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

项目总结摘要 细胞衰老是细胞对环境应激源或肿瘤的反应而进入的获得性状态 逃避。衰老细胞停止增殖，增强反应和调节能力 微环境通过衰老相关分泌音型(SASP)。另一个共享 细胞衰老的特征是肿瘤抑制因子细胞周期蛋白抑制因子p16INK4a表达上调。使用 随着p16INK4a表达的上调，衰老细胞在组织中聚集。 虽然使用遗传老鼠工具移除表达p16INK4a的细胞可以延缓衰老，但也会带来不利的影响 在损伤修复过程中影响伤口愈合，提示p16INK4a在动态平衡和创伤修复中的作用相互矛盾。 伤害反应。我们产生了一个超敏感的p16INK4a报告鼠系名称，命名为Ink4a H2B-GFP 串联报道者，或INKBRITE，以进一步了解体内p16INK4细胞。在INKBRITE成人肺中， P16INK4细胞主要存在于免疫细胞和成纤维细胞中。P16INK4成纤维细胞的表达 衰老的特征包括多倍体、细胞体积增大、低增殖能力和 促进损伤后呼吸道上皮细胞的生长。我的主要目标是确定是否有能力促进 再生仅限于呼吸道p16INK4和上皮细胞或其他区域特异性的p16INK4表达 成纤维细胞可以支持上皮细胞的生长。促进上皮再生的能力是否仅限于 呼吸道p16INK4成纤维细胞或其他空间上确定的成纤维细胞亚群，如肺泡成纤维细胞可以 通过SASP促进上皮生长，目前尚不清楚。为了填补这一知识空白，我将分离肺泡 P16INK4a成纤维细胞并测定其在急性上皮损伤后促进上皮生长的能力1) 体外3D共培养实验，2)通过RNA测序鉴定转录的SASP，3)体内使用 已知的敏感剂达沙替尼和栎素(D&Q)。我们的p16INK4a诱导和敲除研究表明 P16INK4a对已知SASP因子如IL6、Ereg、Ccl8表达的要求。另一个方面是 在体内对SASP因子p16INK4的识别和依赖性表达的研究还很少。对于 R00阶段，我将进一步探索p16INK4的表达如何能够重新编程 SASP支持组织修复，特别是上皮再生。我会用我们的工具从功能上归纳和 去除成纤维细胞中的p16INK4，1)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依赖p16INK4a的SASP因子。我对工作有广泛的了解 使用INKBRITE和操作p16 INK4a表达式的工具，这将使我能够继续研究建议的 轻松工作。在彭博士和谢泼德博士的额外培训下，我将扩大我目前对 肺生物学与细胞衰老--建立体内分泌蛋白鉴定的新方法 这将成为我过渡到自己的实验室的独特技能。这些研究将为我们的 了解SASP因子在动态平衡和损伤中的不同作用。

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.