cAMP Control of Endothelial Barrier and T Cell Migration

cAMP 控制内皮屏障和 T 细胞迁移

• 批准号: 7753048
• 负责人: Tanya N Mayadas
• 金额: $ 46.08万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7753048
• 关键词: 1,2-diacylglycerol; A kinase anchoring protein; AKAP9 gene; Actins; Address; Adhesions; Adhesives; Binding; Biochemical; Biological Assay; Blood Vessels; CD47 gene; Calcium; Cell Adhesion; Cell Line; Cells; Centrosome; Collaborations; Complex; Contact hypersensitivity; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Modeling; Cytoskeleton; Data; Dermal; Diglycerides; Disease; Down-Regulation; Edema; Encephalomyelitis; Endothelial Cells; Endothelium; Event; Experimental Autoimmune Encephalomyelitis; Extravasation; Fluorescence Resonance Energy Transfer; Functional disorder; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Gene Targeting; Generations; Golgi Apparatus; Golgi Targeting; Growth; Guanine; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Homeostasis; Human; Image; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Integrin-mediated Cell Adhesion Pathway; Integrins; Intercellular adhesion molecule 1; Isoproterenol; Leukocytes; Life; Link; Lymphoma; Mediating; Microscopic; Microtubule-Organizing Center; Microtubules; Modeling; Molecular; Mus; Ovalbumin; Pathway interactions; Peptides; Permeability; Physiological; Process; Property; Proteomics; Publishing; Regulation; Relative (related person); Reporting; Resistance; Role; Scaffolding Protein; Signal Transduction; Site; Stromal Cell-Derived Factor 1; Structure; T cell regulation; T-Lymphocyte; Testing; Vascular Permeabilities; adhesion receptor; beta-2 Adrenergic Receptors; cadherin 5; cell motility; chemokine; in vivo; intravital microscopy; migration; phosphoric diester hydrolase; receptor; scaffold; spatiotemporal; sphingosine 1-phosphate; trafficking

Hallmarks of the inflammatory response are the rapid recruitment of leukocytes and vascular leakage. We defined a role for the Rap GTPases and its guanine exchange factors (GEFs) in both these processes. In human T cells, RaplGTase and its calcium and diacylglycerol responsive GEF CalDAG-GEF1 promoted chemokine induced LFA-1-integrin mediated adhesion, while cAMP induced PKA activation downregulated this process. In human endothelial cells, activation of the cAMP inducible GEF Epad enhanced barrier properties: Epad activation of Rap GTPases led to enhanced cortical actin, while Epad adaptor functions promoted microtubule (MT) growth. There is emerging evidence that compartmentalization of cAMP signaling to distinct subcellular sites by the multivalent scaffold proteins, A-kinase anchoring proteins (AKAPs), controls its diverse intracellular functions. Our hvpothesis is that a specific microtubule associated AKAP, AKAP9 selectively relays cAMP signals that regulate endothelial cell barrier function and leukocyte CD18-inteqrin mediated adhesion. Our preliminary data show that AKAP9 co-localizes with Epad at the Golgi and the centrosome of human endothelial cells. Its silencing selectively abrogates the Epad induced enhancement of barrier properties. This was associated with reduced MT growth but intact Rap activation, cortical actin and junctional VE-cadherin. In a published report, delocalization of AKAP9 at the centrosome interfered with LFA-1 mediated polarization of a T lymphoma cell line. Here we show that in primary human T cells, AKAP9 colocalized with LFA-1 in SDF-1a stimulated cells migrating on ICAM-1. Furthermore, a peptide that disrupts PKA interaction with AKAPs enhanced SDF-induced, LFA-1 integrin-mediated cell adhesion. The proposed specific aims will define a molecular framework for AKAP9 function in endothelial cells and T cells and delineate its role in regulating vascular permeability and T cell migration in vivo. In SPECIFIC AIM 1, studies will examine the molecular mechanisms underlying the role of AKAP9 in Epad-mediated reduction in permeability by exploiting AKAP9 silencing, structure-function analysis and proteomic approaches in primary human endothelial cells. We will also explore how Epac-AKAP9-microtubule growth and the Epac-RapGTPase-cortical actin pathway integrate to modulate endothelial barrier properties. In SPECIFIC AIM 2, we will examine the contribution of AKAP9 to chemokine induced LFA-1 integrin function in human T cells. For this, a detailed analysis of the spatiotemporal distribution of AKAP9, relative to MTs, LFA-1 and cAMP gradients in SDF stimulated adherent T cells will be investigated. The approach will include live cell, FRET and confocal microscopic analyses. The effect of AKAP9 silencing, or AKAP anchored PKA in T cell integrin function, GTPase activation, cytoskeletal reorganization and cAMP generation will be assessed using imaging and biochemical approaches coupled with in vitro adhesion assays. In SPECIFIC AIM 3, conditional gene targeting approaches will be used to define the function of AKAP9 in the endothelium and T cells in vascular permeability and T cell recruitment observed in a dermal model of vascular permeability (Miles assay) and intravital microscopy respectively. The pathophysiological relevance of AKAP9 will be assessed by examining its contribution to T cell migration and permeability in a model of contact hypersensitivity and in experimental autoimmune encephalomyelitis. We anticipate that completion of these aims will provide a greater understanding of cAMP dependent mechanisms that stabilize endothelial junctions and reduce T cell migration. This could provide a rationale for enhancing these mechanisms to counteract inflammation induced edema and leukocyte diapedesis in inflammatory diseases.

炎症反应的标志是白细胞的快速募集和血管渗漏。我们定义了Rap GTP酶及其鸟嘌呤交换因子（GEF）在这两个过程中的作用。在人T细胞中，RaplGT酶及其钙和二酰甘油响应性GEF CalDAG-GEF 1促进趋化因子诱导的LFA-1-整联蛋白介导的粘附，而cAMP诱导的PKA活化下调这种粘附。 过程在人内皮细胞中，cAMP诱导型GEF Epad的激活增强了屏障特性： Rap GTP酶的Epad激活导致皮层肌动蛋白增强，而Epad适配器功能促进微管（MT）生长。有新的证据表明，cAMP信号传导到不同的亚细胞位点的多价支架蛋白，A-激酶锚定蛋白（AKAP），控制其多样性。 细胞内功能我们的假设是，AKAP，AKAP 9选择性地传递cAMP信号，调节内皮细胞屏障功能和白细胞CD 18-inteqrin介导的粘附。我们的初步数据表明，AKAP 9与Epad共定位于高尔基体和人内皮细胞的中心体。其沉默选择性地消除Epad诱导的屏障增强， 特性.这与MT生长减少但Rap激活、皮质肌动蛋白和连接VE-钙粘蛋白完整相关。在已发表的报告中，AKAP 9在中心体的离域干扰了LFA-1介导的T淋巴瘤细胞系的极化。在这里，我们表明，在原代人类T细胞，AKAP 9与LFA-1共定位在SDF-1a刺激细胞迁移ICAM-1。此外，破坏PKA相互作用的肽 AKAP增强SDF诱导的LFA-1整合素介导的细胞粘附。提出的具体目标将定义AKAP 9在内皮细胞和T细胞中功能的分子框架，并描述其在体内调节血管通透性和T细胞迁移中的作用。在具体目标1中，研究将检查 在原代人内皮细胞中，通过利用AKAP 9沉默、结构-功能分析和蛋白质组学方法，研究AKAP 9在Epad介导的渗透性降低中的潜在作用的分子机制。 我们还将探讨Epac-AKAP 9-微管生长和Epac-RapGT-皮质肌动蛋白通路如何整合以调节内皮屏障特性。在特异性目的2中，我们将检查AKAP 9对趋化因子诱导的人T细胞中LFA-1整联蛋白功能的贡献。为此，详细分析了SDF刺激的粘附细胞中AKAP 9相对于MT、LFA-1和cAMP梯度的时空分布。 将研究T细胞。该方法将包括活细胞，FRET和共聚焦显微镜分析。AKAP 9沉默或AKAP锚定的PKA在T细胞整联蛋白功能、GT3活化、细胞骨架重组和cAMP产生中的作用将使用成像和生物化学方法结合免疫组织化学来评估。 体外粘附测定。在SPECIFIC AIM 3中，将使用条件性基因靶向方法来定义AKAP 9在内皮和T细胞中的功能，分别在血管渗透性的皮肤模型（Miles测定）和活体显微镜中观察到血管渗透性和T细胞募集。AKAP 9的病理生理学相关性将通过检查其对T细胞迁移的贡献来评估， 在接触性超敏反应模型和实验性自身免疫性脑脊髓炎中的渗透性。我们预计，这些目标的完成将提供一个更好的了解cAMP依赖的机制，稳定内皮连接和减少T细胞迁移。这可以为增强这些机制以对抗炎症诱导的水肿和炎性细胞中的白细胞渗出提供理论基础。 疾病