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

描述（由申请人提供）：拟议研究的总体目标是解决凝血酶的机制，凝血酶是一种丝氨酸蛋白酶，在败血症或血管损伤引起的凝血过程中释放，调节内皮细胞中粘附蛋白细胞间粘附分子-1 （ICAM-1; CD54）的表达，以及这一事件如何促进肺部多形核白细胞（PMN）的隔离和迁移，从而促进肺血管损伤的发展。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)中释放出来，随后将其转运到细胞核中。尽管其释放的机制已经被阐明，但调控RelAp65向细胞核易位的细胞质事件仍然难以捉摸。我们之前的研究表明，RhoA/ROCK的激活和凝血酶诱导的肌动蛋白细胞骨架的动态变化对NF- B的激活和ICAM-1的表达至关重要。我们现在有证据表明，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RelA/p65的核易位，在RhoA-actin通路中介导ICAM-1表达占据中心位置。有趣的是，LIM激酶1 (LIMK1)，一种cofilin激酶和slingshot (SSH1L)，一种cofilin磷酸酶，也调节ICAM-1的表达。此外，MLCK及其靶肌球蛋白IIA在凝血素诱导的NF-(B)活化中起重要作用。基于这些发现，我们假设凝血酶参与LIMK1和SSH1L以及MLCK调节肌动蛋白-肌球蛋白相互作用，进而促进内皮细胞中RelA/p65的核易位和ICAM-1的表达。我们还将通过这一机制验证MLCK信号介导的icam -1依赖性内皮粘附性对小鼠肺PMN隔离和PMN介导的肺血管损伤和组织水肿的影响。我们将追求以下具体目标来检验这一假设。特异性Aim 1将确定LIMK1和SSH1L在调节肌动蛋白细胞骨架变化导致内皮细胞中RelA/p65核易位和ICAM-1表达中的作用。特异性目标2将解决MLCK在调节肌动蛋白-肌球蛋白相互作用中的作用，导致内皮细胞中RelA/p65的核转运和ICAM-1的表达。特异性Aim 3将评估内皮细胞MLCK在调节凝血酶诱导的ICAM-1表达、肺PMN浸润和PMN介导的小鼠肺血管损伤中的体内作用。我们将使用多学科的方法，从生物化学，细胞和分子生物学到肺生理学来开展这些研究。根据获得的信息，我们相信通过抑制与血管内凝血和随之而来的炎症相关的控制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。

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. PUBLIC HEALTH RELEVANCE: These studies will provide novel insights into the mechanisms regulating translocation of RelA/p65 into the nucleus and thereby expression of ICAM-1, and its consequences on lung PMN sequestration and lung vascular injury. The information gained may lead to the development of strategies for interfering with specific signaling events controlling ICAM-1 expression and thereby preventing or limiting lung PMN uptake and lung vascular injury associated with inflammatory disease states as Acute Respiratory Distress Syndrome (ARDS).