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

基本信息

批准号：
10471912
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
项目类别：
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/10471912
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 Categories Cell Adhesion Cells Cellular biology Chondrocytes Collaborations Coronary Cytoplasmic Tail Data Distal Embryo Endothelial Cells Enterobacteria phage P1 Cre recombinase Gene Expression Genes Human Integrin beta Chains Integrins Kidney Knock-out Knowledge Ligands Location Macrophage-1 Antigen Malignant Neoplasms Mediating Membrane Mus Mutation Mutation Analysis Patients Pattern Pericytes Permeability Publishing RNA Role Signal Transduction Site Smooth Muscle Myocytes Specificity Structure of beta Cell of islet Structure-Activity Relationship Systemic Lupus Erythematosus Talin Tamoxifen Testing Tissues Tube Vascular Permeabilities angiogenesis animal tissue atherogenesis cell type extracellular human disease in vivo insight kidney biopsy knock-down migration mutant nephrogenesis novel postnatal response therapeutic target transcriptome sequencing wound healing

项目摘要

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.

项目概要 Kindlin-2 (K2) 是三种哺乳动物 Kindlin 中分布最广泛的，存在于成纤维细胞中。软骨细胞和心肌细胞，其整体敲除具有胚胎致死性，如图所示，K2 具有胚胎致死性。在血管、内皮细胞、平滑肌细胞和周细胞三大关键细胞中高表达，然而，我们关于 K2 在这些细胞中的作用的信息。细胞的功能还很初级，我们将寻求填补许多知识空白。 K2 与其在整合素激活中的重要作用有关，但由于有 20 多个已识别的结合配偶体，K2 可能很好地连接到多个信号传导和细胞骨架节点，因此，K2 的功能假设。在目标 1 中，可细分为整合素依赖性和整合素非依赖性。该命题将通过表达野生型或 K2Q614W615/AA (K2QW/AA) 突变体进行测试，其中禁用其与整合素 β 亚基的高亲和力结合，并评估三个中的特定功能反应在目标 2 中，将在体内评估 K2 在 EC、SMC 和 PC 中的作用。使用细胞类型特异性、有条件的 K2KO 来评估血管反应，包括渗透性、血管生成、这些研究应该清楚地阐明 K2 在脉管系统中的功能。在与人类疾病相关的研究中，系统性红斑狼疮肾脏中 K2 的表达模式 (SLE) 患者和冠状血管将在目标 3 中进行结构与功能关系的检查。这些研究将包括研究 K2 介导细胞反应的机制。精确定位两个独立于整合素的功能（K2 中的肌动蛋白和连环蛋白结合位点）。结构分析表明第二个 K2 结合位点存在于膜近端区域（如与整合素 β 细胞质尾部中其充分表征的膜远端位点区分开来。将研究该位点在整合素之间引发 K2 依赖性信号传导的特异性，并且存在 Kindlin-3 同源位点在 αMβ2 激活和信号传导中的功能也将被确定。总的来说，这些研究应该证实 K2 是血管细胞生物学的主要调节因子，并定义了在体内发挥这一作用的机制。