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kindlin中分布最广的一种。它存在于成纤维细胞中， 软骨细胞和心肌细胞，并且其整体敲除是胚胎致死的。如本文所示，K2是 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， 在那里它有助于这些细胞的功能反应。然而，我们关于K2在这些中的作用的信息 细胞是初级的，有许多知识空白，我们将寻求填补。最广为接受的功能是 K2与其在整联蛋白活化中的重要作用有关，但由于有超过20种已鉴定的结合伴侣，K2可能 很好地连接到多个信号和细胞骨架节点。因此，假设K2的功能 可细分为整联蛋白依赖性和整联蛋白非依赖性。在目标1中， 将通过表达野生型或K2 Q614 W 615/AA（K2 QW/AA）突变体来检验这一命题， 使其与整合素β亚基的高亲和力结合丧失，并评估三种细胞中的特异性功能反应。 血管细胞的主要类别。在目的2中，将在体内评价K2在EC、SMC和PC中的作用 使用细胞类型特异性条件性K2 KO。待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在血管系统中的功能。 在与人类疾病相关的研究中，系统性红斑狼疮患者肾脏中的K2表达模式 (SLE)患者和冠状血管，并将进行检查。在目标3中，结构-功能关系 并将研究K2介导细胞反应的机制。这些研究包括努力 精确定位两个整合素独立的功能（肌动蛋白和连环蛋白结合位点K2）。令人惊讶的是，我们 结构分析表明，第二个K2结合位点存在于膜近端区域（如 区别于其充分表征的膜远端位点）。的功能和 将研究该位点在引发跨整联蛋白的K2依赖性信号传导中的特异性，并且将研究该位点的存在。 还将确定Kindlin-3的同源位点在活化和跨αMβ2的信号传导中的功能。 总的来说，这些研究应该确定K2是血管细胞生物学的主要调节因子，并确定K2在血管形成中的作用。 在体内发挥这种作用的机制。