REGULATION OF ATROPHY-INDUCED PROGENITOR CELLS IN THE GASTRIC CORPUS

项目总结/摘要 该项目旨在确定干细胞分化为酸泵的分子机制 胃上皮中的壁细胞。与壁细胞丰度和活性相关的异常是 与多种胃病相关，包括消化性溃疡，胃食管反流病， 自身免疫性胃炎和幽门螺杆菌感染伴慢性萎缩性胃炎和幽门化生。 尽管有一系列与壁细胞功能障碍有关的人类健康状况， 壁细胞在体内平衡时或在壁细胞丢失的疾病中从干细胞中出现。期间 在该项目的前一个资助期，我们已经证明壁细胞死亡需要IFNγ和IL-17信号转导。 AMPK和AMPK调节的蛋白质如PGC 1 α和CD 36对壁细胞分化至关重要 和功能在这里，我们将显示初步的数据，核激素转录因子雌激素- 相关受体γ（ERRγ，由基因Esrrg编码）首先在壁祖细胞中表达， 它们从诱导的干细胞分化，并且Esrrg的缺失消除了壁细胞分化。因此我们 假设ERRγ对于壁细胞命运选择和从干细胞分化是关键和充分的。 我们提出了三个目标来检验我们的假设。在目标1中，我们将描述分子和细胞步骤 参与小鼠发育期间和壁细胞消融后干细胞向壁细胞分化 使用ERRγ与其他胃谱系标志物、免疫染色和电子显微镜 接近。我们将使用新产生的ERRγ-RFP小鼠，以流式细胞术分选ERRγ+壁细胞祖细胞， 消融后的不同时间点，并通过RNA-Seq鉴定PC分化的新分子调节剂。我们 将在具有不同生长因子的2D/3D条件下生长ERRγ+分选和全体类器官， 已经优化了维持干细胞或产生壁细胞的能力。在目标2中，我们将确定如何和 当ERRγ是壁细胞分化所必需时，使用Esrrg-floxed小鼠与表达 干细胞、祖细胞和成熟壁细胞中的可诱导Cre重组酶，包括新产生的 EsrrgCreERT 2线。我们还将进行ChIP-seq以鉴定ERRγ遗传靶点，并对共 免疫沉淀蛋白质以鉴定共激活剂和调节剂。在目标3中，我们将检查ERRγ， 其他胃谱系标志物，以揭示意外存在丰富的壁细胞祖细胞， 由于自身免疫性胃炎而缺乏成熟壁细胞的患者。我们将ERRγ植入小鼠体内， 人类器官和/或用ERRγ活化和抑制药物处理类器官和小鼠模型， 评估ERRγ是否足以产生壁细胞，并建立一个管道，以确定可以治疗 通过调节壁细胞分化来治疗壁细胞疾病。我们预计这项工作将产生一个更强大的 了解壁细胞分化，并导致治疗壁细胞的新治疗方法 紊乱

PROJECT SUMMARY/ABSTRACT This project seeks to define the molecular mechanisms that stem cells use to differentiate into acid-pumping parietal cells in the gastric epithelium. Abnormalities related to parietal cell abundance and activity are associated with a variety of stomach conditions, including peptic ulcers, gastroesophageal reflux disease, autoimmune gastritis, and Helicobacter pylori infection with chronic atrophic gastritis and pyloric metaplasia. Despite the array of human health conditions related to parietal cell dysfunction, little is known about how parietal cells emerge from stem cells at homeostasis or in diseases where parietal cells are lost. During the prior funding period for this project, we have shown that parietal cell death requires IFNγ and IL-17 signaling and that AMPK and AMPK-regulated proteins like PGC1α and CD36 are critical for parietal cell differentiation and function. Here, we will show preliminary data that the nuclear hormone transcription factor Estrogen- related receptor gamma (ERRγ, encoded by the gene Esrrg) is first expressed in parietal progenitor cells as they differentiate from induced stem cells, and deletion of Esrrg abrogates parietal cell differentiation. Thus, we hypothesize that ERRγ is critical and sufficient for parietal cell fate choice and differentiation from stem cells. We propose three aims to test our hypothesis. In Aim 1, we will characterize the molecular and cellular steps involved in stem cell differentiation to parietal cells during mouse development and after parietal cell ablation using ERRγ in combination with other gastric lineage markers, immunostaining, and electron microscopic approaches. We will use newly generated ERRγ-RFP mice to flow sort ERRγ+ parietal cell progenitors at various time points after ablation and identify novel molecular regulators of PC differentiation via RNA-Seq. We will grow ERRγ+ sorted and whole-corpus organoids in 2D/3D conditions with varying growth factors, which we have optimized for maintaining stem cells or generating parietal cells. In Aim 2, we will determine how and when ERRγ is necessary for parietal cell differentiation using Esrrg-floxed mice crossed with strains expressing inducible Cre recombinase in stem cells, progenitors, and mature parietal cells, including a newly generated EsrrgCreERT2 line. We will also perform ChIP-seq to identify ERRγ genetic targets and mass spectrometry of co- immunoprecipitated proteins to identify co-activators and modulators. In Aim 3, we will examine ERRγ and other gastric lineage markers to reveal the unexpected presence of abundant parietal cell progenitors in patients lacking mature parietal cells due to autoimmune gastritis. We will transduce ERRγ into mouse and human organoids and/or treat organoids and mouse models with ERRγ activating and inhibiting drugs to assess sufficiency of ERRγ to generate parietal cells and establish a pipeline to identify agents that can treat parietal cell disorders by regulating parietal cell differentiation. We expect this work will generate a more robust understanding of parietal cell differentiation and lead to novel therapeutic approaches for treating parietal cell disorders.