Microtubule involvement in lung endothelial pathobiology

内皮细胞(EC)屏障调节障碍是肺部炎症的标志，严重依赖于EC细胞骨架(微丝和微管)的重排。我们先前已经证明肌动球蛋白驱动的微丝收缩和肌球蛋白轻链(MLC)磷酸化增加参与了受体激动剂介导的通透性。相比之下，有关微管网络在EC屏障调节中的作用的信息有限。我们的新观察表明，凝血酶和TGFb介导的EC屏障功能障碍(部分)依赖于微管重构，因为微管网络的稳定显着降低了凝血酶和TGFb诱导的通透性。我们的 数据表明，激动剂诱导的微管溶解与几种微管相关蛋白(MAP)的磷酸化增加密切相关，其中包括钙调蛋白(CaM)依赖的激酶II(CaMKII)靶标、tau和细丝蛋白，暗示MAP和CaMKII和MLC Kinase(MLCK)等CaM依赖的酶在受体激动剂介导的微管重建中可能发挥作用。我们最近发现微管抑制剂显着增加EC MLC的磷酸化和通透性，表明微管在维持EC屏障中的重要性。微管溶解引起的屏障功能障碍可通过抑制Rho和p38 MAPK通路而显著减轻。总之，这些数据清楚地传达了微管介导的通路在EC通透性调节中的复杂参与。这项提案的总体目标是研究分子 在静态和周期性拉伸刺激条件下，微管促进受体激动剂介导的EC屏障功能障碍的机制。SA#1将研究CaMKII在凝血酶和TGFb诱导的微管溶解中的作用，SA#2将研究MLCK参与激动剂介导的微管重建，SA#3将研究Rho通路在微管介导的MLC磷酸化和通透性增加中的作用，SA#4将确定p38 MAPK通路参与微管介导的EC收缩和屏障功能障碍，SA#5将在鞘氨醇1-磷酸屏障保护信号和受体激动剂介导的微管溶解之间建立联系。这些研究将提供对参与肺内皮细胞屏障调节的新的信号通路的理解。

Disturbances in endothelial cell (EC) barrier regulation is a hallmark of lung inflammation and are critically dependent upon rearrangements in the EC cytoskeleton (microfilaments and microtubules). We have previously shown the involvement of actomyosin-driven microfilament contraction and increase in myosin light chain (MLC) phosphorylation in the receptor agonists-mediated permeability. In contrast, information about the role of microtubule network in EC barrier regulation is limited. Our novel observations indicate that thrombin- and TGFb-mediated EC barrier dysfunction is dependent (in part) upon microtubule remodeling as stabilization of microtubule network significantly attenuates thrombin- and TGFb-induced permeability. Our data demonstrate strong correlation between agonist-induced microtubule dissolution and increased phosphorylation of several microtubule-associated proteins (MAPs) including calmodulin (CaM)-dependent kinase II (CaMKII) targets, tau and filamin, implicating a possible role of MAPs and CaM-dependent enzymes such as CaMKII and MLC kinase (MLCK)in receptor agonist-mediated microtubule remodeling. We have recently shown that microtubule inhibitors significantly increases EC MLC phosphorylation and permeability indicating the importance of microtubules in maintaning the EC barrier. Barrier dysfunction induced by microtubule dissolution is significantly attenuated by Rho and p38 MAP kinase pathways inhibition. Together, these data clearly convey the complex involvement of microtubule-mediated pathways in the regulation of EC permeability. The overall objective of this proposal is to examine the molecular mechanisms by which microtubules contribute to the receptor agonist-mediated EC barrier dysfunction under static and cyclic stretch-stimulating conditions. SA#1 will examine the role of CaMKII in thrombin and TGFb-induced microtubule dissolution, SA#2 will characterize the involvement of MLCK in agonist-mediated microtubule remodeling, SA#3 will examine the role of Rho pathway in microtubule-mediated increases in MLC phosphorylation and permeability, SA#4 will identify p38 MAPK pathways involved in microtubulemediatedEC contractility and barrier dysfunction, SA#5 will established the link between sphingosine 1-phosphate barrier-protective signaling and receptor agonist-mediated microtubule dissolution. These studies will provide an understanding of novel signaling pathways involved in lung EC barrier regulation.