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。电导率受收缩力和栓系力之间的平衡调节，并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，炎症细胞因子如TGF-B在体外增加EC的通透性，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，进而激活控制靶基因转录的特定SMAD蛋白，从而对内皮细胞产生影响。然而，ALK/SMAD信号在tgf - b诱导的细胞骨架重排和通透性中的作用几乎未被探索。我们新的初步数据表明，ALK5和SMAD4蛋白的特异性消耗或抑制显著减弱TGF-B在跨内皮电阻（TER）中的下降，这表明ALK/SMAD信号参与了TGF-B诱导的EC屏障损害。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 -肌动蛋白应激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 - b诱导的EC渗透性涉及收缩机制。tgf - b诱导的EC细胞骨架变化严重依赖于Rho GTPase活性和微管重塑，而不是Ca2+信号或MLC激酶激活。cAMP激活可减弱tgf - b诱导的TER下降和MLC磷酸化的增加，支持cAMP/PKA参与tgf - b诱导的EC通透性的屏障保护。此外，tgf - b诱导的EC刺激激活了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非依赖性EC收缩。然而，ALK/SMAD信号的激活与EC收缩性的激活之间的联系尚不清楚。在本提案中，我们将探讨SMAD依赖和独立通路在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性SMAD蛋白磷酸化和Rho-和p38 mapk介导的EC细胞骨架重排和通透性的激活有关。在Specific Aim 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Specific Aim 2中，我们将研究参与tgf - b诱导EC屏障功能障碍的p38 mapk依赖通路与ALK/SMAD信号传导之间的联系。在Specific Aim 3中，我们将探讨cAMP/PKA保护tgf - b诱导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。这些研究将为细胞因子介导的肺EC屏障调节提供新的信号通路，并为肺部疾病的治疗提供新的方向和靶点。