Microtubule-associated regulation of acute lung injury

 描述(申请人提供)：肺部炎症和内皮细胞(EC)通透性改变在急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)的病理生理学中起主要作用，这两种疾病与高死亡率相关。目前治疗的低效率可能部分是由于专注于旨在预防ALI发病的药物治疗，而内源性反馈机制虽然在临床上更相关，但对减轻炎症激活和EC屏障功能障碍的机制仍知之甚少。我们在前一个周期的研究中发现了微管(MT)外周网络在控制肺内皮细胞屏障功能中的新作用。我们发现，屏障增强型激动剂如肝细胞生长因子刺激MT外周生长，促进Rac GTPase依赖的信号转导，减弱Rho GTPase依赖的信号转导，从而导致血管泄漏的下调。我们定义了一种新的范式，即MT相关的鸟嘌呤核苷酸交换因子ASEF和Global-H1对Rac和Rho通路的双重调控，并证明了MT在Rac-Rho串扰和内皮通透性控制机制中的重要作用。然而，MT依赖调节ALI发病和消退的整个机制，特别是MT依赖的炎症级联反应的调节机制，仍然知之甚少。在筛选对照和炎症肺内皮细胞中与MT相关的潜在信号蛋白时，我们发现细胞因子信号抑制因子SOCS1与MT组分有关。这一偶然发现提示了MT细胞骨架与控制内皮炎性激活和与EC炎症反应相关的通透性变化的新联系。MT在调节EC对细菌壁化合物、细胞因子等的反应中所起的作用仍然是未知的。我们假设，调节炎症反应的细胞反馈机制关键需要MT辅助的SOCS1靶向亚膜室，在那里它与其细胞因子受体和TLR相关的蛋白靶点相互作用。这一假设将在四个具体目标上得到检验。AIM-1将在细胞、体外和体内内毒素诱导的ALI模型中表征SOCS1对内毒素诱导的炎症的调节作用。AIM-2将研究微管参与SOCS1抗炎功能的调控。AIMS 3和4将确定MT辅助的SOCS1细胞内靶向的分子机制。AIM-3将研究MT马达在SOCS1运送到细胞外周中的作用。AIM-4将研究MT+末端蛋白在SOCS1锚定在细胞外围的作用。该项目的结果将描绘新的MT依赖的调节肺屏障功能障碍和炎症的机制，这可能导致发现一组新的治疗ALI/ARDS的药物分子。

 DESCRIPTION (provided by applicant): Lung inflammation and alterations in endothelial cell (EC) permeability play a major role in the pathophysiology of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), the conditions associated with high mortality rates. Low efficiency of current therapies may be explained in part by focus on drug treatments aimed at prevention of the onset of ALI, while endogenous feedback mechanisms which subside inflammatory activation and EC barrier dysfunction, although much more clinically relevant, remain poorly understood. Our studies in the previous cycle identified a novel role of microtubule (MT) peripheral network in the control of lung EC barrier function. We discovered that stimulation of MT peripheral growth by barrier enhancing agonists, such as hepatocyte growth factor, promoted Rac GTPase-dependent and attenuated Rho GTPase-dependent signaling, thus leading to downregulation of vascular leak. We defined a novel paradigm of dual regulation of Rac and Rho pathways by MT-associated guanine nucleotide exchange factors Asef and GEF-H1 and demonstrated essential role of MT in the mechanisms of Rac-Rho crosstalk and control of endothelial permeability. However, the entire mechanism of MT-dependent regulation of onset and resolution of ALI, and specifically, MT-dependent modulation of inflammatory cascades, remains poorly understood. During the screening of potential signaling proteins associated with MT in control and inflamed pulmonary EC we discovered association of Suppressor Of Cytokine Signaling, SOCS1, with the MT fraction. This serendipity finding suggested a novel link of MT cytoskeleton to the control of endothelial inflammatory activation and changes in permeability associated with EC inflammatory response. The role of MT in the modulation of EC response to bacterial wall compounds, cytokines, etc., remains virtually unknown. We hypothesize that that cellular feedback mechanisms modulating inflammatory response critically require MT- assisted SOCS1 targeting to the submembrane compartment, where it interacts with its cytokine receptor- and TLR-associated protein targets. This hypothesis will be tested in four Specific Aims. Aim-1 will characterize regulation of LPS-induced inflammation by SOCS1 in cell, ex vivo and in vivo models of LPS-induced ALI. Aim- 2 will investigate involvement of microtubules in control of SOCS1 anti-inflammatory function. Aims 3 and 4 will identify molecular mechanisms of MT-assisted SOCS1 intracellular targeting. Aim-3 will examine the role of MT motors in SOCS1 delivery to the cell periphery. Aim-4 will investigate the role MT plus-end proteins in SOCS1 anchoring at the cell periphery. The results of this project will delineate novel MT-dependent mechanisms regulating lung barrier dysfunction and inflammation, which may lead to discovery of a new group of pharmacological molecules for the treatment of ALI/ARDS.