Regulation of lung pathobiology by endothelial cell MLCK

内皮细胞 MLCK 对肺病理学的调节

• 批准号: 7347543
• 负责人: Joe G. N. Garcia
• 金额: $ 44.62万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7347543
• 关键词: ARHGEF5 gene; Achievement; Actins; Actomyosin; Acute Lung Injury; Agonist; Apoptosis; Apoptotic; Attenuated; Baculoviruses; Binding; Binding Sites; Biological Assay; Blood Vessels; Calmodulin; Caspase; Cell physiology; Cells; Cessation of life; Cloning; Complex; Condition; Cytoskeleton; DNA Sequence Rearrangement; Data; Disruption; Endothelial Cells; Endothelium; Exhibits; Fiber; Funding; Generations; Human; Hybrids; Hypoxia; Inflammatory; Laboratories; Localized; Lung; Mechanical ventilation; Mechanics; Mediating; Migration Inhibitory Factor; Molecular Chaperones; Molecular Weight; Motor Activity; Movement; Myosin ATPase; Myosin Light Chain Kinase; Nucleotides; Numbers; Object Attachment; Participant; Phosphorylation; Physiological; Play; Principal Investigator; Process; Protein Isoforms; Proto-Oncogene Protein pp60 (c-src); RNA Splicing; Regulation; Role; Site; Site-Directed Mutagenesis; Smooth Muscle; Stimulus; Stress Fibers; Stretching; Structure; Structure of parenchyma of lung; Syndrome; Tissues; Tyrosine; Tyrosine Phosphorylation; Up-Regulation; Variant; Vascular Endothelium; Yeasts; caspase-3; clinically relevant; cytokine; fluid flow; human EMS1 protein; human ESD protein; human tissue; in vivo; link protein; mRNA Precursor; migration; novel; novel therapeutics; phenylpyruvate tautomerase; polymerization; programs; response; shear stress; therapeutic target; trafficking; vasoconstriction

Pulmonary endothelial cells (EC) function as a complex tissue with the ability to respond to external stimuli by regulating fluid flow, trafficking inflammatory cells into the lung parenchyma or airways, creating new blood vessels, and undergoing apoptotic death. EC are continuously exposed to shear stress and cyclic stretch, mechanical forces dramatically enhanced in the presence of acute lung injury and mechanical ventilation. The Project Leader has shown each of these key EC processes to involve the critical participation of the myosin light chain kinase (MLCK)-driven actin cytoskeleton. MLCK phosphorylates MLCs on Ser19 and Thr18 producing spatially-localized tension through actomyosin contraction. Studies from the Garcia laboratory first revealed that EC express a unique high molecular weight MLCK isoform. In our initial PPG (1998-2003), we cloned EC MLCK to understand its structure and function. We identified four alternatively splice variants, which yield dramatic splice-specific cellular function. For example, EC MLCK1 but not EC MLCK2, is a substrate for p60src and when Tyr phosphorylated, exhibits unique upregulation of enzymatic activity. Furthermore, Tyr phosphorylation of EC MLCK1 enhances its stable association with the actin-linking protein and p60src substrate, cortactin. We have shown cortactin to localize with EC MLCK in cortical structures such as lamellipodia, and to participate in EC cytoskeletal rearrangement. Additional strategies (yeast 2 hybrid assay) yielded the clinically-relevant cytokine macrophage migration inhibitory factor as an EC MLCK binding partner, providing for potentially exciting pathophysiologic regulation, particularly in the context of acute lung injury. Finally, we have shown EC MLCK to be a critical participant in TNF-induced EC apoptosis with compelling data suggesting EC MLCK as a target for caspase cleavage. To investigate these key MLCK interactions, SA #1 will explore p60src-EC MLCK interaction in cytoskeletal rearrangements in human lung endothelium subjected to biophysical mechanical forces. SA #2 will characterize cortactin-EC MLCK interaction in lung endothelial cytoskeletal rearrangement. SA #3 will define EC MLCK-macrophage migration inhibitory factor (MIF) interaction and its role in human lung endothelial pathobiology. SA #4 will define the role of EC MLCK in both the initiation and execution of lung EC apoptosis.

肺内皮细胞（EC）充当复杂的组织，能够通过调节液体流动，将炎症细胞运输到肺实质或气道，产生新的血管以及凋亡死亡，对外部刺激做出反应。在存在急性肺损伤和机械通气的情况下，EC不断暴露于剪切应力和循环拉伸，机械力显着增强。项目负责人已经显示了这些关键EC过程中的每一个，以涉及肌球蛋白轻链激酶（MLCK）驱动的肌动蛋白细胞骨架的关键参与。 MLCK通过肌动球蛋白收缩在Ser19和Thr18上磷酸化MLC，从而产生空间定位的张力。 Garcia实验室的研究首先表明EC表达了独特的高分子量MLCK同工型。 在我们的 初始PPG（1998-2003），我们克隆了EC MLCK以了解其结构和功能。我们确定了四个替代剪接变体，它们产生了戏剧性的剪接特异性细胞功能。例如，EC MLCK1而不是EC MLCK2是P60SRC的底物，当Tyr磷酸化时，表现出酶活性的独特上调。此外，EC MLCK1的Tyr磷酸化增强了其与肌动蛋白 - 链接蛋白和p60SRC底物Cortactin的稳定关联。 我们已经显示了cortactin以与EC MLCK本地化 皮质结构，例如层状脂蛋白，并参与EC细胞骨架重排。其他策略（酵母2混合分析）产生了与临床上与临床相关的细胞因子巨噬细胞迁移抑制因子作为EC MLCK结合伴侣，从而提供了潜在的令人兴奋的病理生理调节调节，尤其是在急性肺损伤的情况下。最后，我们已经显示EC MLCK是TNF诱导的EC凋亡的关键参与者，并具有令人信服的数据，这表明EC MLCK是caspase裂解的靶标。为了研究这些关键的MLCK相互作用，SA＃1将探索在受到生物物理机械力的人肺内皮中细胞骨架重排的P60SRC-EC MLCK相互作用。 SA＃2将表征肺内皮细胞骨架重排中Cortactin-EC MLCK相互作用。 SA＃3将定义 EC MLCK巨噬细胞迁移抑制因子（MIF）相互作用及其在人肺内皮病理生物学中的作用。 SA＃4将定义EC MLCK在肺部凋亡的起始和执行中的作用。