Regulation of Lung Barrier Function by Endothelial Cell (EC) MLCK

内皮细胞 (EC) MLCK 对肺屏障功能的调节

• 批准号: 8070458
• 负责人: Joe G. N. Garcia
• 金额: $ 41.04万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-10 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8070458
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Acute Lung Injury; Affect; Agonist; Alternative Splicing; Atomic Force Microscopy; Binding; Biological; Biological Assay; Blood Vessels; Candidate Disease Gene; Cells; Chimeric Proteins; Code; Complex; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Cytoskeleton; Development; Edema; Endothelial Cells; Endothelium; Engineering; Enzymes; Event; Exons; Focal Adhesions; Gene Proteins; Genes; Genetic Polymorphism; Genetically Engineered Mouse; Human; In Situ; In Vitro; Inflammation; Inflammatory; Injury; Lead; Length; Locales; Lung; MYLK gene; Maps; Mass Spectrum Analysis; Measures; Mechanics; Mediating; Membrane; Membrane Microdomains; Modeling; Modification; Molecular; Movement; Mus; Muscle; Myosin Light Chain Kinase; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post-Translational Protein Processing; Predisposition; Principal Investigator; Property; Protein Binding; Protein Isoforms; Proteomics; Proto-Oncogene Protein pp60 (c-src); RNA Splicing; Reagent; Recovery; Regulation; Role; Site; Stress Fibers; Stretching; Structure; Testing; Transgenic Mice; Transgenic Organisms; Translational Research; Variant; Vascular Permeabilities; Work; actin kinase; base; cadherin 5; human EMS1 protein; in vivo; insight; lung injury; non-muscle myosin; novel; paxillin; polymerization; programs; promoter; response; restoration; src-Family Kinases; therapeutic target; trafficking; translational study

Project #1 studies have demonstrated that the gene encoding the multi-functional cytoskeletal protein, myosin light chain kinase (MLCK), contains coding polymorphisms which are highly associated with susceptibility to acute lung injury (ALI). The non-muscle isoform, nmMLCK, is a critical cytoskeletal effector which regulates the participation of the EC actin cytoskeleton in vascular barrier disruption, in barrier restoration, in lung inflammatory cell trafficking and in vascular responses to mechanical stretch. Following edemagenic agents, MLCK phosphorylates MLCs on Ser19 and Thr18, producing barrier-disrupting cytoplasmic stress fibers, spatially-localized actomyosin contraction and paracelular gaps. In contrast, EC barrier-protective agonists induce the rapid translocation of MLCK to lamellipodial membrane protrusions (to close paracellular gaps and restore barrier integrity) and to cortical actin networks (to enhance linkage to junctional complexes and increase barrier properties). The mechanism by which the full length nmMLCKI (and its five alternatively spliced variants) is targeted to specific cellular sites is entirely unknown. Furthermore, the influence of ALIassociated nmMLCK coding SNPs (Pro21His, Pro147Ser, Val261Ala) on MLCK structure/function are similarly unknown. We hypothesize that site-specific nmMLCK regulation involves post-translational modifications (PTMs) and results in variant- and SNP-specific MLCK activities. Specific Aim (SA) #1 will conduct studies to characterize nmMLCK (nmMLCKI, nmMLCK splice variants, MLCK-coding SNPs) utilizing kinase and actin polymerization assays, GFP/YFP-MLCK fusion proteins and cytoskeletal binding assays. SA #2 will examine the influence of kinase-mediated PTMs (Src, Abl, ERK and PKA) on site-specific MLCK responses (nmMLCKI, nmMLCK-variants, nmMLCK-SNPs) utilizing mass spectroscopy, phosphopeptide mapping, GFP-MLCK fusion proteins, and binding partner assays. SA #3 will examine MLCK regulation of actin polymeriza-tion and focal adhesion remodeling in EC lamellipodia (critical to paracellular gap closure) using GFP-MLCK- and paxillin fusion proteins coupled to atomic force microscopy. SA #4 will utilize available and novel genetically-engineered mice to further define the in vivo role of nmMLCK splice variants (+/- SNPs) in lung inflammatory injury. These translational studies integrate across our entire PPG and lead to mechanistic insights into EC barrier regulation and the development of novel edema-reducing therapies.

项目#1 研究表明，编码多功能细胞骨架蛋白肌球蛋白的基因 轻链激酶 (MLCK)，包含与易感性高度相关的编码多态性 急性肺损伤（ALI）。非肌肉亚型 nmMLCK 是一种关键的细胞骨架效应子，可调节 EC肌动蛋白细胞骨架参与血管屏障破坏、屏障恢复、肺 炎症细胞运输和血管对机械拉伸的反应。使用消水肿剂后， MLCK 在 Ser19 和 Thr18 上磷酸化 MLC，产生破坏屏障的细胞质应激纤维， 空间局部肌动球蛋白收缩和细胞旁间隙。相比之下，EC 屏障保护激动剂 诱导 MLCK 快速易位至板状足膜突起（以闭合细胞旁间隙） 并恢复屏障完整性）和皮质肌动蛋白网络（以增强与连接复合物和 增加阻隔性能）。全长 nmMLCKI（及其五个可选的 剪接变体）是否针对特定的细胞位点是完全未知的。此外，ALI相关的影响 MLCK 结构/功能上的 nmMLCK 编码 SNP（Pro21His、Pro147Ser、Val261Ala）类似 未知。我们假设位点特异性 nmMLCK 调控涉及翻译后修饰 (PTM) 并导致变体和 SNP 特异性 MLCK 活性。具体目标 (SA) #1 将进行研究 利用激酶表征 nmMLCK（nmMLCKI、nmMLCK 剪接变体、MLCK 编码 SNP） 和肌动蛋白聚合测定、GFP/YFP-MLCK 融合蛋白和细胞骨架结合测定。 SA #2 将检查激酶介导的 PTM（Src、Abl、ERK 和 PKA）对位点特异性 MLCK 的影响 利用质谱、磷酸肽进行响应（nmMLCKI、nmMLCK 变体、nmMLCK-SNP） 作图、GFP-MLCK 融合蛋白和结合伴侣测定。 SA #3 将检查 MLCK 监管 EC板状伪足中的肌动蛋白聚合和粘着斑重塑（对细胞旁间隙闭合至关重要） 使用 GFP-MLCK- 和桩蛋白融合蛋白与原子力显微镜耦合。 SA #4 将利用 可用的新型基因工程小鼠，以进一步确定 nmMLCK 剪接变体的体内作用 （+/- SNP）在肺部炎症损伤中的作用。这些转化研究整合了我们整个 PPG 并引领 对 EC 屏障调节的机制见解和新型减轻水肿疗法的开发。