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

• 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
• 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

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 细胞衰老是细胞响应于环境应激或肿瘤而进入的一种获得性状态 逃避衰老细胞停止其增殖能力，并增强其反应和调节能力 衰老相关分泌型（SASP）。另一共享 细胞衰老的特征是肿瘤抑制细胞周期蛋白抑制剂p16 INK 4a的上调。与 随着年龄的增长，衰老细胞在组织中沿着积累，p16 INK 4a表达上调。 虽然使用遗传小鼠工具去除表达p16 INK 4a的细胞可以减缓衰老，但它也会产生不利影响。 在损伤修复过程中影响伤口愈合，这表明p16 INK 4a在体内平衡过程中的矛盾作用， 伤害反应。我们产生了一个超敏感的p16 INK 4a报告基因小鼠品系，命名为INK 4A H2 B-GFP 串联报告员或INKBRITE，以进一步了解体内p16 INK 4+细胞。在INKBRITE成人肺中， p16 INK 4+细胞主要在免疫和成纤维细胞群中。p16 INK 4+成纤维细胞表达 衰老的特征包括多倍性、细胞大小增加、低增殖能力和 促进损伤后气道上皮细胞生长。我的主要目标是确定 再生仅限于气道p16 INK 4+和上皮细胞或其他区域特异性表达p16 INK 4的细胞。 成纤维细胞可以支持上皮生长。促进上皮再生的能力是否限于 气道p16 INK 4+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，我将分离肺泡 p16 INK 4a+成纤维细胞，并通过以下方法测定它们在急性上皮损伤后促进上皮生长的能力：1） 离体3D共培养测定，2）通过RNA测序鉴定转录的SASP，和3）体内使用 已知的衰老清除剂达沙替尼和槲皮素（D&Q）。我们的p16 INK 4a诱导和敲低研究表明， p16 INK 4a表达已知SASP因子如IL 6、Ereg、Ccl 8的需要。另一方面， 在体内SASP因子的身份和依赖性p16 INK 4表达方面，为 R 00阶段的工作，我将进一步探讨p16 INK 4的表达是如何能够重新编程的。 SASP支持组织修复，特别是上皮再生。我将用我们的工具功能性地诱导， 去除成纤维细胞中的p16 INK 4，1）评估上皮生长，2）转录组分析，3）蛋白质组学， 捕获分泌蛋白以鉴定p16 INK 4a依赖性SASP因子。我有丰富的工作知识 用INKBRITE和工具来操纵p16 INK 4a表达，这将使我能够继续提出 轻松地工作。通过彭博士和谢泼德博士的额外培训，我将扩大我目前的知识， 肺生物学和细胞衰老，同时建立一个新的协议，确定分泌蛋白在体内 这将成为我过渡到自己实验室的独特技能。这些研究将为我们的 了解SASP因子在稳态和损伤过程中的不同作用。