cAMP Control of Endothelial Barrier and T Cell Migration

炎症反应的特点是白细胞迅速募集和血管渗漏。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，RaplGTase及其钙和二酰甘油反应性环境基金CalDAG-GEF1促进趋化因子诱导的LFA-1-整合素介导的黏附，而cAMP诱导的PKA激活则下调这种黏附 进程。在人内皮细胞中，cAMP诱导的gef EPad的激活增强了屏障特性： EPad激活Rap GTP酶导致皮质肌动蛋白增强，而ePad适配器功能促进微管(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信号分配到不同的亚细胞位置，控制其不同的 细胞内功能。我们的假设是，一种与AKAP相关的特定微管AKAP9选择性地传递cAMP信号，调节内皮细胞屏障功能和白细胞CD18-INTERQRIN介导的黏附。我们的初步数据显示，AKAP9与EPAD共同定位于人内皮细胞的高尔基体和中心体。它的沉默选择性地取消了EPAD诱导的屏障增强 属性。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。在一份已发表的报告中，AKAP9在中心体的非局部化干扰了LFA-1介导的T淋巴瘤细胞系的极化。在这里，我们发现在原代人类T细胞中，AKAP9与LFA-1共定位于SDF-1a刺激的细胞在ICAM-1上迁移。此外，一种破坏PKA相互作用的多肽 AKAP可增强SDF诱导、LFA-1整合素介导的细胞黏附。提出的特定目标将定义AKAP9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细胞迁移的作用。在具体的目标1中，研究将检查 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，AKAP9在EPAD介导的通透性降低中的作用的分子机制。 我们还将探索EPAC-AKAP9-微管生长和EPAC-RapGTP酶-皮质肌动蛋白通路如何结合来调节内皮屏障属性。在特定的AIM 2中，我们将研究AKAP9在趋化因子诱导的人T细胞LFA-1整合素功能中的作用。为此，详细分析了AKAP9在SDF刺激的贴壁细胞中相对于MTS、LFA-1和cAMP梯度的时空分布 将对T细胞进行研究。该方法将包括活细胞、FRET和共聚焦显微镜分析。AKAP9沉默，或AKAP锚定的PKA在T细胞整合素功能、GTP酶激活、细胞骨架重组和cAMP生成中的作用将通过成像和生化方法结合 体外黏附实验。在特定的AIM 3中，将使用条件基因打靶方法来确定AKAP9在血管通透性和T细胞在血管通透性和T细胞募集中的作用，分别在真皮血管通透性模型(Miles实验)和活体显微镜下观察到。AKAP9的病理生理学相关性将通过检测其对T细胞迁移和 接触性超敏反应模型和实验性自身免疫性脑脊髓炎的渗透性。我们预计，这些目标的完成将提供对cAMP依赖的机制的更多了解，这些机制稳定内皮细胞连接和减少T细胞迁移。这可能为加强这些机制以对抗炎症引起的水肿和炎症中的白细胞渗出提供了理论基础。 疾病。

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.