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

项目总结/摘要 近几十年来，全基因组关联研究（GWAS）已经确定了与以下相关的遗传变异： 各种性状和疾病，这是了解分子机制的关键第一步， 是常见健康状况的基础。然而，在许多情况下，GWAS基因的功能， 以及它们的功能障碍的后果，或者它们的功能障碍表现为 临床上显著的表型仍然知之甚少。GWAS基因DSP编码一个连接蛋白 在桥粒中发现，桥粒为经历机械损伤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体， rs 2076295与肺纤维化和慢性阻塞性肺病有关 GWAS中的易感性。近年来，我们和其他人已经开发出了区分，成熟， 在来源于iPSC的人AT 2（iAT 2）中的模型疾病，并且在准备该提议时，我们 调整该模型系统以询问与肺部疾病相关的GWAS发现。我们假设 通过rs 2076295介导的DSP的减少使桥粒不稳定，从而通过以下途径调节AT 2表型： 在稳态或损伤AT 2分化的损伤环境中调节Wnt/Tcf信号传导 能力并诱导促纤维化间充质表型。为了验证这一假设，我们将 测试DSP通过基于CRISPR的敲低调节iAT 2成熟的机制，或 DSP或其结合伴侣的过表达。我们将确定减少DSP表达的贡献， iAT 2转分化能力和过渡状态细胞的潜在出现。然后我们将测试 DSP对AT 2再生能力和相关的纤维化肺损伤的贡献 博莱霉素损伤小鼠Dsp缺失的研究。最后，我们将利用LTRC数据来确定 rs 2076295调节基因表达的机制，然后验证这些预测。 患者iAT 2。

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.