ALK/SMAD Signaling in TGF beta-induced EC Permeability

描述(申请人提供)：内皮细胞(EC)屏障调节障碍是肺部炎症、血管生成和癌症的标志。EC的通透性受收缩和牵引力之间的平衡调节，关键依赖于肌动蛋白和微管细胞骨架的协调重排。越来越多的证据表明，炎性细胞因子如转化生长因子-β在体外增加了EC的通透性，并参与了体内肺通透性的增加。转化生长因子-β通过与转化生长因子-βI型受体ALK1和ALK5结合，激活控制靶基因转录的特定SMAD蛋白，从而对内皮细胞产生细胞效应。然而，ALK/SMAD信号在转化生长因子-B诱导的细胞骨架重排和通透性中的作用几乎还没有被探索过。我们新的初步数据显示，Alk5和Smad4蛋白的特异性缺失或抑制显着减弱了转化生长因子-B的跨内皮细胞电阻(TER)的降低，表明ALK/SMAD信号转导通路参与了转化生长因子-B诱导的内皮屏障破坏。我们最近的数据还表明，转化生长因子-β诱导的TER减少和细胞旁间隙的形成与F-肌动蛋白应力纤维的形成和肌球蛋白轻链(MLC)磷酸化的增加密切相关，这表明收缩机制参与了转化生长因子-β诱导的内皮通透性。转化生长因子-β诱导的内皮细胞骨架的改变严重依赖于Rho GTP酶活性和微管重构，而不是钙信号或MLC激酶的激活。CAMP激活可减弱转化生长因子-β诱导的TER减少和MLC磷酸化增加，支持cAMP/PKA参与屏障保护对转化生长因子-β诱导的内皮细胞通透性的影响。此外，转化生长因子-B诱导的内皮细胞激活p38 MAP激酶通路，这也可能通过磷酸化关键的细胞骨架蛋白，如caldesmon和HSP-27，参与非Rho依赖性的内皮细胞收缩。然而，ALK/SMAD信号的激活与EC收缩活性的激活之间的联系尚不清楚。在这项研究中，我们将探索SMAD依赖和独立的通路在转化生长因子-B诱导的EC屏障功能障碍中的作用。ALK1和ALK5受体的参与将与调节SMAD蛋白的磷酸化以及Rho-和p38MAPK介导的EC细胞骨架重排和通透性的激活暂时联系在一起。在具体目标1中，我们将研究转化生长因子-B诱导的Rho激活、ALK/SMAD信号转导与EC通透性之间的关系。在特定的目标2中，我们将研究参与转化生长因子-B诱导的EC屏障功能障碍的p38 MAPK依赖的通路和ALK/SMAD信号之间的联系。在具体目标3中，我们将以Smads、Rho、p38和细胞骨架蛋白为潜在的PKA靶点，探讨cAMP/PKA对转化生长因子-B诱导的EC屏障失效的保护作用的分子机制。这些研究将提供对细胞因子介导的肺内皮细胞屏障调节的新的信号通路的理解，并有望为肺部疾病的治疗提供新的方向和靶点。

DESCRIPTION (provided by applicant): Disturbances in endothelial cell (EC) barrier regulation are a hallmark of lung inflammation, angiogenesis and cancer. EC permeability is regulated by a balance between contractile and tethering forces and critically depends upon the coordinate rearrangement of actin and microtubule cytoskeleton. Growing evidence indicates that inflammatory cytokines like TGF-B increase EC permeability in vitro and are involved in the increase in lung permeability in vivo. TGF-B elicits cellular effects on endothelium by engagement of TGF-B type I receptors, ALK1 and ALK5, following by activation of specific SMAD proteins that control the transcription of target genes. However, the involvement of ALK/SMAD signaling in TGF-B-induced cytoskeletal rearrangement and permeability are virtually unexplored. Our novel preliminary data indicated that specific depletion or inhibition of ALK5 and SMAD4 proteins significantly attenuated TGF-B decrease in transendothelial electrical resistance (TER) indicating the involvement of ALK/SMAD signaling in TGF-B-induced EC barrier compromise. Our recent data also indicate that TGF-B-induced decrease in TER and formation of paracellular gaps is tightly linked to F-actin stress fiber formation and increases in myosin light chain (MLC) phosphorylation indicating the involvement of contractile mechanisms in TGF-B-induced EC permeability. TGF-B-induced changes in EC cytoskeleton are critically dependent upon Rho GTPase activity and microtubule remodeling, but not Ca2+ signaling or MLC kinase activation. cAMP activation attenuates both TGF-B-induced decreases in TER and increases in MLC phosphorylation supporting the involvement of cAMP/PKA in barrier protection against TGF-B-induced EC permeability. In addition, TGF-B-induced EC stimulation activates p38 MAP kinase pathway, which also potentially can be involved in Rho-independent EC contractility via phosphorylation of key cytoskeletal proteins, like caldesmon and HSP-27. However, the link between activation of ALK/SMAD signaling and activation of EC contractility is unknown. In this proposal, we will explore the role of SMAD dependent and independent pathways involved in TGF-B-induced EC barrier dysfunction. Engagement of ALK1 and ALK5 receptors will be temporally linked with regulatory SMAD proteins phosphorylation and activation of Rho- and p38 MAPK-mediated EC cytoskeletal rearrangement and permeability. In Specific Aim 1, we will examine the link between TGF-B-induced Rho activation, ALK/SMAD signaling and EC permeability. In Specific Aim 2, we will examine the link between p38 MAPK-dependent pathways involved in TGF-B-induced EC barrier dysfunction and ALK/SMAD signaling. In Specific Aim 3, we will explore the molecular mechanisms by which cAMP/PKA protects against TGF-B-induced EC barrier failure focusing on the SMADs, Rho, p38 and cytoskeletal proteins as potential PKA targets. These studies will provide an understanding of novel signaling pathways involved in cytokine-mediated lung EC barrier regulation and promise new directions and targets for treatment of lung disorders.