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

项目总结/摘要 TGFβ和Wnt信号通路参与青光眼。然而，他们之间的交流在跨- 视网膜网（TM）和眼内压（IOP）的调节尚不清楚。这个项目的目标是， 探讨TGFβ和Wnt信号通路在POAG发病中的作用。我们的目标是确定 TM和IOP调节中的这种串扰的机制和生物学后果。中央卫生- 假设是TGFβ和Wnt通路之间的相互作用，这是由一种抑制性的转录因子介导的。 Smad 4与β-Catenin形成的脚本复合物调节TM稳态和IOP。我们的理据是 我们对串扰的了解将为治疗青光眼提供一种新的治疗策略， 抑制过度的TGFβ信号传导以及在一些POAG pa-3中升高被抑制的Wnt信号传导， tients。在强有力的初步数据的指导下，这一假设将通过追求3个具体目标进行检验。SA#1：De- 确定TGFβ和Wnt信号通路串扰对IOP调节的影响; SA#2：确定 Wnt通路是否抑制TM细胞中TGFβ诱导的病理变化; SA#3：确定 Smad 4和β-连环蛋白是否形成蛋白质复合物，复合物如何形成，以及它对 信号活动。在SA 1中，我们将使用小鼠模型和人类供体眼睛来确定1）是否存在 Wnt 3a对TGFβ2诱导的小鼠眼内OHT的抑制作用呈剂量依赖性; 2）Wnt 3a是否激活了TGFβ2诱导的OHT， Wnt通路影响TGFβ2诱导的Smad 4和β-Catenin条件性敲除（KO）小鼠眼OHT; 以及3）Wnt通路的激活是否抑制TGFβ2诱导的人前房灌注培养的OHT。 或片段。在SA 2中，我们将确定1）通过使用以下方法相互交叉抑制的基因： RNA测序（RNAseq）和转基因小鼠TM（MTM）细胞株; 2）Wnt信号传导是否抑制 TGFβ2诱导人TM（HTM）和转基因MTM细胞中交联肌动蛋白网络的形成。在 SA 3，我们将1）使用Co-IP测定原代HTM细胞中的Smad 4和β-连环蛋白蛋白复合物， iTRAQ和FRET; 2）使用计算的方法鉴定参与Smad 4-β-连环蛋白结合的结构域， 3）确定Smad 4-β-Catenin相互作用对启动子结合的影响 和转录活性，使用凝胶电泳迁移率变动分析（EMSA），计算分析， 染色质免疫沉淀（ChIP）、荧光素酶测定和qPCR。这个项目意义重大，因为 阐明这种串扰，我们将能够同时操纵两种细胞信号通路。 该策略预期针对导致OHT的TM中POAG的病理学。方法是创新的- 这是因为：1）这两条通路之间的交叉抑制从未被证明过; 2）我们提出 一种独特的分子机制来定义这种交叉抑制。3)RNA测序和反式- 基因MTM细胞; 4）体内（小鼠）、离体（人供体眼）和体外（人和 小鼠原代TM细胞培养物）模型。

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.