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

描述(申请人提供)：拟议研究的总体目标是探讨凝血酶调节内皮细胞黏附蛋白细胞间黏附分子-1(ICAM-1；CD54)表达的机制，以及这一事件如何促进多形核白细胞(PMN)在肺内的隔离和迁移，从而促进肺血管损伤的发展。凝血酶是一种在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。ICAM-1表达的基础涉及转录因子NF-B的relA/p65亚单位的激活。激活relA/p65需要将其从抑制蛋白I(B)中释放出来，并随后将其移位到细胞核。虽然它的释放机制已经被阐明，但调节RelAp65移位到细胞核的细胞质事件仍然难以捉摸。我们先前的研究表明，凝血酶诱导的RhoA/ROCK的激活和肌动蛋白细胞骨架的动态变化对核因子-B的激活和ICAM-1的表达是至关重要的。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的核转位，在RhoA-肌动蛋白途径中居于中心地位，介导ICAM-1的表达。有趣的是，LIM激酶1(LIMK1)，一种粘连蛋白激酶，和弹弓(SSH1L)，一种粘连蛋白磷酸酶，也调节ICAM-1的表达。此外，MLCK及其靶标肌球蛋白IIA在凝血酶诱导的NF-B活化中起重要作用。基于这些发现，我们假设凝血酶与LIMK1和SSH1L以及MLCK结合来调节肌动蛋白-肌球蛋白相互作用，进而促进RelA/p65的核转位和内皮细胞中ICAM-1的表达。我们还将通过这一机制验证MLCK信号对ICAM-1依赖的内皮细胞粘附性的影响，从而促进肺PMN隔离和PMN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说。具体目的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的核转位和ICAM-1在内皮细胞中的表达。具体目标2将阐述MLCK在调节肌动蛋白-肌球蛋白相互作用中的作用，导致RelA/p65的核转运和内皮细胞中ICAM-1的表达。特异性目标3将在体内评价内皮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。有了这些信息，我们相信有可能通过抑制与血管内凝血和随后的炎症相关的控制ICAM-1表达的特定信号事件来阻断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.