Mechanisms of Calmodulin-dependent EC Barrier Regulation

描述(申请人提供)：内皮细胞紊乱(EC) 屏障调节是肺部炎症、血管生成和癌症的标志。 欧盟壁垒的维护受到竞争性收缩的严密监管 以及其效果严重依赖于EC的捆绑力量 细胞角质网重排，并受细胞内信使控制 包括游离钙及其主要的细胞内受体钙调蛋白(CaM)。 大量工作，包括我们自己的工作，已经证实了钙/钙调蛋白复合体 丝氨酸蛋白酶等水肿剂引起的触发通透性 凝血酶。凝血酶受体蛋白降解顺序偶联到 细胞内钙离子增加，钙/钙调素依赖的肌球蛋白激活 链激酶(MLCK)，肌球蛋白轻链(MLC)磷酸化增加， 肌动球蛋白的起始收缩，最终形成间隙和屏障 功能障碍。然而，钙/钙调素靶标(而非MLCK)在EC中的作用 障碍监管还没有完全被理解。我们的初步数据显示 凝血酶介导的内皮细胞通透性是(部分)依赖的 激活钙/钙调素依赖性蛋白激酶II(CaMKII)AS 特异性CaMKII抑制显著减轻凝血酶诱导的应激反应 纤维的形成和渗透性。矛盾的是，凝血酶还激活了 钙/钙调素依赖的磷酸酶2B(PPase 2B或钙调神经磷酸酶) 凝血酶诱导的MLC磷酸化提供一种调节机制 潜在地逆转或限制凝血酶诱导的细胞骨架重排和 渗透性。此外，凝血酶诱导的EC屏障功能障碍 与几种细胞骨架蛋白的磷酸化增加有关 包括钙/钙调素依赖的调节蛋白Caldesmon和CaMKII靶标， 细丝、牛磺酸和波形蛋白。总而言之，这些数据清楚地传达了复杂的 Ca~(2+)/CaM依赖的信号通路参与细胞周期调控 内皮通透性。在这项提案中，我们将探讨 凝血酶介导的EC调节中的Ca~(2+)/CaM靶(非MLCK)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。 蛋白质磷酸化、细胞骨架重排和屏障功能障碍。在……里面 我们将研究PPase 2B活性在EC屏障调节中的作用。在……里面 我们将研究钙调蛋白在激动剂介导的细胞骨架中的作用 重排和屏障调节。这些研究将提供一个 对参与肺内皮细胞屏障的新信号通路的认识 调整和承诺肺治疗的新方向和新靶点 精神错乱。

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.