AMPK activates calcium signaling necessary for endothelial barrier repair

AMPK 激活内皮屏障修复所需的钙信号传导

基本信息

批准号：
8236864
负责人：
Judy Creighton
金额：
$ 29.01万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8236864
关键词：
2&apos -adenylic acid Adenosine Monophosphate Adherens Junction Adhesions Adhesives Adverse effects Alveolar Asthma Atherosclerosis Attenuated Binding Blood Vessels Blood capillaries Cadherins Calcium Calcium Signaling Capillary Permeability Catalytic Domain Cell membrane Cell-Cell Adhesion Cells Chronic Obstructive Airway Disease Complex Cytoskeleton Data Development Edema Endoplasmic Reticulum Endothelial Cells Endothelium Event Feedback Functional disorder G-Protein-Coupled Receptors Health Human Inflammation Inflammatory Injury Intercellular Junctions Location Lung Mediating Mediator of activation protein Membrane Metabolic Metabolic stress Molecular N-Cadherin Pathogenesis Pathology Pathway interactions Permeability Phosphotransferases Process Proteins Pulmonary Circulation Research Role Signal Transduction Stimulus Structural Protein System Techniques Testing Thapsigargin Time Vascular Diseases Vascular Endothelium Vascular Permeabilities body system capillary diabetic in vivo insight mimetics novel protein protein interaction repaired response selective expression sensor small hairpin RNA wound

项目摘要

Project Summary: Endothelial repair subsequent to inflammation-induced vascular damage is a poorly understood process. In this application, we propose a novel role for adenosine monophosphate kinase (AMPK) in promoting cadherin adhesion assembly critical for endothelial repair. We hypothesize that AMPK¿1 and N-cadherin function in tandem as a rapid response mechanism allowing pulmonary microvascular endothelial cells (PMVECs) to repair barrier disruptions quickly and limit increased capillary permeability. AMPK is most frequently described as a metabolic sensor that maintains ATP levels during periods of metabolic stress. However, recent studies indicate that AMPK also acts as a feedback mechanism that counters the destabilizing effects of elevated [Ca2+]i by promoting protein-protein interactions that strengthen cell-cell junctions. Thus, in this parallel signaling manner, an initially barrier disruptive Ca2+ signal can secondarily activate protective mechanisms. This application expands on this idea of time and location dependent Ca2+ signaling to suggest a novel role for AMPK in Ca2+ dependent cadherin adhesion assembly and barrier repair. Our preliminary results indicate that membrane associated AMPK¿1 subsequent to activation by inflammation-induced Ca2+ entry, activates a discrete Ca2+ entry mechanism. This Ca2+ pathway, in contrast to inflammatory Ca2+ signaling, reorganizes the cytoskeleton crucial for N-cadherin adherens' junction assembly and endothelial cell-cell adhesion. Specifically, our preliminary data indicate capillary-derived PMVECs selectively express the AMPK¿1 catalytic subunit, and pro-inflammatory challenges increase its expression in the alveolar/capillary segment in-vivo. We utilized shRNA techniques to inhibit AMPK¿1 activity and observed an attenuated increase in Ca2+ induced by the inflammatory mimetic thapsigargin suggesting AMPK activated a discrete Ca2+ pathway. Moreover, AMPK inhibition blocked wound resealing in PMVECs. AMPK ¿1 co-immunoprecipitates with the adherens junctional protein N-cadherin and co-localizes with N-cadherin to regions of cell-cell contact in PMVECs suggesting AMPK¿1 and N-cadherin function in concert to regulate cytoskeletal mechanisms necessary for establishing lung capillary endothelial integrity. This proposal tests the overall HYPOTHESIS that AMPK ¿1 forms a functional membrane complex with N-cadherin essential for pulmonary endothelial barrier repair. Specific Aims test the related hypotheses that: [1] AMPK¿1 at the membrane forms a functional complex with N-cadherin necessary for establishing cell-cell adhesion in PMVECs.[2] AMPK¿1 activates a discrete Ca2+ entry mechanism that promotes N-cadherin adhesion in PMVECs. [3] Inflammatory stimuli, through Ca2+ signaling, initiate AMPK¿1 mediated PMVEC repair.

项目概要：炎症引起的血管损伤后的内皮修复过程尚不清楚。在本申请中，我们提出了单磷酸腺苷激酶 (AMPK) 在促进钙粘蛋白的形成方面的新作用粘附组装对于内皮修复至关重要。 1和N-钙粘蛋白的功能串联作为一种快速反应机制，允许肺微血管内皮细胞（PMVEC）最常描述的是快速修复屏障破坏并限制毛细血管通透性增加。作为一种代谢传感器，可在代谢应激期间维持 ATP 水平。表明 AMPK 还充当反馈机制，对抗升高的不稳定影响 [Ca2+]i 通过促进蛋白质-蛋白质相互作用来加强细胞-细胞连接。信号方式，最初的屏障破坏性 Ca2+ 信号可以二次激活保护机制。该应用扩展了时间和位置依赖性 Ca2+ 信号传导的想法，为 AMPK 在 Ca2+ 依赖性钙粘蛋白粘附组装和屏障修复中的作用我们的初步结果表明：膜相关AMPK¿ 1 在炎症诱导的 Ca2+ 进入激活后，激活与炎症性 Ca2+ 信号相反，这种 Ca2+ 进入机制会重组细胞。具体来说，细胞骨架对于 N-钙粘蛋白粘附连接组装和内皮细胞-细胞粘附至关重要。我们的初步数据表明毛细血管源性 PMVEC 选择性表达 AMPK¿ 1个催化亚基，和促炎性挑战增加其在体内肺泡/毛细血管段中的表达。抑制 AMPK 的 shRNA 技术¿ 1 活性并观察到 Ca2+ 诱导的减弱增加炎症模拟毒胡萝卜素表明 AMPK 激活了一条离散的 Ca2+ 途径。 AMPK ?? 1 与粘附连接点共免疫沉淀 N-钙粘蛋白蛋白并与 N-钙粘蛋白共定位于 PMVEC 中的细胞-细胞接触区域，表明 AMPK?? 1 和 N-钙粘蛋白协同发挥作用，调节建立必要的细胞骨架机制该提案测试了 AMPK 的整体假设。 1 形成函数式与 N-钙粘蛋白的膜复合物对于肺内皮屏障修复至关重要。相关假设：[1] AMPK¿ 1 在膜上与 N-钙粘蛋白形成必需的功能复合物用于在 PMVEC 中建立细胞间粘附。[2] 1 激活离散的 Ca2+ 进入机制促进 PMVEC 中的 N-钙粘蛋白粘附 [3] 炎症刺激通过 Ca2+ 信号传导启动 AMPK¿ 1 介导 PMVEC 修复。