Genetic regulation of active and reserve intestinal stem cell states

活性和储备肠道干细胞状态的基因调控

• 批准号: 10451524
• 负责人: SCOTT T MAGNESS
• 金额: $ 34.37万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-19 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451524
• 关键词: Ablation; Alleles; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cell Cycle Regulation; Cell Differentiation process; Cell Proliferation; Cell Survival; Cells; Cessation of life; DNA Repair; Data; Dose; Epithelial; Epithelial Cells; Equilibrium; Fluorescence; G1 Arrest; G1 Phase; Generations; Genes; Genetic; Genotoxic Stress; Health; Human; Image; Intestines; Label; Length; Link; Measures; Mediating; Mitosis; Mus; Pathway interactions; Pharmacology; Phase; Phase Transition; Phenocopy; Phenotype; Physiological; Process; Proliferating; Property; Radiation; Radio; Regenerative response; Regulation; Resistance; S Phase Arrest; Secretory Cell; Series; Signal Transduction; System; Testing; Tissues; Ubiquitination; Western Blotting; Work; base; cell type; epithelium regeneration; experimental study; in vivo; intestinal crypt; intestinal epithelium; irradiation; monolayer; notch protein; outcome prediction; overexpression; radiation resistance; response; self-renewal; stem cells; transcription factor

ABSTRACT: Complete physiologic renewal of intestinal epithelium occurs about every week and is driven by actively proliferating ISCs (aISCs) located in the epithelial-crypt base. A rare subset cells in the crypt, still not completely defined but that are collectively known as `reserve' ISCs (rISCs), are `quiescent' or slowly dividing, and can convert into aISCs when certain health conditions or radio- or chemotherapeutic exposures damage and deplete the native aISC pool. The rISC to aISC conversion process is traditionally studied in mice where irradiation (IR) is used to deplete aISCs and induce rISCs. Here, rISCs are defined by their resistance to IR-induced death, and then by their `plastic' ability to generate actively dividing ISC progeny, which replenish the aISC pool and drive subsequent epithelial regeneration. The mechanisms conferring rISC radio-resistance and plasticity are unknown. In prior work we demonstrated that the transcription factor Sox9 is required for the generation and function of rISCs in mice. Lineage tracing with a Sox9CreERT2 driver showed that after IR, all regenerating epithelium is derived from cells that expressing Sox9, and epithelium-specific genetic ablation of Sox9 profoundly impeded epithelial regeneration and cell survival post-IR. These findings indicate that Sox9-dependent mechanisms govern rISC function. Growing evidence in the stem cell and radiation fields suggest that slowing the cell-cycle rate in dividing cells can enhance radioresistance after IR-exposure, and can modulate cell fate commitment versus self-renewal decisions. We have found that elevated Sox9 levels are associated with slowly dividing cells in the crypt, and that Sox9-overexpression in rapidly dividing aISCs can slow or halt their proliferation. We hypothesize that Sox9-expression levels modulate cell-cycle progression to determine and diversify rISC (Sox9HI) and aISC (Sox9LO) functions in the intestinal crypt. If supported by the results of our experiments, this study will uncover the underlying pathways governing rISC radioresistance and plasticity, and could provide a unifying mechanism describing whether and how rISC properties exist among a broad range of cell types in the crypt. Aim 1: Assess the effects of increasing Sox9 levels on cell-cycle progression in ISCs. Aim 2: Determine if Sox9-mediated G1-elongation confers radio-resistance. Aim 3: Determine if Sox9- mediated G1-elongation confers a `secretory precursor' rISC phenotype.

摘要：肠上皮的完全生理性更新大约每周发生一次， 由位于上皮-隐窝基底中的活跃增殖的ISCs（aISCs）驱动。一种罕见的细胞亚群， crypt仍然没有完全定义，但被统称为“保留”ISC（rISC）， “静止”或缓慢分裂，并在某些健康状况或无线电时转换为aISC-或 化学治疗暴露损伤并耗尽天然aISC库。rISC到aISC的转换 这一过程传统上在小鼠中进行研究，其中使用辐射（IR）来消耗aISC并诱导rISC。 在这里，rISCs的定义是它们对IR诱导的死亡的抵抗力，然后是它们的“可塑性”能力， 产生活跃分裂的ISC后代，其补充aISC库并驱动随后的上皮细胞 再生赋予rISC辐射抗性和可塑性的机制尚不清楚。于过往 我们的工作表明，转录因子Sox 9是产生和功能所必需的。 小鼠中的rISC。使用Sox 9 CreERT 2驱动程序进行谱系追踪显示，IR后，所有再生上皮 来源于表达Sox 9的细胞，并且Sox 9的上皮特异性基因切除是一种深刻的 这些发现表明，Sox 9依赖性的细胞凋亡抑制了上皮细胞的再生和细胞存活。 rISC功能的机制。在干细胞和辐射领域越来越多的证据表明， 减缓分裂细胞的细胞周期速率可以增强IR暴露后的辐射抗性，并且可以 调节细胞命运的承诺与自我更新的决定。我们发现Sox 9水平升高 与隐窝中缓慢分裂的细胞有关，而Sox 9过表达在快速分裂的细胞中， aISCs可以减缓或停止它们的增殖。我们假设Sox 9表达水平调节了 细胞周期进程，以确定和多样化rISC（Sox 9 HI）和aISC（Sox 9 LO）的功能， 肠隐窝如果得到我们实验结果的支持，这项研究将揭示 控制rISC辐射抗性和可塑性的潜在途径，并可以提供一个统一的 描述rISC特性是否以及如何存在于细胞中广泛的细胞类型中的机制。 地穴目的1：评估增加Sox 9水平对ISCs细胞周期进程的影响。目标二： 确定Sox 9介导的G1-延长是否赋予辐射抗性。目的3：确定Sox 9- 介导的G1-延长赋予“分泌前体”rISC表型。