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)引起的高眼压(OHT)和青光眼(GIG)发生率约为40%。 人群和约90%的原发性开角型青光眼(POAG)患者。我们已发表的研究和初步报告 数据显示，典型的Wnt信号激活抑制糖皮质激素受体信号转导和GC诱导 啊哈。我们假设，规范的Wnt信号的激活抑制了GIG，并且这种抑制需要核- ARβ-连环蛋白，糖皮质激素受体(GR)，以及表观遗传修饰酶包括HDAC1和MeCP2。 我们的目标是阐明GIG的作用机制。我们的目标是在不损害的情况下抑制GC诱导的OHT- ING GC的抗炎作用。我们的理论基础是WNT激活是预防和 看待OHT/GIG，研究GIG有助于我们更好地理解POAG。在这里，我们提出三个具体的建议 旨在检验我们的核心假设。SA1.探讨Wnt信号如何调节GC诱导的OHT和ANT 对小鼠眼睛的炎症作用。我们将确定SA1.1)是否规范的Wnt信号激活抑制 使用典型的Wnt信号报告小鼠诱导GC诱导的OHT；1)是否需要β-连环蛋白来抑制 使用条件性基因敲除小鼠的GC诱导的OHT；以及SA1.3)如果激活规范的Wnt信号与 用小鼠葡萄膜炎模型研究MISES GC的抗炎作用。SA2.确定规范WNT的作用 人眼对糖皮质激素反应的信号传导。SA2.1)确定Dkk1(WNT信令- 抑制物)升高，而规范的Wnt信号被抑制。我们将收集房水和小梁 研究DKK1、β-连环蛋白和Axin2的水平。我们 预计GIG患者的DKK1水平较高，但β-连环蛋白和Axin2水平较低。SA2.2)确定是否规范 WNT信号影响人供眼的GC反应性。我们将用DEX给配对的供体眼灌流 确定响应性。对于响应者的眼睛，我们将用CHIR(一种小分子Wnt信号-)联合治疗一只眼睛。 ING激活剂)，预计它将缓解OHT。对于无反应眼，我们将与Dkk1和Dkk1共同治疗一只眼 预计它会诱发OHT。SA3.确定规范的Wnt信号和β-的分子机制 连环蛋白在GC反应和TM动态平衡中的作用。SA3.1)确定Wnt信号诱导的基因ex- GC反应中的表达和相关的染色质可及性。我们将确定WNT信令是否已 利用RNA-SEQ和转座酶可及染色质检测对GC诱导基因的差异调控 高通量测序(ATAC-SEQ)。SA3.2)确定β-连环蛋白-GR/HDAC1/MeCP2 nu-的作用 清除GR信号调节中的复合体。这个复合体的组成和结合模式将被阻止- 利用CoIP、MASS-SPEC、FLIM-FRET和CHIP-QPCR进行挖掘。总之，我们提出了一种新的方法来 消除GC副作用，而不影响其治疗效果。我们测试的化合物可能是 GIG未来药物开发的先导化合物，它将使GC的长期、不受干扰的使用成为可能 对于各种眼病来说，这项研究取得了成功。