Investigating the Role of IGF2 mRNA-binding protein 1 (IMP1) In Regulating Reserve Intestinal Stem Cell Survival In Response to DNA Damage

研究 IGF2 mRNA 结合蛋白 1 (IMP1) 在调节储备肠干细胞响应 DNA 损伤的存活中的作用

• 批准号: 10468297
• 负责人: Louis Parham
• 金额: $ 1万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468297
• 关键词: Ablation; Adult; Adverse effects; Affect; Alleles; Antioxidants; Apoptosis; Architecture; Autophagocytosis; Binding Proteins; CASP3 gene; Cell Count; Cell Cycle; Cell Death; Cell Maintenance; Cells; Cessation of life; Cysteine; DNA Damage; Data; Development; Embryonic Development; Epithelial; Epithelial Cells; Exhibits; Flow Cytometry; Foundations; Frequencies; Gene Expression; Genes; Homeostasis; Hour; IGF2 gene; Immunohistochemistry; Impairment; In Situ Nick-End Labeling; Injury; Intestines; Knock-out; Knockout Mice; Knowledge; Label; Malignant Neoplasms; Measures; Mediating; Membrane Potentials; Messenger RNA; Mitochondria; Mus; Natural regeneration; Organ; Pathologic; Pathway interactions; Perinatal mortality demographics; Phenocopy; Play; Polyadenylation; Population; RNA Splicing; RNA-Binding Proteins; Radiation; Radiation Dose Unit; Radiation therapy; Reactive Oxygen Species; Recovery; Regenerative response; Regulator Genes; Reporter; Role; Syndrome; Tamoxifen; Testing; Therapeutic; Transcript; Vesicle; Whole-Body Irradiation; base; cell behavior; cell regeneration; cell type; epithelium regeneration; experimental study; gastrointestinal; intestinal crypt; intestinal epithelium; intestinal homeostasis; irradiation; loss of function; mitotracker green FM; novel therapeutics; radiation resistance; radioresistant; regenerative; resilience; response; self renewing cell; stem cell model; stem cell survival; stem cells; tissue injury; tissue regeneration; transcriptome sequencing

The intestinal epithelium undergoes complete turnover in 3-5 days. This is achieved through the presence of active intestinal stem cells (a-ISCs) that are self-renewing and can give rise to all differentiated epithelial cell types. Following irradiation, which ablates cycling cells in the intestine, cells that survive irradiation referred to as reserve intestinal stem cells (r-ISCs) are able to re-enter the cell cycle and contribute to tissue regeneration. Although mechanisms regulating the dormancy of these cells are beginning to be elucidated, their radioresistance and subsequent re-activation is still unclear, representing a gap in our broader knowledge of intestinal epithelial regeneration. RNA-binding proteins (RBP) are regulators of gene expression that facilitate cellular adaptation during homeostasis or pathological states. Deletion of the RBP IGF2 mRNA-binding protein 1 (IMP1) in the intestinal epithelium is associated with enhanced regenerative response following irradiation, a result that is phenocopied in mice with Imp1 deleted specifically in r-ISCs, highlighting a putative role for Imp1 in r-ISCs. Imp1 loss in the intestinal epithelium leads to an increase in autophagic vesicle content at homeostasis, suggesting that Imp1 may function as a negative regulator of the autophagy pathway. Combined deletion of Imp1 and Atg7 in the intestinal epithelium abrogates the enhanced regeneration observed with Imp1 deletion, suggesting that Imp1’s regulatory roles are dependent on the autophagy pathway. Studies show that mice lacking the autophagy gene Atg5 in intestinal epithelial cells exhibit a marked decrease in a-ISCs and impaired regeneration following irradiation. This is attributed to accumulation of dysfunctional mytochondria and a subsequent increase in reactive oxygen species levels (ROS). The role played by autophagy in radioresistant r- ISCs during homeostasis and regeneration remains unknown. I hypothesize that Imp1 modulates r-ISC survival in response to DNA-damage by enhancing autophagy mediated clearing of reactive oxygen species. In Aim 1, we will evaluate r-ISC frequency at homeostasis as well as cell death and DNA damage post- irradiation with or without Imp1 deletion. We will use a reporter mouse that labels r-ISCs and progeny with RFP following tamoxifen administration for lineage-tracing. We will use 12Gy whole-body irradiation and evaluate RFP+ cells using flow cytometry and immunohistochemistry. For Aim 2 we will use r-ISC specific Atg7 knockout as well as Atg7 and Imp1 double knockout mice to determine whether enhanced regeneration observed in the absence of Imp1 is dependent on autophagy. Regenerative microcolony frequency will be measured via immunohistochemistry. Furthermore, we will evaluate how Imp1 loss affects mitochondrial content using Mitotracker Green FM, transmembrane potential using rhodamine123, and ROS using CellROX via flow cytometry. Lastly, we will evaluate how ROS modulation affects regenerative microcolony formation with or without the antioxidant N-acetyl-L-cysteine. These studies will uncover roles played by RBPs in regulating stem cell activity and will provide a mechanistic understanding of stem cell survival following DNA damage.

肠上皮细胞在3-5天内完全更新。这是通过以下方式实现的 活跃的肠干细胞（a-ISCs）能够自我更新，并能产生所有分化的上皮细胞， 类型在照射后，其消融肠中的循环细胞，在照射后存活的细胞被称为 因为储备肠干细胞（r-ISCs）能够重新进入细胞周期并有助于组织再生。 虽然调节这些细胞休眠的机制开始被阐明，但它们的 辐射抗性和随后的再激活仍然不清楚，这代表了我们对辐射抗性的更广泛认识的空白。 肠上皮再生RNA结合蛋白（RBP）是基因表达的调节因子， 在体内平衡或病理状态下的细胞适应。RBP IGF 2 mRNA结合蛋白缺失 1（IMP 1）与辐射后增强的再生反应有关， 在r-ISCs中特异性缺失Imp 1的小鼠中表型模拟的结果，突出了Imp 1在 r-ISC。肠上皮中的IMP 1缺失导致稳态时自噬囊泡含量的增加， 提示Imp 1可能是自噬途径的负调节因子。合并缺失Imp 1 并且肠上皮中的Atg 7消除了Imp 1缺失所观察到的增强的再生， 这表明Imp 1的调节作用依赖于自噬途径。研究表明，小鼠 在肠上皮细胞中缺乏自噬基因Atg 5表现出a-ISCs的显著减少和受损， 辐照后再生。这是由于功能障碍性肌萎缩和 随后活性氧水平（ROS）增加。自噬在抗辐射受体中的作用- ISCs在体内平衡和再生过程中的作用仍不清楚。我假设Imp 1调节r-ISC 通过增强自噬介导的活性氧清除应答DNA损伤的存活 物种在目标1中，我们将评估稳态时的r-ISC频率以及细胞死亡和DNA损伤后， 辐照与或不与Imp 1缺失。我们将使用一个报告小鼠标记r-ISCs和后代与RFP 在他莫昔芬给药后进行谱系追踪。我们将使用12 Gy全身照射并评估 流式细胞术和免疫组织化学检测RFP+细胞。对于目标2，我们将使用r-ISC特异性Atg 7敲除 以及Atg 7和Imp 1双敲除小鼠，以确定是否在小鼠中观察到增强的再生。 Imp 1的缺失依赖于自噬。再生小菌落频率将通过 免疫组化此外，我们将评估Imp 1损失如何影响线粒体含量， Mitotracker绿色FM，使用罗丹明123的跨膜电位，和使用CellROX的ROS通过流动 细胞仪最后，我们将评估ROS调节如何影响再生小菌落的形成， 没有抗氧化剂N-乙酰-L-半胱氨酸。这些研究将揭示RBPs在调节干细胞中所起的作用 细胞活性，并将提供DNA损伤后干细胞存活的机制理解。