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

项目摘要/摘要 纤维化是细胞外基质(ECM)蛋白的过度分泌，导致组织僵硬和 器官功能减退。成纤维细胞和肌成纤维细胞是细胞外基质的主要细胞类型 证词。特发性肺纤维化(IPF)是一种病因不明的进行性纤维性疾病。的作用 通过TGFb信号通路的生化信号是IPF和其他纤维化的公认驱动因素 疾病。机械信号在调节细胞外基质沉积中也发挥着新的和不断发展的作用。 成纤维细胞和纤维化的进展。机械输入的分子机制 结合生化信号来调节纤维化仍是个谜。我们实验室的工作表明 核骨架和细胞骨架(LINC)复合体的连接物作为一种新的纤维化调节因子；LINC的消融 SUN2-/-小鼠模型中的复杂成分SUN2在肥大偶联的心脏中具有保护作用 纤维化和损伤性肺纤维化。鉴于LINC复合体在机械传递中的既定作用 从细胞表面到细胞核的作用力，一个令人信服的假设是LINC复合体有助于 纤维化进展过程中的机械转导。这项提案的总体目标是定义 SUN2缺失导致抗纤维化的分子机制。 这项拟议的研究将从两个具体目标来探讨这一目标。目标1将定义整合 生化(调节TGFb和TGFb受体)和机械(调节细胞底物硬度)输入 在调节促肝纤维化基因表达和获得促肝纤维化表型中的作用 小鼠肺成纤维细胞。SUN蛋白影响促肝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。目标2将定义太阳蛋白质丢失对两种染色质状态的影响 以及已建立的TGFb信号转导，Smad转录因子家族，使用一种组合 基因组技术和成像方法。这项提案将涉及基因的基本方面。 监管，因为它具体涉及肺纤维化的进展，符合世界卫生组织的使命 国家心肺血液研究所。 学员将沉浸在支持、协作和跨学科的环境中，同时 在发起人/共同发起人的大力支持和指导下，最终完成拟议的项目 让她通过翻译的视角来处理基本问题。她会提高自己的水平 实验设计技能，在严格分析实验数据方面获得培训，并扩展她的技术 技能包括从基因组学到组织成像的方法。实习生还将有足够的机会访问 提高口头陈述、写作、指导和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.