The role of Wnt signaling in treating glucocorticoid-induced glaucoma

Wnt信号在治疗糖皮质激素诱发的青光眼中的作用

• 批准号: 10904116
• 负责人: Weiming Mao
• 金额: $ 9.9万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10904116
• 关键词: ATAC-seq; Affect; Antiinflammatory Effect; Aqueous Humor; Binding; Biological Assay; Cattle; Cells; Chromatin; Complex; DNA; Data; Development; Dexamethasone; Disease; Endotoxins; Enhancers; Enzymes; Epigenetic Process; Eye; Eye diseases; Fluorescence Resonance Energy Transfer; Future; Gene Expression; General Population; Genes; Glaucoma; Glucocorticoid Receptor; Glucocorticoids; Goals; HDAC1 gene; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Inflammatory; Knock-out; Knockout Mice; Knowledge; Lead; Methyl-CpG-Binding Protein 2; Mission; Modeling; Modification; Molecular; Mus; Nuclear; Ocular Hypertension; Pathogenesis; Pathway interactions; Patients; Pattern; Perfusion; Physiologic Intraocular Pressure; Primary Open Angle Glaucoma; Public Health; Publications; Publishing; Receptor Signaling; Regulation; Reporter; Research; Response Elements; Role; Signal Induction; Signal Transduction; Testing; Therapeutic Effect; Therapeutic Glucocorticoid; Tissues; Trabecular meshwork structure; Transgenic Organisms; Transposase; Uveitis; WNT Signaling Pathway; arm; autoimmune uveitis; beta catenin; conditional knockout; drug development; histone modification; inhibitor; novel strategies; prevent; recruit; responders and non-responders; response; side effect; small hairpin RNA; small molecule; success; therapeutic target; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Glucocorticoid (GC)-induced ocular hypertension (OHT) and glaucoma (GIG) occur in ~40% of the general population and ~90% primary open angle glaucoma (POAG) patients. Our published studies and preliminary data show that canonical Wnt signaling activation inhibits glucocorticoid receptor signaling and GC-induced OHT. We hypothesize that activation of canonical Wnt signaling inhibits GIG, and this inhibition requires nucle- ar β-catenin, glucocorticoid receptor (GR), and epigenetic modification enzymes including HDAC1 and MeCP2. Our goal is to elucidate the mechanism of GIG. Our objective is to inhibit GC-induced OHT without compromis- ing GC’s anti-inflammatory effects. Our rationale is that Wnt activation is a potential approach to prevent and treat OHT/GIG, and studying GIG will help us to better understand POAG. Here, we propose three specific aims to test our central hypothesis. SA1. Explore how Wnt signaling modulates GC-induced OHT and anti- inflammatory effects in mouse eyes. We will determine SA1.1) If canonical Wnt signaling activation inhibits GC-induced OHT using canonical Wnt signaling reporter mice; SA1.2) If β-catenin is necessary for inhibiting GC-induced OHT using conditional knockout mice; and SA1.3) If activated canonical Wnt signaling compro- mises GC’s anti-inflammatory effects using mouse uveitis models. SA2. Determine the role of canonical Wnt signaling in glucocorticoid responsiveness in human eyes. SA2.1) Determine if Dkk1 (a Wnt signaling in- hibitor) is elevated while canonical Wnt signaling is inhibited. We will collect aqueous humor and trabecular meshwork tissues from GIG eyes as well as from non-GIG eyes to study Dkk1, β-catenin and axin2 levels. We expect to see higher Dkk1 levels but lower β-catenin and axin2 in GIG eyes. SA2.2) Determine if canonical Wnt signaling affects GC responsiveness in human donor eyes. We will perfuse paired donor eyes with DEX to identify responsiveness. For responder eyes, we will co-treat one eye with CHIR (a small molecule Wnt signal- ing activator) and expect it will relieve OHT. For non-responder eyes, we will co-treat one eye with Dkk1 and expect it will induce OHT. SA3. Determine the molecular mechanism of canonical Wnt signaling and β- catenin in GC responsiveness and TM homeostasis. SA3.1) Determine Wnt signaling induced gene ex- pression and associated chromatin accessibility in GC response. We will determine whether Wnt signaling has differential regulation of GC-induced genes using RNA-seq and Assay for Transposase-Accessible Chromatin with high-throughput Sequencing (ATAC-seq). SA3.2) Determine the role of β-catenin-GR/HDAC1/MeCP2 nu- clear complex in regulating GR signaling. The components and binding pattern of this complex will be deter- mined using Co-IP, Mass-Spec, FLIM-FRET, and ChIP-qPCR. In summary, we propose a novel approach to remove GC side effects without compromising its therapeutic effects. The compound that we test may serve as a lead compound in future drug development for GIG, and it would enable long-term, undisrupted use of GCs for various eye diseases upon the success of this study.

项目总结/摘要 糖皮质激素（GC）诱导的高眼压（OHT）和青光眼（GIG）发生在约40%的一般人群中。 人群和约90%的原发性开角型青光眼（POAG）患者。我们发表的研究和初步的 数据显示典型的Wnt信号传导激活抑制糖皮质激素受体信号传导和GC诱导的 好的。我们假设经典Wnt信号的激活抑制了GIG，这种抑制需要核- ar β-连环蛋白、糖皮质激素受体（GR）和表观遗传修饰酶，包括HDAC 1和MeCP 2。 我们的目标是阐明GIG的机制。我们的目标是抑制GC诱导的OHT而不损害- GC的抗炎作用。我们的理由是，Wnt激活是一种潜在的方法，以防止和 治疗OHT/GIG，研究GIG有助于我们更好地了解POAG。在此，我们提出三个具体的 旨在检验我们的核心假设。SA 1.探索Wnt信号传导如何调节GC诱导的OHT和抗 小鼠眼睛的炎症作用。我们将确定SA 1.1）如果典型的Wnt信号传导激活抑制 使用经典Wnt信号转导报告小鼠的GC诱导的OHT; SA1.2）如果β-连环蛋白对于抑制 使用条件性敲除小鼠的GC诱导的OHT;和SA1.3）如果激活经典Wnt信号传导， 使用小鼠葡萄膜炎模型研究GC的抗炎作用。SA 2.确定规范Wnt的作用 在人眼中糖皮质激素反应性的信号传导。SA2.1）确定Dkk 1（- 抑制剂）升高，而经典Wnt信号传导被抑制。我们将收集房水和小梁 GIG眼以及非GIG眼的网状组织以研究Dkk 1、β-catenin和axin 2水平。我们 在GIG眼中，Dkk 1水平较高，而β-catenin和axin 2水平较低。SA2.2）确定是否为规范 Wnt信号传导影响人供体眼中的GC反应性。我们将用DEX灌注成对的供体眼睛， 识别响应性。对于反应者眼睛，我们将用CHIR（小分子Wnt信号- 并期望它能缓解OHT。对于无反应的眼睛，我们将用Dkk 1联合治疗一只眼睛， 希望它能引起OHT。SA 3.确定经典Wnt信号传导和β- 连环蛋白在GC反应性和TM稳态中的作用。SA3.1）确定Wnt信号传导诱导的基因表达。 GC反应中的表达和相关的染色质可及性。我们将确定Wnt信号是否具有 使用RNA-seq和转座酶可降解染色质的测定对GC诱导的基因进行差异调节 高通量测序（ATAC-seq）SA3.2）确定β-连环蛋白-GR/HDAC 1/MeCP 2 nu- 在调节GR信号传导中具有明显的复合物。确定了该复合物的组成和结合模式。 使用Co-IP、Mass-Spec、FLIM-FRET和ChIP-qPCR进行挖掘。总之，我们提出了一种新的方法， 消除GC副作用，同时不影响其治疗效果。我们测试的化合物可以作为 这是未来GIG药物开发的先导化合物，它将使GCs的长期，不间断使用成为可能。 在这项研究成功的基础上，