ALK/SMAD Signaling in TGF beta-induced EC Permeability

TGFβ 诱导的 EC 通透性中的 ALK/SMAD 信号转导

• 批准号: 7446642
• 负责人: ALEXANDER D VERIN
• 金额: $ 34.85万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7446642
• 关键词: Actins; Acute Lung Injury; Alveolar; Attenuated; Binding; Biochemical; Blood Vessels; Cell surface; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeletal Modeling; Cytoskeletal Proteins; Cytoskeleton; DNA Sequence Rearrangement; Data; Electrical Resistance; Endothelial Cells; Endothelium; Equilibrium; F-Actin; Failure; Figs - dietary; Floods; Functional Imaging; Functional disorder; Gene Targeting; Genetic Transcription; Histamine; Hypoxemia; In Vitro; Inflammatory; Link; Liquid substance; Lung; Lung Inflammation; Lung diseases; MADH4 gene; MAP Kinase Gene; MAPK14 gene; Malignant Neoplasms; Mediating; Mediator of activation protein; Microtubules; Molecular; Morbidity - disease rate; Myosin Light Chain Kinase; Myosin Light Chains; Pathway interactions; Permeability; Phosphorylation; Principal Investigator; Proteins; Regulation; Role; Signal Pathway; Signal Transduction; Stress Fibers; Structure; TGF-beta type I receptor; Thrombin; Tissues; Transcriptional Activation; Transforming Growth Factor beta; Vascular Endothelial Cell; Vascular Permeabilities; Work; angiogenesis; caldesmon; cytokine; human MAPK14 protein; in vivo; macromolecule; mitogen-activated protein kinase p38; monolayer; mortality; novel; programs; receptor; rho; rho GTP-Binding Proteins

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.

描述（由申请人提供）：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 通透性由收缩力和束缚力之间的平衡调节，并且关键取决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，TGF-B 等炎症细胞因子在体外可增加 EC 通透性，并参与体内肺通透性的增加。 TGF-B 通过与 TGF-B I 型受体、ALK1 和 ALK5 结合，引发对内皮细胞的影响，然后激活控制靶基因转录的特定 SMAD 蛋白。然而，ALK/SMAD 信号传导在 TGF-B 诱导的细胞骨架重排和通透性中的参与实际上尚未被探索。我们的新初步数据表明，ALK5 和 SMAD4 蛋白的特异性去除或抑制可显着减弱 TGF-B 跨内皮电阻 (TER) 的降低，表明 ALK/SMAD 信号传导参与 TGF-B 诱导的 EC 屏障受损。我们最近的数据还表明，TGF-B 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，表明收缩机制参与了 TGF-B-诱导EC通透性。 TGF-B 诱导的 EC 细胞骨架变化主要依赖于 Rho GTPase 活性和微管重塑，而不是 Ca2+ 信号传导或 MLC 激酶激活。 cAMP 激活可减弱 TGF-B 诱导的 TER 减少和 MLC 磷酸化增加，支持 cAMP/PKA 参与屏障保护以对抗 TGF-B 诱导的 EC 通透性。此外，TGF-B 诱导的 EC 刺激可激活 p38 MAP 激酶途径，该途径也可能通过关键细胞骨架蛋白（如 caldesmon 和 HSP-27）的磷酸化参与 Rho 独立的 EC 收缩性。然而，ALK/SMAD 信号传导激活与 EC 收缩性激活之间的联系尚不清楚。在本提案中，我们将探讨 SMAD 依赖和独立途径在 TGF-B 诱导的 EC 屏障功能障碍中的作用。 ALK1 和 ALK5 受体的参与将暂时与调节 SMAD 蛋白磷酸化以及 Rho 和 p38 MAPK 介导的 EC 细胞骨架重排和通透性激活相关。在具体目标 1 中，我们将研究 TGF-B 诱导的 Rho 激活、ALK/SMAD 信号传导和 EC 通透性之间的联系。在具体目标 2 中，我们将研究参与 TGF-B 诱导的 EC 屏障功能障碍的 p38 MAPK 依赖性通路与 ALK/SMAD 信号传导之间的联系。在具体目标 3 中，我们将探讨 cAMP/PKA 防止 TGF-B 诱导的 EC 屏障衰竭的分子机制，重点关注作为潜在 PKA 靶标的 SMAD、Rho、p38 和细胞骨架蛋白。这些研究将提供对参与细胞因子介导的肺 EC 屏障调节的新信号通路的理解，并为治疗肺部疾病提供新的方向和目标。