ALK/SMAD Signaling in TGF beta-induced EC Permeability

描述（由申请方提供）：内皮细胞（EC）屏障调节紊乱是肺部炎症、血管生成和癌症的标志。EC的渗透性是由收缩力和束缚力之间的平衡调节的，并且严重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TGF-β在体外增加EC通透性，并参与体内肺通透性的增加。TGF-β通过与TGF-β I型受体ALK 1和ALK 5结合，随后激活控制靶基因转录的特异性SMAD蛋白，从而增强对内皮的细胞效应。然而，ALK/SMAD信号转导在TGF-β诱导的细胞骨架重排和通透性中的参与几乎未被探索。我们的新的初步数据表明，特异性耗竭或抑制ALK 5和SMAD 4蛋白显著减弱了TGF-β跨内皮电阻（TER）的降低，表明ALK/SMAD信号转导参与了TGF-β诱导的EC屏障受损。我们最近的数据还表明，TGF-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TGF-β诱导的EC通透性的收缩机制的参与。TGF-β诱导的EC细胞骨架的变化主要依赖于Rho GT3活性和微管重塑，而不是Ca 2+信号或MLC激酶激活。cAMP激活减弱了TGF-β诱导的TER降低和MLC磷酸化增加，支持cAMP/PKA参与了针对TGF-β诱导的EC渗透性的屏障保护。此外，TGF-β诱导的EC刺激激活p38 MAP激酶途径，其也可能通过关键细胞骨架蛋白如钙调蛋白和HSP-27的磷酸化参与Rho非依赖性EC收缩性。然而，ALK/SMAD信号传导的激活与EC收缩性的激活之间的联系尚不清楚。在本研究中，我们将探讨SMAD依赖性和非依赖性通路在TGF-β诱导的EC屏障功能障碍中的作用。ALK 1和ALK 5受体的参与将与调节SMAD蛋白磷酸化和Rho和p38 MAPK介导的EC细胞骨架重排和渗透性的激活在时间上相关。在具体目标1中，我们将研究TGF-β诱导的Rho激活，ALK/SMAD信号转导和EC通透性之间的联系。在具体目标2中，我们将研究TGF-β诱导的EC屏障功能障碍和ALK/SMAD信号转导中涉及的p38 MAPK依赖性通路之间的联系。在具体目标3中，我们将探索cAMP/PKA保护TGF-β诱导的EC屏障失效的分子机制，重点关注SMADs，Rho，p38和细胞骨架蛋白作为潜在的PKA靶点。这些研究将提供一个新的信号转导途径参与尼古丁介导的肺EC屏障调节的理解，并承诺新的方向和肺部疾病的治疗目标。

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.