Mechanistic studies of the genetic contribution of desmoplakin to pulmonary fibrosis in alveolar type 2 cells

桥粒斑蛋白对肺泡2型细胞肺纤维化的遗传贡献机制研究

基本信息

批准号：
10736228
负责人：
ANDREW A WILSON
金额：
$ 79.94万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10736228
关键词：
6p24 AGTR2 gene ATAC-seq Adherens Junction Adult Affect Alveolar Binding Bioinformatics Biological Assay Biological Models Biology Bleomycin CISH gene CRISPR interference CRISPR-mediated transcriptional activation Cell Differentiation process Cell Maturation Cells Chronic Obstructive Pulmonary Disease Clustered Regularly Interspaced Short Palindromic Repeats Complex Data Desmosomes Disease Disease model Disease susceptibility Distal Epithelial Cells Epithelium Family member Functional disorder Gene Expression Genes Genetic study Genome Health Heart Homeostasis Human Impairment Injury Intercellular Junctions Knock-out Label Link Linkage Disequilibrium Lung Lung diseases Mechanical Stress Mediating Mesenchymal Modeling Molecular Mus Nuclear Organoids Participant Pathogenesis Pathway Analysis Pathway interactions Patients Phenotype Physiological Play Preparation Probability Proteins Protocols documentation Publishing Pulmonary Fibrosis Regenerative capacity Respiratory Disease Risk Role Sampling Signal Transduction Skin Structure of parenchyma of lung Testing Tissues Variant alveolar epithelium armadillo proteins causal variant cell type cigarette smoke clinically significant desmoplakin disorder risk experience fibrotic lung genetic variant genome wide association study human subject in vivo induced pluripotent stem cell knock-down lung injury overexpression plakoglobin regenerative cell response risk variant single cell sequencing stem cell function stem cell model trait transcription factor transcriptome sequencing transcriptomics transdifferentiation transgene expression translational potential

项目摘要

Project Summary/Abstract In recent decades, genome-wide association studies (GWAS) have identified genetic variants associated with a variety of traits and diseases, a critical first step towards understanding the molecular mechanisms that underlie common health conditions. In many cases, however, the functions of GWAS genes, the mechanisms and consequences of their dysfunction, or the relevant cell types in which their dysfunction manifests a clinically significant phenotype remain poorly understood. The GWAS gene DSP encodes a junctional protein found in desmosomes that provide structural integrity to epithelial cells in tissues that experience mechanical stress, such as skin, heart, and lung. A variant associated with DSP expression in lung but not other tissues, rs2076295, has been linked to both pulmonary fibrosis and chronic obstructive pulmonary disease susceptibility in GWAS. In recent years, we and others have developed protocols to differentiate, mature, and model disease in human AT2s derived from iPSCs (iAT2s) and in preparation for this proposal we have adapted this model system to interrogate lung disease-relevant GWAS discoveries. We hypothesize that reductions in DSP mediated through rs2076295 destabilize desmosomes to regulate AT2 phenotypes through modulation of Wnt/Tcf signaling either at homeostasis or in the setting of injury that impairs AT2 differentiation capacity and induces a profibrotic mesenchymal phenotype. To test this hypothesis in this proposal, we will test the mechanisms through which DSP regulates iAT2 maturation through CRISPR-based knockdown or overexpression of DSP or its binding partners. We will identify the contribution of reduced DSP expression on iAT2 transdifferentiation capacity and potential emergence of cells in a transitional state. We will then test the contribution of DSP to AT2 regenerative capacity and associated fibrotic lung injury in vivo using AT2-specific Dsp deletions in mice in combination with bleomycin injury. Finally, we will leverage LTRC data to determine mechanisms through which rs2076295 regulates gene expression and then validate those predictions in patient iAT2s.

项目概要/摘要近几十年来，全基因组关联研究（GWAS）已经确定了与多种性状和疾病，这是了解其分子机制的关键的第一步是常见健康状况的基础。然而，在许多情况下，GWAS 基因的功能、机制及其功能障碍的后果，或其功能障碍表现出的相关细胞类型具有临床意义的表型仍然知之甚少。 GWAS基因DSP编码连接蛋白存在于桥粒中，为经历机械作用的组织中的上皮细胞提供结构完整性压力，如皮肤、心脏和肺。一种与肺中 DSP 表达相关但与其他组织无关的变异， rs2076295，与肺纤维化和慢性阻塞性肺病有关 GWAS 的易感性。近年来，我们和其他人开发了协议来区分、成熟和源自 iPSC (iAT2) 的人类 AT2 模型疾病，在为该提案做准备时，我们调整该模型系统来询问与肺部疾病相关的 GWAS 发现。我们假设通过 rs2076295 介导的 DSP 减少会破坏桥粒的稳定性，从而通过以下方式调节 AT2 表型在稳态或损伤 AT2 分化的情况下调节 Wnt/Tcf 信号传导能力并诱导促纤维化间充质表型。为了检验本提案中的这一假设，我们将测试 DSP 通过基于 CRISPR 的敲除来调节 iAT2 成熟的机制，或 DSP 或其结合伙伴的过度表达。我们将确定 DSP 表达减少对 iAT2 转分化能力和细胞处于过渡状态的潜在出现。然后我们将测试使用 AT2 特异性的 DSP 对 AT2 再生能力和体内相关纤维化肺损伤的贡献小鼠中 Dsp 缺失与博来霉素损伤相结合。最后，我们将利用 LTRC 数据来确定 rs2076295 调节基因表达的机制，然后验证这些预测患者 iAT2。