Regulation of Endothelial ICAM-1 and PMN-Mediated Lung Injury by Actin Dynamics

肌动蛋白动力学对内皮 ICAM-1 和 PMN 介导的肺损伤的调节

• 批准号: 8446994
• 负责人: Fabeha Fazal
• 金额: $ 29.12万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 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/8446994
• 关键词: Actin-Binding Protein; Actins; Acute Lung Injury; Address; Adhesions; Adhesives; Adult Respiratory Distress Syndrome; Binding; Biochemical; Blood Vessels; Cell Nucleus; Cell surface; Coagulation Process; Cytoplasm; Cytoskeleton; Data; Development; Disease; Edema; Emigrations; Endothelial Cells; Endothelium; Event; Genetic; ICAM1 gene; Immigration; Infiltration; Inflammation; Inflammatory; Injury; Intercellular adhesion molecule 1; LIM Domain Kinase 1; Lead; Lung; MYLK gene; Mediating; Modeling; Molecular and Cellular Biology; Mus; Myosin ATPase; Myosin Light Chain Kinase; Myosin Type II; Nonmuscle Myosin Type IIA; Nuclear; Nuclear Import; Nuclear Translocation; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Play; Positioning Attribute; Proteins; Regulation; Role; Secondary to; Sepsis; Serine Protease; Signal Transduction; Testing; Thrombin; Tissues; base; cofilin; depolymerization; in vivo; insight; interdisciplinary approach; intraperitoneal; lung injury; lung vascular injury; migration; mouse model; neutrophil; novel; nucleocytoplasmic transport; p65; prevent; promoter; public health relevance; trafficking; transcription factor; uptake

DESCRIPTION (provided by applicant): The overall objective of the proposed studies is to address the mechanisms by which thrombin, a serine protease released during clotting initiated by sepsis or vascular injury, regulates the expression of adhesive protein intercellular adhesion molecule-1 (ICAM-1; CD54) in endothelial cells, and how this event promotes sequestration and migration of polymorphonuclear leukocytes (PMN) in the lung and thus contributes to development of lung vascular injury. The basis of ICAM-1 expression involves activation of RelA/p65 subunit of the transcription factor NF-(B. Activation of RelA/p65 requires its release from the inhibitory protein I(B( in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the mechanisms of its release have been elucidated, the cytoplasmic events regulating the translocation of RelAp65 to the nucleus remain elusive. We previously showed that activation of RhoA/ROCK and the dynamic changes in actin cytoskeleton induced by thrombin are crucial for NF-(B activation and ICAM-1 expression. We now have evidence that cofilin, an actin binding protein that promotes actin depolymerization, occupies a central position in RhoA-actin pathway mediating ICAM-1 expression by virtue of facilitating the nuclear translocation of RelA/p65. Interestingly, LIM kinase 1 (LIMK1), a cofilin kinase, and slingshot (SSH1L), a cofilin phosphatase, also regulate ICAM-1 expression. Additionally, MLCK and its target myosin IIA play an important role in thrombin-induced NF-(B activation. Based upon these findings, we hypothesize that thrombin engages LIMK1 and SSH1L as well as MLCK to regulate actin-myosin interaction, which in turn facilitates nuclear translocation of RelA/p65, and expression of ICAM-1 in endothelial cells. We will also test the hypothesis that MLCK signaling of ICAM-1-dependent endothelial adhesivity by this mechanism contributes to lung PMN sequestration and PMN-mediated lung vascular injury and tissue edema in mice. We will pursue the following specific aims to test this hypothesis. Specific Aim 1 will determine the role of LIMK1 and SSH1L in regulating the changes in the actin cytoskeleton leading to nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells. Specific Aim 2 will address the role of MLCK in regulating actin-myosin interaction leading to nuclear transport of RelA/p65 and expression of ICAM-1 in endothelial cells. Specific Aim 3 will evaluate the in vivo role of endothelial MLCK in regulating thrombin-induced ICAM-1 expression, lung PMN infiltration, and PMN-mediated lung vascular injury in mice. We will use multidisciplinary approaches ranging from biochemical, cellular, and molecular biology to lung physiology to carry out these studies. With the information gained, we believe that it will be possible to block PMN-mediated lung vascular injury by inhibiting the specific signaling events controlling ICAM-1 expression associated with intravascular coagulation and consequent inflammation.

描述（由申请人提供）：拟议研究的总体目标是解决凝血酶（脓毒症或血管损伤引发的凝血过程中释放的丝氨酸蛋白酶）调节内皮细胞中粘附蛋白细胞间粘附分子-1（ICAM-1；CD54）表达的机制，以及该事件如何促进多形核白细胞的隔离和迁移 (PMN) 存在于肺部，从而导致肺血管损伤。 ICAM-1 表达的基础涉及转录因子 NF-(B) 的 RelA/p65 亚基的激活。RelA/p65 的激活需要其从细胞质中的抑制蛋白 I(B() 中释放，随后转位至细胞核。虽然其释放机制已阐明，但调节 RelAp65 转位至细胞核的细胞质事件 仍然难以捉摸。我们之前表明，凝血酶诱导的RhoA/ROCK激活和肌动蛋白细胞骨架的动态变化对于NF-(B激活和ICAM-1表达至关重要。现在我们有证据表明，cofilin是一种促进肌动蛋白解聚的肌动蛋白结合蛋白，通过促进肌动蛋白核转位，在介导ICAM-1表达的RhoA-肌动蛋白通路中占据中心位置。 RelA/p65。有趣的是，LIM 激酶 1 (LIMK1)（一种肌动蛋白丝切蛋白激酶）和弹弓 (SSH1L)（一种肌动蛋白丝切蛋白磷酸酶）也调节 ICAM-1 表达。此外，MLCK 及其靶标肌球蛋白 IIA 在凝血酶诱导的 NF-(B 激活) 中发挥重要作用。根据这些发现，我们假设凝血酶与 LIMK1 结合， SSH1L 和 MLCK 调节肌动蛋白-肌球蛋白相互作用，进而促进 RelA/p65 的核转位以及内皮细胞中 ICAM-1 的表达。我们还将测试以下假设：通过这种机制，ICAM-1 依赖性内皮粘附的 MLCK 信号传导有助于小鼠肺中性粒细胞隔离以及中性粒细胞介导的肺血管损伤和组织水肿。 我们将追求以下具体目标来检验这一假设。具体目标 1 将确定 LIMK1 和 SSH1L 在调节肌动蛋白细胞骨架变化中的作用，从而导致 RelA/p65 核转位和内皮细胞中 ICAM-1 表达。具体目标 2 将解决 MLCK 在调节肌动蛋白-肌球蛋白相互作用中的作用，从而导致 RelA/p65 的核转运和 ICAM-1 的表达 内皮细胞。具体目标 3 将评估内皮 MLCK 在调节小鼠凝血酶诱导的 ICAM-1 表达、肺 PMN 浸润和 PMN 介导的肺血管损伤中的作用。我们将使用从生化、细胞和分子生物学到肺生理学的多学科方法来开展这些研究。根据所获得的信息，我们相信 通过抑制控制与血管内凝血和随后的炎症相关的 ICAM-1 表达的特定信号事件，将有可能阻断 PMN 介导的肺血管损伤。