Mechanisms of Calmodulin-dependent EC Barrier Regulation

描述（由申请方提供）：内皮细胞（EC）紊乱 屏障调节是肺部炎症、血管生成和癌症的标志。 EC屏障的维持受到竞争性收缩因子的密切调节， 和束缚力，其影响严重依赖于EC 在细胞内信使的控制下， 包括游离Ca 2+及其主要的细胞内受体钙调素（CaM）。 大量的工作，包括我们自己的工作，已经证实了Ca 2 +/钙调素复合物， 由水肿剂如丝氨酸蛋白酶诱导的触发渗透性 凝血酶凝血酶受体蛋白水解依次偶联到 细胞内Ca ~（2+）增加，激活Ca ~（2+）/Ca ~（2+）依赖性肌球蛋白发光 链激酶（MLCK），肌球蛋白轻链（MLC）磷酸化增加， 开始肌动球蛋白收缩，最后形成间隙和屏障 功能障碍然而，Ca ~（2+）/CaM靶点（MLCK除外）在EC中的作用可能与细胞内Ca ~（2+）/CaM靶点（MLCK）的表达有关。 屏障调节尚未完全理解。我们的初步数据显示 凝血酶介导的内皮细胞通透性依赖于（部分） 在激活Ca 2 +/CaM依赖性蛋白激酶II（CaMKII）后， 特异性CaMKII抑制显著减弱凝血酶诱导的应激 纤维形成和渗透性。巧合的是，凝血酶也激活了 Ca 2 +/CaM依赖性磷酸酶2B（PPase 2B或钙调神经磷酸酶）， 凝血酶诱导的MLC磷酸化提供了一种调节机制， 可能逆转或限制凝血酶诱导的细胞骨架重排， 磁导率此外，凝血酶诱导的EC屏障功能障碍， 与几种细胞骨架蛋白磷酸化增加相关 包括Ca 2 +/CaM依赖性调节蛋白钙调蛋白和CaMKII靶， 细丝蛋白、tau蛋白和波形蛋白。总之，这些数据清楚地传达了复杂的 Ca 2 +/CaM依赖性信号通路参与调节 内皮通透性在本提案中，我们将探讨 凝血酶介导的EC调节中的Ca 2 +/CaM靶点（MLCK除外）。在SA 1中 我们将研究CaMKII活性在激动剂诱导的细胞骨架中的作用， 蛋白磷酸化、细胞骨架重排和屏障功能障碍。在 我们将研究PPase 2B活性在EC屏障调节中的作用。在 我们将研究钙调素在激动剂介导的细胞骨架蛋白中的作用。 重排和屏障调节。这些研究将提供一个 了解参与肺EC屏障的新信号通路 调节并为肺部治疗提供新的方向和靶点 紊乱

DESCRIPTION (provided by applicant): Disturbances in endothelial cell (EC) barrier regulation is a hallmark of lung inflammation, angiogenesis and cancer. Maintaining of EC barrier is under close regulation by competing contractile and tethering forces whose effects are critically dependent upon EC cytoskelatal rearrangement and is under control of intracellular messengers including free Ca2+ and its major intracellular receptor, calmodulin (CaM). Substantial work, including our own, has verified that Ca2+/calmodulin complex triggered permeability induced by edemagenic agents such as the serine protease thrombin. Thrombin receptor proteolysis is sequentially coupled to intracellular Ca2+ increases, activation of Ca2+/CaM-dependen myosin light chain kinase (MLCK), increases in myosin light chain (MLC) phosphorylation, initiation of actomyosin contraction and finally gap formation and barrier dysfunction. However, the role of Ca2+/CaM targets (other than MLCK) in EC barrier regulation is not completely understood. Our preliminary data indicate that thrombin-mediated endothelial cell permeability is dependent (in part) upon activation of the Ca2+/CaM dependent protein kinase II (CaMKII) as specific CaMKII inhibition significantly attenuates thrombin-induced stress fiber formation and permeability. Paradoxically, thrombin also activates the Ca2+/CaM-dependent phosphatase 2B (PPase 2B or calcineurin) which decreases thrombin-induced MLC phosphorylation providing a regulatory mechanism to potentially reverse or limit thrombin-induced cytoskeletal rearrangement and permeability. In addition, thrombin-induced EC barrier dysfunction is correlated with increased phosphorylation of several cytoskeletal proteins including Ca2+/CaM-dependent regulatory protein caldesmon and CaMKII targets, filamin, tau and vimentin. Together, these data clearly convey the complex involvement of Ca2+/CaM-dependent signaling pathways in the regulation of endothelial permeability. In this proposal, we will explore the role of Ca2+/CaM targets (other than MLCK) in thrombin-mediated EC regulation. In SA 1 we will examine the role of CaMKII activity in agonist-induced cytoskeletal protein phosphorylation, cytoskeletal rearrangement and barrier dysfunction. In SA 2 we will examine the role of PPase 2B activity in EC barrier regulation. In SA 3 we will examine the role of caldesmon in agonist-mediated cytoskeletal rearrangement and barrier regulation. These studies will provide an understanding of novel signaling pathways involved in lung EC barrier regulation and promise new directions and targets for treatment of lung disorders.