Defining the role of the nuclear lamina in the mechanical regulation of lung fibrosis

项目总结/摘要 纤维化是细胞外基质（ECM）蛋白的过度分泌，其导致组织硬化， 器官功能下降。成纤维细胞和肌成纤维细胞是负责ECM的主要细胞类型 证词特发性肺纤维化（IPF）是一种原因不明的进行性纤维化疾病。的作用 通过TGF β信号传导途径的生化信号传导是IPF和其他纤维化疾病的公认驱动因素。 疾病机械信号在ECM沉积的调节中也有一个新兴的和不断发展的作用 和纤维化的进展。机械输入的分子机制 与生物化学信号整合来调节纤维化仍然是个谜。我们实验室的研究表明 核骨架和细胞骨架连接体（LINC）复合物作为一种新的纤维化调节剂; LINC消融 复合组分SUN2在Sun2-/-小鼠模型中在肥厚性偶联心脏病的背景下具有保护作用。 纤维化和损伤诱导的肺纤维化。鉴于LINC复合物在传递机械信号中的既定作用， 力从细胞表面到细胞核，一个令人信服的假设是，LINC复合物有助于 纤维化进展过程中的机械转导。本提案的总体目标是界定 SUN2缺失导致纤维化保护的分子机制。 拟议的研究将在两个具体目标中调查这一目标。目标1将定义集成 生物化学（调节TGF β和TGF β受体）和机械（调节细胞基质硬度）输入 在原代人中调节促纤维化基因表达和获得促纤维化表型， 小鼠肺成纤维细胞。SUN蛋白影响促纤维化整合的背景和机制 调控基因表达的信号将使用基于CRISPR的基因编辑、转录组 分析和成像技术。目的2将定义SUN蛋白的丢失对染色质状态的影响 和已建立的TGF β信号转导子，转录因子的Smad家族，使用组合 基因组技术和成像技术的结合。该提案将解决基因的基本方面 由于其特别涉及肺纤维化的进展，因此与肺纤维化的使命一致， 国家心肺血液研究所。 学员将沉浸在一个支持，协作和跨学科的环境中， 最终在赞助商/共同赞助商的大力支持和指导下完成拟议项目 使她能够通过一个平移的透镜来处理基本问题。她会改善她的 实验设计技能，获得严格的实验数据分析培训，并扩大她的技术 包括从基因组学到组织成像的方法。学员还将有足够的机会， 提高口头陈述、写作、指导、科学推广技能的机会。

PROJECT SUMMARY / ABSTRACT Fibrosis is the excessive excretion of extracellular matrix (ECM) proteins, which leads to tissue stiffening and decreased organ function. Fibroblasts and myofibroblasts are the primary cell types responsible for ECM deposition. Idiopathic Pulmonary Fibrosis (IPF) is a progressive fibrotic disease of unknown origin. The role of biochemical signaling through the TGFb signaling pathway is a well-established driver of IPF and other fibrotic disease. There is also an emerging and evolving role for mechanical signaling in the regulation of ECM deposition in fibroblasts and the progression of fibrosis. The molecular mechanism by which mechanical inputs are integrated with biochemical signals to regulate fibrosis remains enigmatic. Work from our lab has implicated the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex as a new regulator of fibrosis; ablation of the LINC complex component SUN2 in a Sun2-/- mouse model is protective in the context of hypertrophy-coupled cardiac fibrosis and injury-induced lung fibrosis. Given the established role of LINC complexes in transmitting mechanical force from the cell surface to the nucleus, a compelling hypothesis is that LINC complexes contribute to mechanotransduction during fibrosis progression. The overall objective of this proposal is to define the molecular mechanisms by which loss of SUN2 leads to protection from fibrosis. The proposed research will investigate this objective in two specific aims. Aim 1 will define the integration of biochemical (modulating TGFb and TGFb receptor) and mechanical (modulating cell substrate stiffness) inputs in the regulation of pro-fibrotic gene expression and attainment of pro-fibrotic phenotypes in primary human and mouse lung fibroblasts. The contexts and mechanism by which SUN proteins influence integration of pro-fibrotic signals to regulate gene expression will be interrogated using CRISPR-based gene editing, transcriptome analysis, and imaging techniques. Aim 2 will define the impact of loss of SUN proteins on both chromatin state and the established transducers of TGFb signaling, the Smad family of transcription factors, using a combination of genomic techniques and imaging approaches. This proposal will address fundamental aspects of gene regulation as it pertains specifically to the progression of lung fibrosis, in line with the mission of the National Heart, Lung, and Blood Institute. The trainee will be immersed in a supportive, collaborative and interdisciplinary environment while completing the proposed project under the rich support and mentorship of the sponsor/co-sponsor, ultimately positioning her to approach fundamental questions through a translational lens. She will improve her experimental design skills, obtain training in rigorous analysis of experimental data, and expand her technical skillset to include methods from genomics to imaging of tissue. The trainee will also have ample access to opportunities for improving oral presentation, writing, mentorship, scientific outreach skills.