人类疾病相关的研究中，k2的表达模式在全身性红斑狼疮的肾脏中 （SLE）患者和冠状动脉血管，将检查。在AIM 3中，结构功能关系 以及K2介导细胞反应的机制。这些研究包括 为了精确定位两个独立的函数（K2中的肌动蛋白和catenin结合位点）。令人惊讶的是，我们的 结构分析表明，膜代理区域中存在第二个K2结合位点（AS 与整合素β细胞质尾部中的特征良好的膜远端位点区分开。功能和 将研究该站点在启发整个整合素的K2依赖性信号传导中的特异性，并存在 还将确定Kindlin-3在激活和跨αMβ2的信号传导中的同源位点的功能。 总体而言，这些研究应确定K2是血管细胞生物学的主要调节剂，并定义 在体内发挥作用的机制。