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: 10298637
• 负责人: Weiming Mao
• 金额: $ 50.61万
• 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/10298637
• 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; Physiologic Intraocular Pressure; Primary Open Angle Glaucoma; Public Health; Publications; Publishing; Receptor Signaling; Regulation; Reporter; Research; Response Elements; Role; Signal Transduction; Testing; Therapeutic Effect; Therapeutic Glucocorticoid; Tissues; Trabecular meshwork structure; Transgenic Organisms; Transposase; Uveitis; WNT Signaling Pathway; arm; autoimmune uveitis; base; beta catenin; conditional knockout; drug development; histone modification; inhibitor/antagonist; 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.

项目摘要/摘要 糖皮质激素（GC）诱导的眼高血压（OHT）和青光眼（GIG）发生在〜40％的一般性中 人口和〜90％的初级开角素（POAG）患者。我们发表的研究和初步 数据表明，规范Wnt信号传导激活抑制了糖皮质激素受体信号传导和GC诱导的 OHT。我们假设典型的Wnt信号传导的激活抑制了GIG，并且这种抑制需要核 - ARβ-catenin，糖皮质激素受体（GR）和包括HDAC1和MECP2在内的表观遗传修饰酶。 我们的目标是阐明演出的机制。我们的目标是抑制GC诱导的OHT而不妥协 - GC的抗炎作用。我们的理由是，Wnt激活是预防和 对待OHT/演出，学习演出将有助于我们更好地理解POAG。在这里，我们提出了三个特定的 旨在检验我们的中心假设。 SA1。探索Wnt信号如何调节GC诱导的OHT和抗 - 小鼠眼中的炎症作用。我们将确定sa1.1）如果典型的Wnt信号激活抑制 GC诱导的OHT使用经典Wnt信号报告者小鼠； SA1.2）如果β-catenin对于抑制需要 GC诱导的OHT使用有条件的基因敲除小鼠； SA1.3）如果激活了经典的Wnt信号传导妥协 - MISES GC使用小鼠葡萄膜炎模型的抗炎作用。 SA2。确定规范Wnt的作用 人眼中的糖皮质激素反应性中的信号传导。 sa2.1）确定dkk1是否（wnt信号传导in- Hibitor）升高，而抑制规范的Wnt信号传导。我们将收集幽默和小梁 从演出眼睛以及从非gig眼到研究dkk1，β-catenin和axin2水平的网状组织。我们 期望看到较高的DKK1水平，但β-catenin和axin2降低了。 SA2.2）确定是否规范 WNT信号会影响人眼中的GC反应性。我们将灌注配对的供体的眼睛与dex to 确定响应能力。对于响应者的眼睛，我们将与Chir共同治疗一只眼睛（一个小分子Wnt信号 - 激活器）并期望它将营救OHT。对于无反应的眼睛，我们将与DKK1共同处理一只眼睛 期望它会引起OHT。 SA3。确定规范Wnt信号和β-的分子机制 Catenin在GC反应性和TM稳态中。 SA3.1）确定Wnt信号传导诱导的基因 GC响应中的处方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和分析酶可访问的染色质对GC诱导的基因的差异调节 带有高通量测序（ATAC-SEQ）。 SA3.2）确定β-catenin-gr/hdac1/mecp2 nu-的作用 在调节GR信号传导时清除复合物。该复合物的成分和结合模式将被确定 - 使用Co-IP，Mass-Spec，Flim-Fret和Chip-QPCR开采。总而言之，我们提出了一种新颖的方法 消除GC副作用而不会损害其治疗效果。我们测试的化合物可能是 演出未来药物开发中的铅含量，它将能够长期使用GCS 对于本研究成功的各种眼科疾病。