Integrin Mechanotransduction and caveolae-based signaling complexes

整合素机械转导和基于小窝的信号复合物

基本信息

批准号：
8290346
负责人：
VICTOR J. RIZZO
金额：
$ 37.13万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-15 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8290346
关键词：
1-Phosphatidylinositol 3-Kinase Actins Address Adenoviruses Area Arterial Fatty Streak Atherosclerosis Attenuated Beryllium Biological Assay Biomechanics Blocking Antibodies Blood Vessels Blood flow Brain Capsid Proteins Caveolae Caveolins Cell Culture Techniques Cell Cycle Inhibition Cell Cycle Regulation Cell Fractionation Cell Proliferation Cell membrane Cell physiology Cell surface Cells Cellular Structures Cholesterol Complex Cytoskeleton Data Detection Development Elements Endothelial Cells Event Family member Focal Adhesion Kinase 1 Focal Adhesions GTPase-Activating Proteins Gene Expression Goals Health Heart Immunofluorescence Immunologic In Situ In Vitro Individual Infection Integrins Knockout Mice Laboratories Lead Ligands Link Liquid substance Location Measures Mechanics Mediating Membrane Membrane Microdomains Methodology Methods Microscopic Mitogen-Activated Protein Kinase 3 Mitogen-Activated Protein Kinases Modeling Morphology Myocardial Infarction Nitric Oxide Organ Pathology Pathway interactions Peptides Phosphorylation Play Process Production Property Proteins Receptor Signaling Regulation Reporting Research Role Second Messenger Systems Signal Transduction Signaling Molecule Silicon Dioxide Site Small Interfering RNA Specificity Stroke Structure Surface Techniques Technology Testing Tyrosine Phosphorylation Western Blotting Work base caveolin 1 hemodynamics human NOS3 protein inhibiting antibody luminal membrane member novel paxillin pressure research study response second messenger shear stress src-Family Kinases

项目摘要

DESCRIPTION (provided by applicant): The long-range goal of our laboratory is to describe meaningful elements of the mechanotransduction process in endothelial cells to further understand the role of hemodynamics in atherosclerosis. Our proposal will test the specific idea that caveolae are sites for the formation of a mechanosensory signaling complex which serves to coordinate biomechanical inputs and induce signaling events that modify endothelial cell structure and function. The working hypothesis is that shear stress-induced mechanotransduction involves a caveolae-based mechanosensory signaling complex that forms when mechanically sensitive integrins translocate to caveolae. We will test this hypothesis by using subcellular fractionation and immunoelectron microscopic techniques to track the distribution of activated integrins following shear stress. We hypothesize that the functional significance of integrin transposition to caveolae is to allow integrins to association with downstream signaling partners. Specifically, integrins associate with a caveolae-based phospho-caveolin/Src/Csk complex which governs RhoA activity and reorganization of the actin cytoskeleton and inhibition of cell cycle in response to shear stress. To test this hypothesis, endothelial cell cultures will be pretreated with integrin inhibiting antibodies, Csk siRNA and caveolin-1 phospho-peptide prior to shear stress. Additionally, specific reduction of caveolae through siRNA knockdown of caveolin-1 and cholesterol sequestering compounds will be used to evaluate the role of rafts/caveolae in integrin-mediated mechanotransduction. In vitro results will be validated in caveolin-1 null mice. Integrins also regulate shear-induced NO production. However, our understanding of how integrin-mediated mechanotransduction processes elicit specific cellular responses to fluid mechanical forces is unclear. We propose that signal specificity is conferred through integrin association with caveolae domains containing specific subsets of signaling molecules which are segregated between focal contacts and the luminal cell surface. Here, we will examine eNOS activity at the luminal surface and within purified caveolae in endothelial cells pretreated with integrin blocking antibodies. We propose that integrins expressed on the luminal endothelial cell surface translocate to caveolae following shear stress where they interact with a SFK/caveolin/PI3-Kinase/Akt complex to activate eNOS. PUBLIC HEALTH RELEVANCE The development of plaques within a blood vessel can impede normal blood flow to organs such as the heart and brain and lead to heart attack or stroke, respectively. Interestingly, atherosclerotic plaques are most often found where blood vessels bifurcate. The flow of blood within these regions is chaotic and turbulent, which alters the normal functioning of the cells which line the blood vessel wall, called endothelial cells. The long-range goal of our research is to understand the role of hemodynamics in atherosclerosis and describe meaningful elements of the mechanotransduction process in endothelial cells. The rationale that underlies this research is that, once a clear understanding of the mechanotransduction pathways is achieved, relevant components may be targeted to attenuate plaque formation in hemodynamic sensitive areas of the vasculature.

描述（由申请人提供）：我们实验室的长期目标是描述内皮细胞中机械转导过程的有意义的元素，以进一步了解血流动力学在动脉粥样硬化中的作用。我们的提案将测试这一具体想法，即小穴是形成机械感觉信号复合体的部位，该复合体用于协调生物力学输入并诱导改变内皮细胞结构和功能的信号事件。工作假设是，剪切应力诱导的机械转导涉及基于小凹的机械感觉信号复合物，当机械敏感的整合素易位到小凹时形成。我们将通过使用亚细胞分级分离和免疫电子显微镜技术来跟踪剪切应力后激活的整合素的分布来检验这一假设。我们假设整合素转座到小凹的功能意义是允许整合素与下游信号传导伙伴结合。具体来说，整合素与基于小窝的磷酸小窝蛋白/Src/Csk 复合物结合，该复合物控制 RhoA 活性和肌动蛋白细胞骨架的重组以及响应剪切应力的细胞周期抑制。为了检验这一假设，在施加剪切应力之前，将用整合素抑制抗体、Csk siRNA 和 Caveolin-1 磷酸肽对内皮细胞培养物进行预处理。此外，通过siRNA敲低caveolin-1和胆固醇隔离化合物来特异性减少小窝将用于评估筏/小窝在整合素介导的机械转导中的作用。体外结果将在 Caveolin-1 缺失小鼠中得到验证。整合素还调节剪切诱导的 NO 产生。然而，我们对整合素介导的机械转导过程如何引发对流体机械力的特定细胞反应的理解尚不清楚。我们提出信号特异性是通过整合素与含有特定信号分子子集的小凹结构域关联而赋予的，这些信号分子在焦点接触和管腔细胞表面之间分离。在这里，我们将检查用整合素阻断抗体预处理的内皮细胞的管腔表面和纯化的小凹内的 eNOS 活性。我们提出，在管腔内皮细胞表面表达的整合素在剪切应力后易位到小凹，在那里它们与 SFK/小凹蛋白/PI3-激酶/Akt 复合物相互作用以激活 eNOS。公众健康相关性血管内斑块的形成会阻碍正常血液流向心脏和大脑等器官，并分别导致心脏病发作或中风。有趣的是，动脉粥样硬化斑块最常见于血管分叉处。这些区域内的血液流动是混乱和湍流的，这改变了排列在血管壁上的细胞（称为内皮细胞）的正常功能。我们研究的长期目标是了解血流动力学在动脉粥样硬化中的作用，并描述内皮细胞中机械转导过程的有意义的元素。这项研究的基本原理是，一旦对机械传导途径有了清晰的了解，就可以针对相关成分来减少脉管系统血流动力学敏感区域的斑块形成。