Microtubule involvement in lung endothelial pathobiology

内皮细胞（EC）屏障调节的紊乱是肺部炎症的标志，并且严重依赖于EC细胞骨架（微丝和微管）的重排。我们以前已经表明，肌动球蛋白驱动的微丝收缩和增加肌球蛋白轻链（MLC）磷酸化受体激动剂介导的渗透性的参与。相反，关于微管网络在EC屏障调节中的作用的信息是有限的。我们的新的观察表明，凝血酶和TGF β介导的EC屏障功能障碍是依赖于（部分）微管重塑的微管网络的稳定显着减弱凝血酶和TGF β诱导的渗透性。我们 数据表明激动剂诱导的微管溶解和几种微管相关蛋白（MAP）的磷酸化增加之间有很强的相关性，所述MAP包括钙调蛋白（CaM）依赖性激酶II（CaMKII）靶、tau和细丝蛋白，暗示MAP和CaM依赖性酶如CaMKII和MLC激酶（MLCK）在受体激动剂介导的微管重塑中的可能作用。我们最近发现，微管抑制剂显着增加EC MLC磷酸化和渗透性，表明微管在维持EC屏障的重要性。通过Rho和p38 MAP激酶途径抑制，微管溶解诱导的屏障功能障碍显著减弱。总之，这些数据清楚地传达了复杂的参与微管介导的途径在EC渗透性的调节。本提案的总体目标是检查分子 微管在静态和周期性牵张刺激条件下促进受体激动剂介导的EC屏障功能障碍的机制。SA#1将检查CaMKII在凝血酶和TGF β诱导的微管溶解中的作用，SA#2将表征MLCK参与激动剂介导的微管重塑，SA#3将检查Rho途径在微管介导的MLC磷酸化和通透性增加中的作用，SA#4将鉴定参与微管介导的EC收缩性和屏障功能障碍的p38 MAPK途径，SA#5将建立1-磷酸鞘氨醇屏障保护信号与受体激动剂介导的微管溶解之间的联系。这些研究将提供一个新的信号通路参与肺EC屏障调节的理解。

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.