REGULATION OF ATROPHY-INDUCED PROGENITOR CELLS IN THE GASTRIC CORPUS

项目摘要/摘要 该项目试图确定干细胞用于分化为抽酸的分子机制。 胃上皮内的壁细胞。与壁细胞丰度和活性相关的异常有 与各种胃病有关，包括消化性溃疡、胃食道反流病、 自身免疫性胃炎，幽门螺杆菌感染合并慢性萎缩性胃炎和幽门上皮化生。 尽管有一系列与壁细胞功能障碍有关的人类健康问题，但人们对此知之甚少。 壁细胞在体内稳态状态下或在壁细胞丢失的疾病中从干细胞中分化出来。在.期间 这个项目之前的资助期，我们已经证明了壁细胞死亡需要干扰素γ和IL-17信号 AMPK和AMPK调节的蛋白如Pgc1、α和CD36对壁细胞分化起关键作用 和功能。在这里，我们将展示初步数据，核激素转录因子雌激素- 相关受体γ(ERRγ，由ESRRG基因编码)首先在壁祖细胞中表达为 它们从诱导的干细胞分化而来，ESRRG的缺失取消了壁细胞的分化。因此，我们 假设Errγ对壁细胞的命运选择和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 在小鼠发育过程中和壁细胞切除后参与干细胞向壁细胞的分化 ERRγ与其他胃谱系标记物、免疫染色和电子显微镜的联合应用 接近了。我们将使用新生成的ERRγ-rfp小鼠来流式分选Errγ+壁细胞前体细胞 消融后的不同时间点，并通过RNA-Seq鉴定PC分化的新的分子调节因子。我们 将在2D/3D条件下生长Errγ+分类和整个语料库的有机化合物，并具有不同的生长因素，我们 已经为维持干细胞或产生壁细胞进行了优化。在目标2中，我们将确定如何和 当ERRγ是壁细胞分化所必需的时，将ESRG-FLOL小鼠与表达 干细胞、祖细胞和成熟壁细胞中可诱导的Cre重组酶，包括新生成的 EsrrgCreERT2线。我们还将进行CHIP-SEQ以识别Errγ遗传靶点，并对co. 免疫沉淀蛋白质以识别共激活剂和调节剂。在目标3中，我们将检查Errγ和 其他胃谱系标记物揭示意外地存在丰富的壁细胞前体细胞 因自身免疫性胃炎而缺乏成熟壁细胞的患者。我们将把Errγ转化为小鼠并 人类有机物和/或用ERRγ激活和抑制药物治疗类有机物和小鼠模型 评估ERRγ产生壁细胞的充分性，并建立一条管道来鉴定可以治疗的药物 通过调节壁细胞分化而引起的壁细胞紊乱。我们预计这项工作将产生更强大的 了解壁细胞分化并引导治疗壁细胞的新方法 精神错乱。

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.