Notch Pathway Regulation of Intestinal Epithelial Cell Homeostasis

肠上皮细胞稳态的Notch通路调节

• 批准号: 8915683
• 负责人: LINDA C. SAMUELSON
• 金额: $ 32.98万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-18 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8915683
• 关键词: Adult; Apoptosis; Biological Assay; Biological Models; Cell Count; Cell Differentiation process; Cell Fate Control; Cell Lineage; Cell Proliferation; Cell physiology; Cells; Chronic; Columnar Cell; Computer Simulation; Data; Endocrine; Enterocytes; Epithelial Cells; Gene Expression Profiling; Genes; Genetic; Genetic Models; Growth; Health; Homeostasis; Human; Immunohistochemistry; In Vitro; Intestines; Kinetics; Laboratory Research; Maintenance; Malignant Neoplasms; Measures; Modeling; Mus; Notch Signaling Pathway; Organoids; Pathway interactions; Play; Population; Population Dynamics; Process; Radiation; Radiation Injuries; Receptor Signaling; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Secretory Cell; Signal Pathway; Signal Transduction; Source; Stem cells; System; Testing; Time; Tissues; Toxic effect; Villus; base; cell type; design; genetic manipulation; human disease; human stem cells; intestinal epithelium; mathematical model; mouse model; notch protein; progenitor; repaired; response; stem; stem cell fate; stem cell population; targeted treatment; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): This revised new R01 application focuses on pathways regulating intestinal epithelial cell homeostasis. The project investigates the role of th Notch signaling pathway for regulation of intestinal stem cells, examining both active and quiescent stem cell populations marked by Lgr5, Olfm4, Bmi1, and Lrig1. Notch is well established as a critical regulator of cell fate via regulation of the secretory lineage- specific transcription factor Atoh1. Our recent studies have shown that Notch also plays a distinct role in the maintenance of intestinal stem cells in an Atoh1-independent mechanism and this finding serves as the underlying rationale for this application. We have established that chronic Notch inhibition in adult mice results in decreased progenitor cell proliferation and a marked decrease in expression of the crypt base columnar cell (CBC) marker Olfm4. Furthermore, these studies demonstrated that the actively cycling crypt base columnar stem cell (CBC) is a direct cellular Notch target and that disruption of Notch signaling results in loss of CBCs. The overriding hypothesis for this proposal is: Notch signaling regulates the transition from ISC to fated progenitor cells and loss of Notch signaling leads to depletion of the stem cell pool and activation of quiescent stem cells to replenish the pool. To test this hypothesis studies will examine intact mouse models (genetic and pharmacologic), mouse intestinal organoids and, importantly, a new human organoid model. This project will, for the first time, test whether Notch regulation of human intestine parallels the findings in the mouse. Genetic manipulation and marking of ISC cell populations in mouse will take advantage of Cre drivers specific for active or quiescent ISCs. Strains of floxed-gene mice will allow deletion or activation of Notch pathway components in specific ISC cell populations. RNA-Seq gene expression profiling will be used to identify Notch-responsive genes and computational modeling will inform our understanding of progenitor cell population dynamics. Three specific aims are proposed: (1) Test the hypothesis that CBC stem cells are dynamically regulated by Notch signaling; (2) Test the hypothesis that Notch regulates the ability of quiescent stem cells to repopulate the active cycling stem and progenitor cell pool; (3) Test the hypothesis that Notch signaling regulates cellular differentiatin and stem cell function in human in vitro derived intestinal organoids. These studies are important to further our understanding of the function of different intestinal stem cell population and to characterize the importance of Notch signaling for human intestinal epithelial cell homeostasis. Since current human disease therapies are under design to target the Notch pathway, it is crucial to understand the function of this pathway in the intestine to avoid the intestinal toxicity that can result from systemic disruption of this pathway.

描述（由申请人提供）：该修订后的新R 01申请侧重于调节肠上皮细胞稳态的途径。该项目研究了Notch信号通路在调节肠道干细胞中的作用，检查了由Lgr 5，Olfm 4，Bmi 1和Lrig 1标记的活性和静止干细胞群。Notch通过调节分泌谱系特异性转录因子Atoh 1被公认为细胞命运的关键调节因子。我们最近的研究表明，Notch在Atoh 1独立机制中对肠道干细胞的维持也起着独特的作用，这一发现是该应用的基本原理。我们已经确定，成年小鼠中的慢性Notch抑制导致祖细胞增殖降低和隐窝基底柱状细胞（CBC）标记物Olfm 4的表达显著降低。此外，这些研究表明，活跃循环的隐窝基底柱状干细胞（CBC）是直接的细胞Notch靶标，并且Notch信号传导的破坏导致CBC的损失。该提议的最重要的假设是：Notch信号传导调节从ISC到命运祖细胞的转变，并且Notch信号传导的丧失导致干细胞池的耗尽和静止干细胞的激活以补充池。为了验证这一假设，研究将检查完整的小鼠模型（遗传和药理学），小鼠肠道类器官，重要的是，一种新的人类类器官模型。该项目将首次测试Notch对人类肠道的调节是否与小鼠的发现相似。小鼠中ISC细胞群的遗传操作和标记将利用对活性或静止ISC特异的Cre驱动子。加氟基因小鼠品系将允许特定ISC细胞群中Notch途径组分的缺失或激活。RNA-Seq基因表达谱将用于识别Notch反应基因，计算建模将告知我们对祖细胞群体动力学的理解。提出了三个具体目标：（1）检验CBC干细胞由Notch信号传导动态调节的假设;（2）检验Notch调节静止干细胞重新填充活跃的循环干细胞和祖细胞池的能力的假设;（3）检验Notch信号传导调节人体外衍生的肠类器官中的细胞分化和干细胞功能的假设。这些研究对于我们进一步了解不同肠干细胞群的功能以及表征Notch信号对人肠上皮细胞稳态的重要性具有重要意义。由于目前的人类疾病治疗正在设计中靶向Notch途径，因此了解该途径在肠道中的功能以避免该途径的全身性破坏可能导致的肠道毒性至关重要。