Microtubules in lung endothelial cell barrier regulation

描述（由申请人提供）：内皮细胞（EC）屏障的破坏是急性肺损伤（ALI）的一个突出特征。EC的渗透性受收缩力和栓系力之间的平衡调节，并依赖于细胞骨架中相关元件的功能协调，即微丝（MF）和微管（MT）。我们的新数据表明，MT重塑直接参与凝血酶诱导的EC屏障损害。与凝血酶类似，脂多糖（LPS）诱导MT网络的部分溶解，这与体外EC通透性的增加有关。我们新的初步数据表明，单次静脉注射MT稳定剂紫杉醇，可以显著降低lps诱导的炎症性肺损伤的多个指标，这表明MT重塑参与了脓毒症诱导的ALI。凝血酶诱导的信号通路包括异源三聚体g蛋白G12和G13的激活。我们已经证明，这些蛋白亚基的缺失会减弱凝血素诱导的EC渗透性，而它们的过表达会导致MT解体和大量应力纤维形成，这表明收缩。抑制Rho和p38 MARK通路可减弱凝血酶对MT结构的影响，提示这些通路参与MT重塑。我们的数据表明，大量的Rho激酶和p38 MARK与MT密切相关。凝血酶诱导几种MT和MF相关的调节蛋白磷酸化，包括caldesmon （CaD）、HSP-27和tau，它们可能是导致凝血酶诱导的MT和MF结构变化的原因。我们假设凝血酶诱导的G12和G13的激活导致Rho和p38 MARK信号的激活，细胞骨架调节蛋白的磷酸化，协调MT和MF重塑，最终导致屏障妥协。特异性目标1将定义G12和G13在凝血酶诱导的MT分解和EC渗透性中的作用。特异性目的2将研究MT-和mf结合调节蛋白tau、CaD和热休克蛋白27在凝血酶诱导的MT重塑和屏障衰竭中的作用。特异性目的3将使用小鼠ALI模型研究MT重塑在脓毒症诱导的肺损伤中的作用。这些研究将探讨MT参与EC屏障调节的基本分子机制，并为治疗肺部疾病提供新的方向和靶点。

DESCRIPTION (provided by applicant): Disruption of the endothelial cell (EC) barrier is a prominent feature of acute lung injury (ALI). EC permeability is regulated by a balance between contractile and tethering forces and is dependent on the functional coordination of interrelated elements of the cytoskeleton, namely microfilaments (MF) and microtubules (MT). Our novel data indicate that MT remodeling is directly involved in thrombin-induced EC barrier compromise. Similar to thrombin, lipopolysaccharide (LPS) induces partial dissolution of the MT network, which correlates with an increase in EC permeability in vitro. Our novel preliminary data demonstrated that single intravenous dose of MT stabilizer, taxol, produced significant reductions in multiple indices of LPS-induced inflammatory lung injury suggesting the involvement of MT remodeling in sepsis-induced ALI in vivo. Thrombin-induced signaling involves activation of heterotrimeric G-proteins, G12 and G13. We have demonstrated that depletion of subunits of these proteins attenuates thrombin-induced EC permeability, whereas their overexpression led to MT disassembly and massive stress fiber formation, indicative of contraction. Inhibition of Rho and p38 MARK pathways attenuates the effect of thrombin on MT structure suggesting the involvement of these pathways in MT remodeling. Our data indicate that significant pools of Rho kinase and p38 MARK are tightly associated with MT. Thrombin induces phosphorylation of several MT- and MF-associated regulatory proteins, including caldesmon (CaD), HSP-27 and tau, which are potentially responsible for thrombin-induced changes in MT and MF structure. We hypothesize that thrombin-induced activation of G12 and G13 leads to activation of Rho and p38 MARK signaling, phosphorylation of cytoskeletal regulatory proteins, coordinated MT and MF remodeling and finally to barrier compromise. Specific Aim 1 will define the role of G12 and G13 in thrombin-induced MT disassembly and EC permeability. Specific Aim 2 will examine the role of MT- and MF-binding regulatory proteins, tau, CaD and HSP-27, in thrombin-induced MT remodeling and barrier failure. Specific Aim 3 will examine the role of MT remodeling in sepsis-induced lung injury using a murine model of ALI. These studies will examine basic molecular mechanisms by which MT are involved in EC barrier regulation and promise new directions and targets for treatment of lung disorders.