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

定义核层在肺纤维化机械调节中的作用

• 批准号: 10387049
• 负责人: Emma Carley
• 金额: $ 4.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-16 至 2025-01-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10387049
• 关键词: ATAC-seq; Ablation; Address; Binding; Biochemical; Biology; Cell Nucleus; Cell physiology; Cell surface; Cells; Cellular biology; ChIP-seq; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Complex; Coupled; Cytoskeleton; Data; Data Analyses; Deposition; Disease; Disease model; Excretory function; Experimental Designs; Extracellular Matrix; Extracellular Matrix Proteins; Family; Fibroblasts; Fibrosis; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genomic approach; Genomics; Heart Injuries; Human; Hypertrophy; Image; Imaging Techniques; Injury; Knowledge; Lung; Mechanics; Mentors; Mentorship; Methods; Mission; Modeling; Molecular; Mus; Myofibroblast; National Heart, Lung, and Blood Institute; Nuclear; Nuclear Envelope; Nuclear Lamina; Oral; Organ; Pathway interactions; Phenotype; Phosphorylation; Physiological; Play; Positioning Attribute; Profibrotic signal; Proteins; Publishing; Pulmonary Fibrosis; Regulation; Research; Role; Signal Pathway; Signal Transduction; System; Techniques; Testing; Tissue imaging; Tissues; Training; Transducers; Work; Writing; base; career; cell type; collaborative environment; computerized tools; coronary fibrosis; experimental analysis; experimental study; idiopathic pulmonary fibrosis; imaging approach; improved; insight; lens; mechanical force; mechanical signal; mechanotransduction; mouse model; novel; novel strategies; outreach; overexpression; protective effect; receptor; skills; supportive environment; tool; transcription factor; transcriptome

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.

项目概要/摘要 纤维化是细胞外基质（ECM）蛋白的过度排泄，导致组织僵硬和 器官功能下降。成纤维细胞和肌成纤维细胞是 ECM 的主要细胞类型 沉积。特发性肺纤维化（IPF）是一种来源不明的进行性纤维化疾病。的作用 通过 TGFb 信号通路的生化信号传导是 IPF 和其他纤维化的公认驱动因素 疾病。机械信号在 ECM 沉积调节中也发挥着新兴和不断发展的作用 成纤维细胞和纤维化的进展。机械输入的分子机制 与生化信号整合来调节纤维化仍然是个谜。我们实验室的工作涉及 核骨架和细胞骨架连接体（LINC）复合物作为纤维化的新调节剂； LINC 消融 Sun2-/- 小鼠模型中的复杂成分 SUN2 在心肌肥大相关的情况下具有保护作用 纤维化和损伤引起的肺纤维化。鉴于 LINC 复合体在传递机械力方面的既定作用 从细胞表面到细胞核的力，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。该提案的总体目标是定义 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 生化（调节 TGFb 和 TGFb 受体）和机械（调节细胞基质硬度）输入 在原代人类和人类中促纤维化基因表达的调节和促纤维化表型的获得中 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 将使用基于 CRISPR 的基因编辑、转录组来询问调节基因表达的信号 分析和成像技术。目标 2 将定义 SUN 蛋白丢失对染色质状态的影响 以及已建立的 TGFb 信号传导转导子（转录因子 Smad 家族），使用组合 基因组技术和成像方法。该提案将解决基因的基本方面 监管，因为它具体涉及肺纤维化的进展，符合该组织的使命 国家心、肺和血液研究所。 学员将沉浸在一个支持性、协作性和跨学科的环境中，同时 在赞助商/共同赞助商的大力支持和指导下，最终完成了拟议的项目 让她能够通过转化的视角来解决基本问题。她会提高自己 实验设计技能，获得严格分析实验数据的培训，并扩展她的技术 技能集包括从基因组学到组织成像的方法。受训者还将有足够的机会接触到 提高口头表达、写作、指导、科学推广技能的机会。