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

项目摘要/摘要 转化生长因子β和Wnt信号通路参与了青光眼的发病过程。然而，他们之间的串话在传输- 小梁网络(TM)和眼压(IOP)的调节尚不清楚。这个项目的目标是-- 探讨转化生长因子β和Wnt信号通路在POAG中的作用(S)。我们的目标是确定 TM和IOP调节中这种串扰的机制和生物学后果。中央高铁- 假说是转化生长因子β和WNT通路之间的串扰，它是由一种抑制性的转导- Smad4和β-连环蛋白形成的脚本复合体，调节TM的动态平衡和眼压。我们的理由是 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，通过以下两种方式- 抑制过度的转化生长因子β信号转导和上调受抑制的WnT信号。 蒂恩斯。在强劲的初步数据的指导下，这一假说将通过追求3个具体目标来检验。SA#1：De- 终止转化生长因子β和Wnt信号通路对眼压调节的影响；SA#2：测定 Wnt通路是否抑制转化生长因子β诱导的TM细胞病变；SA#3：确定 Smad4和β-连环蛋白是否形成蛋白质复合体，该复合体是如何形成的，以及它对 信令活动。在SA1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 2)WNT3a对转化生长因子β-2诱导的小鼠眼内羟色胺的激活是否具有剂量依赖性抑制作用 WNT通路影响转化生长因子β-2诱导的Smad4和β-catenin条件性基因敲除(KO)小鼠眼的过敏性羟色胺； 3)Wnt通路的激活是否能抑制转化生长因子β-2诱导的人前动脉灌流过氧化应激 或者是细分市场。在SA2中，我们将确定1)被彼此的途径交叉抑制的基因 RNA测序(RNAseq)和转基因小鼠TM(MTM)细胞株；2)Wnt信号是否抑制 转化生长因子β-2诱导人TM和转基因MTM细胞形成交联型肌动蛋白网络。在……里面 SA3，我们将1)用共IP法检测原代人巨噬细胞中的Smad4和β-连环蛋白复合体， ITRAQ和FRET；2)使用计算方法确定Smad4-β-连环蛋白结合中涉及的结构域 3)Smad4-β-连环蛋白相互作用对启动子结合的影响 和使用凝胶电泳迁移率改变分析(EMSA)的转录活性，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，因为在 阐明这种串扰，我们将能够同时操纵两个细胞信号通路。 这一策略有望针对导致OHT的TM中的POAG的病理。这种方法是创新的- 主动，因为1)这两条通路之间的交叉抑制从未被证明2)我们建议 一个独特的分子机制来定义这种交叉抑制。3)RNA测序和反式-DNA测序相结合。 基因MTM细胞；4)体内(小鼠)、体外(人供体眼)和体外(人和 小鼠原代TM细胞培养)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.