Cross-talk between TGF-beta and Wnt pathways in the trabecular meshwork

小梁网中 TGF-β 和 Wnt 通路之间的串扰

• 批准号: 9310886
• 负责人: Weiming Mao
• 金额: $ 36.5万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9310886
• 关键词: Actins; Affect; Alpha Cell; Anterior; Aqueous Humor; Binding; Biological; Biological Assay; Cattle; Cell Culture Techniques; Cells; Complex; Computer Analysis; Data; Development; Disease; Dose; Electrophoretic Mobility Shift Assay; Extracellular Matrix; Eye; Eye diseases; Feedback; Fluorescence Resonance Energy Transfer; Genes; Genetic Transcription; Glaucoma; Goals; Homeostasis; Human; In Vitro; Knockout Mice; Knowledge; Luciferases; Mediating; Mediator of activation protein; Medicine; Mission; Modeling; Molecular; Mus; Mutation; Nuclear Translocation; Ocular Hypertension; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Perfusion; Physiologic Intraocular Pressure; Physiological; Play; Primary Open Angle Glaucoma; Proteins; Public Health; Publications; Regulation; Research; Risk Factors; Role; Signal Pathway; Signal Transduction; Testing; Trabecular meshwork structure; Transforming Growth Factor beta; Transgenic Mice; WNT Signaling Pathway; beta catenin; chromatin immunoprecipitation; crosslink; gel electrophoresis; in vivo; inhibitor/antagonist; innovation; mouse model; novel strategies; novel therapeutic intervention; novel therapeutics; promoter; protein complex; therapeutic target; transcriptome sequencing; Actins; Affect; Alpha Cell; Anterior; Aqueous Humor; Binding; Biological; Biological Assay; Cattle; Cell Culture Techniques; Cells; Complex; Computer Analysis; Data; Development; Disease; Dose; Electrophoretic Mobility Shift Assay; Extracellular Matrix; Eye; Eye diseases; Feedback; Fluorescence Resonance Energy Transfer; Genes; Genetic Transcription; Glaucoma; Goals; Homeostasis; Human; In Vitro; Knockout Mice; Knowledge; Luciferases; Mediating; Mediator of activation protein; Medicine; Mission; Modeling; Molecular; Mus; Mutation; Nuclear Translocation; Ocular Hypertension; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Perfusion; Physiologic Intraocular Pressure; Physiological; Play; Primary Open Angle Glaucoma; Proteins; Public Health; Publications; Regulation; Research; Risk Factors; Role; Signal Pathway; Signal Transduction; Testing; Trabecular meshwork structure; Transforming Growth Factor beta; Transgenic Mice; WNT Signaling Pathway; beta catenin; chromatin immunoprecipitation; crosslink; gel electrophoresis; in vivo; inhibitor/antagonist; innovation; mouse model; novel strategies; novel therapeutic intervention; novel therapeutics; promoter; protein complex; therapeutic target; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT TGFβ and Wnt signaling pathways are involved in glaucoma. However, the cross-talk between them in the tra- becular meshwork (TM) and intraocular pressure (IOP) regulation is unclear. The goal of this project is to elu- cidate the role(s) of TGFβ and Wnt signaling pathway cross-talk in POAG. Our objective is to determine the mechanisms and the biological consequence of this cross-talk in the TM and IOP regulation. The central hy- pothesis is that the cross-talk between the TGFβ and Wnt pathways, which is mediated by a repressive tran- scriptional complex formed by Smad4 and β-Catenin, regulates TM homeostasis and IOP. Our rationale is that our knowledge of the cross-talk will provide a novel therapeutic strategy in treating glaucoma by both in- hibiting the excessive TGFβ signaling as well as elevating suppressed Wnt signaling in a number of POAG pa- tients. Guided by strong preliminary data, this hypothesis will be tested by pursuing 3 specific aims. SA#1: De- termine the effect of TGFβ and Wnt signaling pathway cross-talk on IOP regulation; SA#2: Determine whether the Wnt pathway inhibits TGFβ-induced pathological changes in TM cells; SA#3: Determine whether Smad4 and β-Catenin form a protein complex, how the complex is formed, and its effect on signaling activities. In SA1, we will use a mouse model and human donor eyes to determine 1) whether there is a dose-dependent inhibition of TGFβ2-induced OHT by Wnt3a in mouse eyes; 2) whether activation of the Wnt pathway affects TGFβ2-induced OHT in Smad4 and β-Catenin conditional knockout (KO) mouse eyes; and 3) whether activation of the Wnt pathway inhibits TGFβ2-induced OHT in perfusion cultured human anteri- or segments. In SA2, we will determine 1) the genes that are cross-inhibited by each other’s pathway using RNA sequencing (RNAseq) and transgenic mouse TM (MTM) cell strains; 2) whether Wnt signaling inhibits TGFβ2-induced formation of cross-linked actin networks in human TM (HTM) and transgenic MTM cells. In SA3, we will 1) determine the Smad4 and β-Catenin protein complex in primary HTM cells using Co-IP, iTRAQ, and FRET; 2) identify the domains that are involved in Smad4-β-Catenin binding using computational analysis and mutational studies; 3) determine the effect of Smad4-β-Catenin interaction on promoter binding and transcriptional activities using gel electrophoresis mobility shift assay (EMSA), computational analysis, Chromatin immunoprecipitation (ChIP), luciferase assay, and qPCR. This project is significant, because upon the elucidation of this cross-talk, we will be able to manipulate both cell signaling pathways simultaneously. This strategy is expected to target the pathology of POAG in the TM that causes OHT. The approach is innova- tive, because 1) The cross-inhibition between the two pathways has never been demonstrated 2) We propose a unique molecular mechanism to define this cross-inhibition.; 3) A combination of RNA sequencing and trans- genic MTM cells; 4) A combination of in vivo (mice), ex vivo (human donor eyes), and in vitro (human and mouse primary TM cell cultures) models.

项目摘要/摘要 TGFβ和WNT信号通路参与青光眼。但是，它们之间的串扰 尚不清楚Becular网状工作（TM）和眼内压（IOP）调节。该项目的目的是Elu- 确定TGFβ和Wnt信号通路在POAG中的作用。我们的目标是确定 TM和IOP调节中这种串扰的机理和生物学后果。中央 pothesis是TGFβ和Wnt途径之间的串扰，该途径是由反射性转移介导的 由Smad4和β-catenin形成的Scriptal复合体调节TM稳态和IOP。我们的理由是 我们对串扰的了解将为两者的治疗方面提供新的热策略 在许多poag pa-中提高过量的TGFβ信号传导以及升高抑制的Wnt信号传导 t。在强大的初步数据的指导下，该假设将通过追求3个具体目标来检验。 SA＃1：DE- 终止TGFβ和Wnt信号通路对IOP调控的影响； SA＃2：确定 Wnt途径抑制TGFβ诱导的TM细胞病理变化。 SA＃3：确定 Smad4和β-catenin是否形成蛋白质复合物，如何形成络合物及其对 信号活动。在SA1中，我们将使用鼠标模型和人类捐赠者的眼睛来确定1）是否存在 是小鼠眼中Wnt3a对TGFβ2诱导的OHT的剂量依赖性抑制作用； 2）激活是否激活 Wnt途径会影响TGFβ2诱导的SMAD4和β-catenin条件敲除（KO）小鼠眼睛的OHT； 3）Wnt途径的激活是否抑制TGFβ2诱导的OHT在灌注培养的人前 - 或细分市场。在SA2中，我们将确定1）使用彼此途径交叉抑制的基因 RNA测序（RNASEQ）和转基因小鼠TM（MTM）细胞菌株； 2）Wnt信号是否抑制 TGFβ2诱导的人TM（HTM）和转基因MTM细胞中交联的肌动蛋白网络的形成。在 SA3，我们将使用Co-IP，确定原代HTM细胞中SMAD4和β-catenin蛋白复合物， itraq和fret; 2）确定使用计算的SMAD4-β-catenin结合所涉及的域 分析和突变研究； 3）确定SMAD4-β-catenin相互作用对启动子结合的影响 和使用凝胶电泳迁移率转移测定法（EMSA），计算分析，计算分析的转录活性 染色质免疫沉淀（CHIP），荧光素酶测定和QPCR。这个项目很重要，因为 阐明了这个串扰，我们将能够简单地操纵两个细胞信号通路。 预计该策略将针对导致OHT的TM中POAG的病理。该方法是创新的 Tive，因为1）从未证明两种途径之间的交叉抑制2）我们提出 定义这种交叉抑制的独特分子机制。 3）RNA测序和反式的结合 基因MTM细胞； 4）体内（小鼠），外体（人类捐赠者）和体外（人类和人类和 小鼠初级TM细胞培养物）模型。