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: 7407780
• 负责人: Joe G. N. Garcia
• 金额: $ 40.95万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7407780
• 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; Closure; Code; Complex; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Proteins; Cytoskeleton; Development; Disruption; 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; Localized; 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; 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的研究已经证明，编码多功能细胞骨架蛋白肌球蛋白的基因