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）：我们实验室的远距离目标是描述内皮细胞中机械转移过程的有意义的要素，以进一步了解血液动力学在动脉粥样硬化中的作用。我们的建议将检验以下特定观念：小窝是形成机械感应信号传导复合物的位点，该机械感应信号传导复合物可用于协调生物力学输入并诱导修改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传导涉及基于小窝的机械感觉信号传导复合物，该机械敏感整合素在转移到小窝时形成。我们将通过使用亚细胞分馏和免疫电子显微镜技术来检验这一假设，以跟踪剪切应力后激活整合素的分布。我们假设整联蛋白向小窝的换位的功能意义是允许整联蛋白与下游信号伴侣的关联。具体而言，整联蛋白与基于小窝的磷酸化 - 卡韦尔蛋白/SRC/CSK复合物相关联，该复合物控制肌动蛋白细胞骨架的RhoA活性和重组，并抑制细胞周期对剪切应力的响应。为了检验该假设，在剪切应力之前，内皮细胞培养物将通过抑制抗体，CSK siRNA和Caveolin-1磷酸肽的整合素预处理。此外，通过小窝蛋白-1和胆固醇隔离化合物的siRNA敲低的特异性减少将用于评估木筏/小窝在整联蛋白介导的机械传导中的作用。体外结果将在小窝-1 NULL小鼠中得到验证。整联蛋白还调节剪切诱导的NO产生。但是，我们对整联蛋白介导的机械转导过程如何引起对流体机械力的特定细胞反应的理解尚不清楚。我们建议信号特异性通过整合素与含有特定信号分子的特定子集的Caveolae结构域赋予信号特异性，这些域在焦点接触和腔内细胞表面之间被隔离。在这里，我们将检查在腔表面和纯化的口腔内的eNOS活性，这些内皮细胞中用整合素阻断抗体预处理的内皮细胞中的活性。我们提出，在剪切应力之后，在腔内皮细胞表面上表达的整联蛋白与SFK/Caveolin/pi3-激酶/Akt复合物相互作用以激活eNOS。公共卫生相关性的血管内斑块的发展会阻碍正常的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，在血管分叉的地方最常发现动脉粥样硬化斑块。这些区域内的血液流动是混乱而湍流的，它改变了排列血管壁（称为内皮细胞）细胞的正常功能。我们研究的远距离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。这项研究基础的基本原理是，一旦对机械转导途径有了清晰的了解，则可以将相关的组成部分用于减轻血管血流动力学敏感区域中的斑块形成。