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

项目摘要/摘要 近几十年来，全基因组关联研究(GWAS)已经确定了与 各种特征和疾病，这是理解分子机制的关键第一步 是常见的健康状况的基础。然而，在许多情况下，Gwas基因的功能、机制 以及其功能障碍的后果，或其功能障碍表现为 临床上有意义的表型仍然知之甚少。Gwas基因dsp编码一种连接蛋白。 发现于桥粒中，为经历机械反应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。一种与肺组织中的DSP表达相关的变体，而不是其他组织， Rs2076295与肺纤维化和慢性阻塞性肺疾病有关。 GWA型的易感性。近年来，我们和其他人开发了协议，以区分、成熟和 源于IPSCs的人类AT2s的模型疾病(IAT2s)，在为这项提议做准备时，我们有 采用这个模型系统来询问与肺部疾病相关的GWAs的发现。我们假设 Rs2076295介导的DSP减少通过破坏桥粒的稳定性来调节AT2表型 Wnt/Tcf信号在动态平衡或损伤背景下对AT2分化的调节 并诱导成纤维细胞间质表型。为了在该提案中检验这一假设，我们将 测试DSP通过基于CRISPR的基因敲除或 过表达DSP或其结合伙伴。我们将确定减少的DSP表达对 IAT2的转分化能力和细胞在过渡状态下的潜在出现。然后我们将测试 在体内利用AT2特异性的DSP在AT2再生能力和相关纤维化肺损伤中的作用 合并博莱霉素损伤的小鼠中的DSP缺失。最后，我们将利用LTRC数据来确定 Rs2076295调节基因表达的机制，然后在 病人2小时急救。

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.