Microtubules in lung endothelial cell barrier regulation

描述（由申请方提供）：内皮细胞（EC）屏障的破坏是急性肺损伤（ALI）的一个显著特征。EC渗透性由收缩力和束缚力之间的平衡调节，并且依赖于细胞骨架的相关元件（即微丝（MF）和微管（MT））的功能协调。我们的新数据表明，MT重塑直接参与凝血酶诱导的EC屏障妥协。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渗透性的增加相关。我们的新的初步数据表明，单次静脉注射MT稳定剂紫杉醇，产生显着减少多个指标的LPS诱导的炎性肺损伤，这表明MT重塑参与脓毒症诱导的ALI在体内。凝血酶诱导的信号传导涉及异源三聚体G蛋白G12和G13的活化。我们已经证明，这些蛋白质的亚基的耗竭减弱凝血酶诱导的EC通透性，而它们的过度表达导致MT拆卸和大量的应力纤维形成，收缩的指示。Rho和p38 MARK通路的抑制减弱了凝血酶对MT结构的影响，表明这些通路参与了MT重塑。我们的数据表明Rho激酶和p38 MARK的显著池与MT紧密相关。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，这些蛋白可能是凝血酶诱导的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信号传导的激活、细胞骨架调节蛋白的磷酸化、协调的MT和MF重塑，并最终导致屏障受损。具体目标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。具体目标2将检查MT和MF结合调节蛋白，tau，CaD和HSP-27，在凝血酶诱导的MT重塑和屏障失效的作用。具体目标3将使用ALI的小鼠模型来检查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.