Integrative Characterization on the function of COPD GWAS gene, HHIP

COPD GWAS 基因 HHIP 功能的综合表征

基本信息

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
项目类别：
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

项目摘要

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）是美国第三大死亡原因受香烟烟（CS）和遗传易感性的强烈影响。 hhip，编码刺猬互动蛋白质一直与对COPD的易感性有关，包括气道重塑和气肿。但是，该关联基础的分子机制仍然不完全理解。我们发表的工作表明，HHIP杂合小鼠（HHIP +/-）概括了多个 COPD病理特征，包括烟雾和年龄相关的肺气肿和气道重塑。我们也是发现HHIP在表达LGR6的气道平滑肌细胞（ASMC）和LGR5-中高度表达表达肺泡间充质细胞；在显示代谢的COPD ASMC中的表达降低从氧化磷酸化转变为与细胞生长增加有关的糖酵解。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（牙槽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，可能会调节两者气道重塑和肺气肿通过两个主要肺中的固有和外在信号传导间充质细胞类型：ASMC和肺泡间充质细胞。在当前的建议中，我们旨在扩展我们以前的研究通过解决与HHIP相关的两个机械问题：1）HHIP的缺陷如何 ASMC通过通过代谢增加气道增厚和细胞增生来促进气道重塑重新编程和2）肺泡间充质细胞中HHOH的缺乏以及如何损害生态位 II细胞生成的支持。这些问题将通过生化的组合解决测定，谱系跟踪，基于CRISPR的基因组编辑和类器官培养模型。在AIM1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，这是一种限制酶糖酵解的最后一步。我们将进一步表征它们在AMSC中的相互作用，并确定影响在CS引起的气道重塑上的HHIP。在AIM 2中，我们假设HHIP缺乏会导致受损 LGR5+肺泡间充质细胞中对AT II细胞再生很重要的利基功能确定肺气肿的敏感性。为了测试这一点，我们选择在LGR5+细胞中有条件地耗尽HHIP，A 已知的肺泡间充质细胞标记物，然后进行CS暴露，随后的测量空域的大小，Wnt和刺猬途径的活性以及两者的增殖和分化 AIM 2.1中的互补方法，牙槽间充质细胞（LGR5+）和在II细胞上进行了II细胞。在AIM 2.2中，我们将使用肺泡类器官共培养模型来剖定HHIP确定利基函数的机制使用来自Hop的小鼠的鼠细胞或由CRISPR/CAS-9编辑的人类原代成纤维细胞靶向HHIP的方法。该项目的成功完成将阐明分子洞察到CS诱导的 COPD通过HHIP确定的敏感性。