p16INK4a+ fibroblasts regulate epithelial regeneration after injury in lung alveoli through the SASP

p16INK4a成纤维细胞通过SASP调节肺泡损伤后的上皮再生

基本信息

批准号：
10643269
负责人：
Nabora Soledad Reyes de Barboza
金额：
$ 17.8万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10643269
关键词：
3-Dimensional Acute Address Adult Age Aging Alleles Alveolar Alveolus Anatomy Architecture Basement membrane Behavior Bioinformatics Biological Assay Biology Biotinylation CDKN2A gene Cell Aging Cell Cycle Cell Size Cells Characteristics Coculture Techniques Cyclin-Dependent Kinase Inhibitor 2A Cyclins DNA Damage Dasatinib Data Dependence Development Epithelium Excision Fibroblasts Foundations Gases Gene Expression Profile Genetic Genetic Transcription Goals Growth Growth Factor Homeostasis IL6 gene Immune Immune system Inflammatory Injury Knowledge Lead Ligands Lung Matrix Metalloproteinases Mediating Metabolic Modeling Mus Names Natural regeneration Nature Oncogenic Organoids Phase Phenotype Play Polyploidy Population Proliferating Protein Secretion Proteomics Protocols documentation Pulmonary alveolar structure Quercetin Reporter Resistance Role Stimulus Stress Surface TP53 gene Techniques Testing Tissues Training Tumor Escape Tumor Suppressor Proteins Up-Regulation Work airway epithelium airway regeneration alveolar epithelium biological adaptation to stress career development cell growth cell type cytokine environmental stressor epithelial injury epithelial stem cell epithelium regeneration experimental study immune cell infiltrate in vivo inhibitor injured airway injury and repair insight knock-down lung regeneration novel programs receptor repaired response response to injury senescence single-cell RNA sequencing skills stem cell growth stem cell proliferation stem cells stressor tissue repair tool transcriptome transcriptome sequencing transcriptomics tumorigenesis wound healing young adult

项目摘要

PROJECT SUMMARY ABSTRACT Cellular Senescence is as an acquired state cells enter in response to environmental stressors or tumor evasion. Senescent cells cease their proliferative capacity and enhance their ability to respond and regulate the microenvironment through the senescence-associated secretory phonotype (SASP). Another shared characteristic of cellular senescence is the upregulation of the tumor suppressor cyclin inhibitor p16INK4a. With age there’s accumulation of senescence cells in tissues along with the upregulation of p16INK4a expression. While removal of p16INK4a expressing cells using genetic mouse tools slows down aging, it also adversely impacts wound healing during injury repair, suggesting contradictory roles of p16INK4a during homeostasis and injury response. We generated an ultra-sensitive p16INK4a reporter mouse line name, named INK4A H2B-GFP Reporter-In-Tandem, or INKBRITE to further understand in vivo p16INK4+ cells. In INKBRITE adult lungs, p16INK4+ cells are predominantly within immune and fibroblasts populations. p16INK4+ fibroblasts express features of senescence including polyploidy, increase in cell size, low proliferation capacity and ability to promote airway epithelial cell growth after injury. My main goal is to determine if the capacity to promote regeneration is restricted to airway p16INK4+ and epithelium or other regionally specific p16INK4 expressing fibroblast can support epithelial growth. Whether the capacity to promote epithelial regeneration is restricted to airway p16INK4+ fibroblasts or other spatially defined fibroblast subpopulations such as alveolar fibroblast can promote epithelial growth through SASP, remains unknown. To fill this knowledge gap, I will isolate alveolar p16INK4a+ fibroblasts and determine their capacity to promote epithelial growth after acute epithelial injury by 1) ex vivo 3D co-culture assay, 2) identify the transcribed SASP through RNA sequencing, and 3) in vivo using known senolytics Dasatinib and Quercetin (D&Q). Our p16INK4a induction and knockdown studies showed the requirement of p16INK4a for expression of known SASP factors such as IL6, Ereg, Ccl8. Another aspect that has been largely underexplored in the identity and dependence p16INK4 expression of SASP factors in vivo. For the R00 phase of my proposed work, I will further explore how the expression of p16INK4 is able to reprogram the SASP to support tissue repair, specifically epithelial regeneration. I will with our tools to functionally induce and remove p16INK4 in fibroblasts and 1) asses epithelial growth, 2) transcriptome analysis, and 3) proteomics to capture secreted proteins to identify p16INK4a-dependent SASP factors. I have extensive knowledge on working with INKBRITE and the tools to manipulate p16 INK4a expression which will allow me to pursue the proposed work with ease. With additional training from Drs. Peng and Sheppard I will expand my current knowledge of lung biology and cellular senescence while establishing a new protocol of identifying secreted proteins in vivo that will serve as a unique skillset for my transition to my own lab. These studies will lay foundation for our understanding of diverse roles of SASP factors during homeostasis and injury.

项目摘要摘要细胞敏感是作为对环境应激源或肿瘤的响应的获得的状态细胞输入的逃避。衰老细胞停止其增殖能力并增强其响应能力和调节的能力通过感应相关的秘书型（SASP）的微环境。另一个共享细胞感应的特征是肿瘤抑制细胞周期蛋白抑制剂P16INK4A的上调。和年龄在组织中积累了感应细胞以及p16Ink4a表达的上调。虽然使用遗传小鼠工具去除表达细胞的P16INK4A会减慢衰老，但也有不利的影响伤害修复过程中的伤口愈合，表明p16ink4a在体内稳态和伤害反应。我们生成了一个超敏感的P16INK4A记者鼠标线名，名为Ink4a H2B-GFP 记者在tandem中或inkbrite，以进一步了解体内P16INK4+细胞。在inkbrite成人肺中， P16INK4+细胞主要在免疫内部，成纤维细胞种群。 P16INK4+成纤维细胞Express 感应的特征，包括多倍体，细胞大小的增加，低增殖能力和能力损伤后促进气道上皮细胞生长。我的主要目标是确定是否有能力促进再生仅限于气道P16INK4+和上皮或其他特定于区域特定的P16INK4表达成纤维细胞可以支持上皮生长。促进上皮再生的能力是否仅限于气道P16INK4+成纤维细胞或其他空间定义的成纤维细胞亚群，例如肺泡成纤维细胞可以通过SASP促进上皮生长仍然未知。为了填补这一知识空白，我将隔离肺泡 P16INK4A+成纤维细胞，并确定其在急性上皮损伤后促进上皮生长的能力1）外体3D共培养分析，2）通过RNA测序识别转录的SASP，3）使用体内使用已知的Senolotics Dasatinib和槲皮素（D＆Q）。我们的P16INK4A诱导和敲低研究表明 p16Ink4a的要求表达已知SASP因子，例如IL6，EREG，CCL8。另一个方面在体内的SASP因子的身份和依赖性p16INK4表达中，大大毫无疑问。为了我提出的工作的R00阶段，我将进一步探讨p16ink4的表达方式如何重新编程 SASP支持组织修复，特别是上皮再生。我将使用我们的工具来诱导和删除成纤维细胞中的p16ink4和1）驴上皮生长，2）转录组分析和3）蛋白质组学捕获分泌的蛋白质以识别p16Ink4a依赖性SASP因子。我有广泛的工作知识使用inkbrite和操纵p16 ink4a表达的工具，这将使我能够追求提议的轻松工作。接受DRS的额外培训。彭和谢泼德，我将扩大我目前的知识肺部生物学和细胞感应，同时建立了一种在体内鉴定分泌蛋白质的新方案这将是我过渡到自己的实验室的独特技能。这些研究将为我们的基础了解SASP因子和受伤期间SASP因素的作用。