cAMP Control of Endothelial Barrier and T Cell Migration

cAMP 控制内皮屏障和 T 细胞迁移

基本信息

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
项目类别：
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

项目摘要

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 GTPases 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，RaplGTase 及其钙和二酰甘油反应性 GEF CalDAG-GEF1 促进趋化因子诱导的 LFA-1-整合素介导的粘附，而 cAMP 诱导的 PKA 激活则下调这种粘附过程。在人内皮细胞中，cAMP 诱导型 GEF Epad 的激活增强了屏障特性： Rap GTPases 的 Epad 激活导致皮质肌动蛋白增强，而 Epad 接头功能促进微管 (MT) 生长。新出现的证据表明，多价支架蛋白 A 激酶锚定蛋白 (AKAP) 将 cAMP 信号传导至不同的亚细胞位点，控制着其多样性。细胞内功能。我们的假设是，与 AKAP、AKAP9 相关的特定微管选择性地传递 cAMP 信号，调节内皮细胞屏障功能和白细胞 CD18-整合蛋白介导的粘附。我们的初步数据表明 AKAP9 与 Epad 共定位于高尔基体和人内皮细胞的中心体。它的沉默选择性地消除了 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-钙粘蛋白完整有关。在一份发表的报告中，AKAP9 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 细胞中，AKAP9 与 SDF-1a 中的 LFA-1 共定位，刺激细胞在 ICAM-1 上迁移。此外，一种破坏 PKA 相互作用的肽 AKAP 增强了 SDF 诱导、LFA-1 整合素介导的细胞粘附。所提出的具体目标将定义内皮细胞和 T 细胞中 AKAP9 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，研究将检验通过在原代人内皮细胞中利用 AKAP9 沉默、结构功能分析和蛋白质组学方法，研究 AKAP9 在 Epad 介导的通透性降低中作用的分子机制。我们还将探讨 Epac-AKAP9-微管生长和 Epac-RapGTPase-皮质肌动蛋白通路如何整合来调节内皮屏障特性。在 SPECIFIC AIM 2 中，我们将研究 AKAP9 对人类 T 细胞中趋化因子诱导的 LFA-1 整合素功能的贡献。为此，详细分析了 AKAP9 相对于 SDF 中的 MT、LFA-1 和 cAMP 梯度的时空分布刺激粘附 T细胞将被研究。该方法将包括活细胞、FRET 和共聚焦显微镜分析。将使用成像和生化方法结合体外粘附测定。在 SPECIFIC AIM 3 中，将使用条件基因靶向方法来定义 AKAP9 在内皮和 T 细胞中的功能，分别在血管通透性真皮模型（Miles 测定）和活体显微镜中观察到血管通透性和 T 细胞募集。 AKAP9 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使人们更好地了解稳定内皮连接和减少 T 细胞迁移的 cAMP 依赖性机制。这可以为增强这些机制以对抗炎症引起的水肿和白细胞渗出提供理论依据。疾病。