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）屏障调节的干扰是肺部炎症，血管生成和癌症的标志。 EC渗透性受收缩力和绑扎力之间的平衡调节，并且批判性地取决于肌动蛋白和微管细胞骨架的坐标重排。越来越多的证据表明，炎性细胞因子（如TGF-B）在体外增加了EC的渗透性，并参与体内肺渗透性的增加。 TGF-B通过激活控制靶基因转录的特定SMAD蛋白来激活特定的SMAD蛋白，从而通过TGF-B型受体ALK1和ALK5的参与对内皮产生影响。但是，实际上未探索ALK/SMAD信号在TGF-B诱导的细胞骨架重排和渗透性中的参与。我们新的初步数据表明，对ALK5和SMAD4蛋白的特异性耗竭或抑制显着减弱了跨内皮电阻（TER）的TGF-B降低，表明ALK/SMAD信号在TGF-B诱导的EC屏障中涉及ALK/SMAD信号的参与。我们最近的数据还表明，TGF-B诱导的TER降低，副细胞间隙的形成与F-肌动蛋白应力纤维的形成密切相关，并增加肌球蛋白光链（MLC）磷酸化，这表明收缩机制参与TGF-B-B诱导的ECEABEBEABIBLIDIO。 TGF-B诱导的EC细胞骨架变化严重取决于Rho GTPase活性和微管重塑，而不是Ca2+信号传导或MLC激酶激活。 CAMP激活减弱了TGF-B诱导的TER降低，MLC磷酸化的增加，支持CAMP/PKA参与抗TGF-B诱导的EC渗透性的屏障保护。此外，TGF-B诱导的EC刺激激活了P38 MAP激酶途径，该途径也有可能通过关键细胞骨架蛋白（如Caldesmon和HSP-27）的磷酸化来参与Rho独立的EC收缩力。但是，ALK/SMAD信号传导的激活与EC收缩力激活之间的联系尚不清楚。在此提案中，我们将探讨与TGF-B诱导的EC屏障功能障碍有关的SMAD依赖和独立途径的作用。 ALK1和ALK5受体的参与将与调节性SMAD蛋白磷酸化以及Rho-和P38 MAPK介导的EC细胞骨架重排和渗透性的激活相关。在特定目标1中，我们将检查TGF-B诱导的RHO激活，ALK/SMAD信号传导和EC渗透性之间的联系。在特定的目标2中，我们将检查与TGF-B诱导的EC屏障功能障碍与ALK/SMAD信号转导有关P38 MAPK依赖性途径之间的联系。在特定的目标3中，我们将探索CAMP/PKA保护TGF-B诱导的EC屏障衰竭的分子机制，该障碍的重点是SMADS，RHO，P38和细胞骨架蛋白作为潜在的PKA靶标。这些研究将提供对细胞因子介导的肺部EC屏障调节中涉及的新型信号通路的理解，并承诺对肺部疾病治疗的新方向和目标。