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)的显著特征。内皮细胞的通透性受收缩和拴系力之间的平衡调节，并依赖于细胞骨架相关元素，即微丝(MF)和微管(MT)的功能协调。我们的新数据表明，MT重塑直接参与了凝血酶诱导的EC屏障破坏。与凝血酶类似，脂多糖(LPS)可导致MT网络的部分溶解，这与体外EC通透性的增加有关。我们新的初步数据显示，单次静脉注射MT稳定剂紫杉醇可显著降低内毒素诱导的炎性肺损伤的多个指标，提示MT重塑参与了体内脓毒症诱导的ALI。凝血酶诱导的信号转导涉及异三聚体G蛋白、G12和G13的激活。我们已经证明，这些蛋白质亚单位的缺失可以降低凝血酶诱导的内皮细胞通透性，而它们的过度表达会导致MT的分解和大量应力纤维的形成，这表明细胞收缩。抑制Rho和p38Mark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p38Mark与MT密切相关。凝血酶诱导几种MT和MF相关调节蛋白的磷酸化，包括钙调蛋白(CAD)、HSP-27和tau，这些蛋白可能与凝血酶诱导MT和MF结构的变化有关。我们假设凝血酶诱导的G12和G13的激活导致Rho和p38Mark信号的激活，细胞骨架调节蛋白的磷酸化，协调的MT和MF重塑，最终导致屏障破坏。具体目标1将确定G12和G13在凝血酶诱导的MT分解和EC通透性中的作用。具体目标2将研究MT和MF结合调节蛋白tau、CAD和HSP-27在凝血酶诱导的MT重塑和屏障破坏中的作用。具体目标3将使用ALI的小鼠模型来研究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，并有望为肺部疾病的治疗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.