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: 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
• 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

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)是三种哺乳动物亲缘关系中分布最广的一种。它存在于成纤维细胞中， 软骨细胞和心肌细胞，它的全局敲除对胚胎是致命的。如图所示，K2为 在血管内皮细胞、平滑肌细胞和周细胞三个关键细胞中高度表达， 它对这些细胞的功能反应有贡献。然而，我们关于K2在这些事件中的作用的信息 细胞是初级的，我们将寻求填补知识中的许多空白。最广为接受的功能是 K2与其在整合素激活中的重要作用有关，但有超过20个已确定的结合伙伴，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‘S起作用 可能细分为整合素依赖和非整合素依赖将被严格评估。在目标1中， 这一命题将通过表达野生型或K2Q614W615/AA(K2QW/AA)突变体来检验。 禁用其与整合素β亚基的高亲和力结合，并评估三个亚基中的特定功能反应 血管细胞的主要类别。在目标2中，将在体内评估K2在EC、SMC和PC中的作用 使用特定的细胞类型，有条件的K2KO。需要评估的血管反应包括通透性、血管生成、 动脉粥样硬化和伤口愈合。这些研究应该清楚地解决K2在血管系统中的功能。 在与人类疾病相关的研究中，K2在系统性红斑狼疮肾脏中的表达模式 对系统性红斑狼疮(SLE)患者和冠状动脉血管进行检查。在目标3中，结构-功能关系 K2介导细胞反应的机制将被研究。这些研究包括一项努力 精确定位两个不依赖于整合素的功能(K2中的肌动蛋白和连接素结合位点)。令人惊讶的是，我们的 结构分析表明，在膜近端区域(AS)存在第二个K2结合部位 与其特征良好的膜远端部位不同)，在整合素β胞浆尾部。功能和 将研究该位点在跨整合素诱导依赖于K2的信号传递中的特异性，并且存在 此外，还将确定Kindlin-3的同源位点在αMβ2的激活和信号传递中的功能。 总体而言，这些研究应该确定K2是血管细胞生物学的主要调节者，并定义 体内这种作用的潜在机制。