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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：
10379283
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 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10379283
关键词：
3-Dimensional Address Age 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 Generations Genes Genetic Determinism Genetic Predisposition to Disease Genotype Glycolysis Growth 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 Protein Deficiency Proteins Public Health Publishing Pulmonary Emphysema Pyruvate Kinase Role SHH gene Signal Transduction Smoke Smooth Muscle Myocytes Testing Tomatoes Work aerobic glycolysis age related aging population airway obstruction airway remodeling alveolar type II cell base 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 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.

慢性阻塞性肺病(COPD)是美国第三大致死原因。它也是受吸烟(CS)和遗传易感性的强烈影响。HHIP，编码刺猬交互蛋白质一直与COPD的易感性有关，包括气道重塑和肺气肿。然而，这种联系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杂合子小鼠(HHIP+/-)可概括为多个 COPD的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高表达。表达肺泡间充质细胞；在表现为代谢的COPD ASMCs中表达降低随着细胞生长的增加，从氧化磷酸化转变为糖酵解。此外，肺泡成纤维细胞来源的HHIP促进肺泡II型细胞增殖模特。这些发现表明，慢性阻塞性肺疾病的关键基因决定因素HHIP可能调节两者两大肺内、外信号互补的气道重塑与肺气肿间充质细胞类型：间质细胞和肺泡间充质细胞。在目前的提案中，我们的目标是延长我们以前的研究通过解决与髋关节相关的两个力学问题：1)髋关节如何在 ASMC通过代谢增加气道增厚和细胞增殖促进气道重塑 2)肺泡间充质细胞HHIP的缺失是否以及如何损害了支持AT II细胞生成。这些问题将通过生物化学的组合来解决分析、谱系追踪、基于CRISPR的基因组编辑和有机化合物共培养模型。在Aim1，我们有发现了HHIP与PKM2(丙酮酸激酶同工酶M2)之间的一种新的相互作用，PKM2是HHIP的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，并确定其影响 HHIP对CS诱导的气道重塑的影响。在目标2中，我们假设髋关节缺陷会导致受损 Lgr5+肺泡间充质细胞的生态位功能对AT II细胞的再生非常重要决定了肺气肿的易感性。为了测试这一点，我们选择有条件地耗尽Lgr5+细胞中的HHIP，a 已知的肺泡间充质细胞标记物和CS暴露后的后续测量空域大小、Wnt和Hedgehog通路的活性以及两者的增殖和分化肺泡间充质细胞(Lgr5+)和AT II细胞在Aim 2.1中的互补方法。在AIM 2.2中，我们将使用肺泡有机化合物共培养模型来剖析HHIP决定生态位功能的机制使用HHIP缺陷小鼠的小鼠细胞或CRISPR/Cas-9编辑的人原代成纤维细胞方法靶向HHIP。该项目的成功完成将阐明对CS诱导的分子洞察力 HHIP法测定COPD易感性。