Integrative Characterization on the function of COPD GWAS gene, HHIP

COPD GWAS 基因 HHIP 功能的综合表征

• 批准号: 10598541
• 负责人: Wenyi Wei
• 金额: $ 65.55万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-05 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10598541
• 关键词: 3-Dimensional; Address; Agonist; Airway Fibrosis; Alveolar; Alveolus; Binding; Biochemical; Biological; Biological Assay; CRISPR/Cas technology; Cause of Death; Cell Differentiation process; Cell Proliferation; Cells; Chromatin; Chronic; Chronic Obstructive Pulmonary Disease; Cigarette smoke-induced emphysema; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Collagen; Communication; Data; Deposition; Development; Differentiation and Growth; Disease susceptibility; Distal; Enzymes; Epithelial Cells; Erinaceidae; Fibroblasts; Foundations; General Population; Generations; Genes; Genetic Determinism; Genetic Predisposition to Disease; Genotype; Glycolysis; Growth; Heterozygote; Human; Hyperplasia; Impairment; In Vitro; Investigation; Isoenzymes; LGR5 gene; Lung; Measurement; Mediating; Mesenchymal; Metabolic; Methods; Modeling; Molecular; Mus; Organoids; Oxidative Phosphorylation; Oxygen Consumption; Pathologic; Pathway interactions; Patients; Predisposition; Proliferating; Protein Deficiency; Protein Kinase Interaction; Proteins; Public Health; Publishing; Pulmonary Emphysema; Pyruvate Kinase; Role; SHH gene; Signal Transduction; Smoke; Smooth Muscle Myocytes; Sorting; Testing; Type II Epithelial Receptor Cell; Work; aerobic glycolysis; age related; aging population; airway obstruction; airway remodeling; alveolar type II cell; cell growth; cell regeneration; cell type; cigarette smoke; cigarette smoke-induced; cigarette smoke-induced COPD; cigarette smoking; disease phenotype; exposure to cigarette smoke; genome editing; genome wide association study; in vivo; inhibitor; insight; mutant; novel; personalized medicine; prevent; protein expression; pulmonary function; pyruvate kinase deficiency; respiratory smooth muscle; risk variant; single-cell RNA sequencing; stem cells; targeted treatment

Chronic obstructive pulmonary disease (COPD), ranks as the third leading cause of death in the U.S. It is also strongly influenced by cigarette smoke (CS) and genetic predisposition. HHIP, encoding Hedgehog interacting protein, has consistently been associated with the susceptibility to COPD including airway remodeling and emphysema. However, the molecular mechanism underlying this association remains incompletely understood. Our published work has demonstrated that Hhip heterozygous mice (Hhip+/-) recapitulated multiple COPD pathological features including smoke- and age-related emphysema and airway remodeling. We also found that HHIP is highly expressed in Lgr6-expressing airway smooth muscle cells (ASMCs) and Lgr5- expressing alveolar mesenchymal cells; has reduced expression in COPD ASMCs that display a metabolic shift from oxidative phosphorylation to glycolysis associated with increased cell growth. Furthermore, alveolar fibroblasts-derived HHIP promotes proliferation of AT II (alveolar type II) cells in alveolar organoid co-culture model. These findings suggested that Hhip, the key genetic determinant for COPD, possibly modulates both airway remodeling and emphysema through complementarily intrinsic and extrinsic signaling in two major lung mesenchymal cell types: ASMCs and alveolar mesenchymal cells. In this current proposal, we aim to extend our previous studies by addressing two mechanistic questions related with HHIP: 1) How deficiency of Hhip in ASMCs promote airway remodeling by increasing airway thickening and cell hyperplasia through metabolic reprograming and 2) whether and how deficiency of Hhip in alveolar mesenchymal cells have impaired niche to support AT II cell generation. These questions will be addressed through the combinations of biochemical assays, lineage tracing, CRISPR-based genome editing and organoid co-culture models. In Aim1, we have identified a novel interaction between HHIP and PKM2 (pyruvate kinase isozyme M2), a rate-limiting enzyme in the last step for glycolysis. We will further characterize their interaction in AMSCs as well as determine impacts of HHIP on CS-induced airway remodeling. In Aim 2, we hypothesize that Hhip deficiency leads to impaired niche function in Lgr5+ alveolar mesenchymal cells that are important for AT II cells regeneration thereby determines emphysema susceptibility. To test this, we choose to conditionally deplete Hhip in Lgr5+ cells, a known alveolar mesenchymal cells marker followed by CS exposure and subsequent measurements on airspace size, the activity of the Wnt and Hedgehog pathway and proliferation and differentiation of both alveolar mesenchymal cells (Lgr5+) and AT II cells by complementary approaches in Aim 2.1. In Aim 2.2., we will use alveolar organoid co-culture model to dissect the mechanism by which Hhip determines niche function using either murine cells from Hhip deficient mice or from human primary fibroblasts edited by CRISPR/Cas-9 method targeting HHIP. Successful completion of this project will illuminate molecular insights into CS-induced COPD susceptibility determined by HHIP.

慢性阻塞性肺疾病（COPD）是美国第三大死因 受香烟烟雾（CS）和遗传倾向的强烈影响。 HHIP，编码 Hedgehog 交互 蛋白质，一直与慢性阻塞性肺病（COPD）的易感性相关，包括气道重塑和 气肿。然而，这种关联背后的分子机制仍然不完全 明白了。我们发表的工作表明，Hhip 杂合小鼠 (Hhip+/-) 重现了多种 COPD 病理特征包括吸烟和年龄相关的肺气肿和气道重塑。我们也 发现 HHIP 在表达 Lgr6 的气道平滑肌细胞 (ASMC) 和 Lgr5 中高表达 表达肺泡间充质细胞； COPD ASMC 中的表达减少，显示代谢 从氧化磷酸化转变为与细胞生长增加相关的糖酵解。此外，肺泡 成纤维细胞衍生的 HHIP 促进肺泡类器官共培养中 AT II（肺泡 II 型）细胞的增殖 模型。这些发现表明 Hhip 是 COPD 的关键遗传决定因素，可能调节这两种疾病 通过两个主要肺部互补的内在和外在信号传导来实现气道重塑和肺气肿 间充质细胞类型：ASMC 和肺泡间充质细胞。在当前的提案中，我们的目标是延长 我们之前的研究通过解决与 HHIP 相关的两个机制问题：1）Hhip 缺陷如何影响 HHIP？ ASMC 通过代谢增加气道增厚和细胞增生，从而促进气道重塑 重编程和 2) 肺泡间充质细胞中 Hhip 的缺陷是否以及如何损害了生态位 支持AT II细胞生成。这些问题将通过生化的结合来解决。 分析、谱系追踪、基于 CRISPR 的基因组编辑和类器官共培养模型。在目标 1 中，我们有 确定了 HHIP 和 PKM2（丙酮酸激酶同工酶 M2）之间的新型相互作用，PKM2 是一种限速酶 糖酵解的最后一步。我们将进一步描述它们在 AMSC 中的相互作用并确定影响 HHIP 对 CS 诱导气道重塑的影响。在目标 2 中，我们假设髋关节缺陷会导致功能障碍 Lgr5+ 肺泡间充质细胞中的生态位功能对于 AT II 细胞再生非常重要 决定肺气肿的易感性。为了测试这一点，我们选择有条件地耗尽 Lgr5+ 细胞中的 Hhip， 已知的肺泡间充质细胞标记物，然后进行 CS 暴露和随后的测量 空腔大小、Wnt 和 Hedgehog 通路的活性以及两者的增殖和分化 通过目标 2.1 中的互补方法培养肺泡间充质细胞 (Lgr5+) 和 AT II 细胞。在目标 2.2 中，我们 将利用肺泡类器官共培养模型来剖析Hhip决定生态位功能的机制 使用来自 Hhip 缺陷小鼠的鼠细胞或通过 CRISPR/Cas-9 编辑的人类原代成纤维细胞 针对 HHIP 的方法。该项目的成功完成将阐明 CS 诱导的分子洞察 COPD 易感性由 HHIP 测定。