BACKGROUND & AIMS In addition to the Notch and Wnt signaling pathways, energy metabolism also regulates intestinal stem cell (ISC) function. Tumor suppressor and kinase STK11 (also called LKB1) regulates stem cells and cell metabolism. We investigated whether loss of LKB1 alters ISC homeostasis in mice. METHODS We deleted LKB1 from ISCs in mice using Lgr5-regulated CRE-ERT2 (Lkb1Lgr5-KO mice) and the traced lineages by using a CRE-dependent TdTomato reporter. Intestinal tissues were collected and analyzed by immunohistochemical and immunofluorescence analyses. We purified ISCs and intestinal progenitors using flow cytometry and performed RNA-sequencing analysis. We measured organoid-forming capacity and ISC percentages using intestinal tissues from Lkb1Lgr5-KO mice. We analyzed human Ls174t cells with knockdown of LKB1 or other proteins by immunoblotting, real-time quantitative polymerase chain reaction, and the Seahorse live-cell metabolic assay. RESULTS Some intestinal crypts from Lkb1Lgr5-KO mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1Lgr5-KO mice contained functional ISCs that expressed increased levels of Atoh1 messenger RNA (mRNA), acquired a gene expression signature associated with secretory cells, and generated more cells in the secretory lineage compared with control mice. Knockdown of LKB1 in Ls174t cells induced expression of Atoh1 mRNA and a phenotype of increased mucin production; knockdown of ATOH1 prevented induction of this phenotype. The increased expression of Atoh1 mRNA after LKB1 loss from ISCs or Ls174t cells did not involve Notch or Wnt signaling. Knockdown of pyruvate dehydrogenase kinase 4 (PDK4) or inhibition with dichloroacetate reduced the up-regulation of Atoh1 mRNA after LKB1 knockdown in Ls174t cells. Cells with LKB1 knockdown had a reduced rate of oxygen consumption, which was partially restored by PDK4 inhibition with dichloroacetate. ISCs with knockout of LKB1 increased the expression of PDK4 and had an altered metabolic profile. CONCLUSIONS LKB1 represses transcription of ATOH1, via PDK4, in ISCs, restricting their differentiation into secretory lineages. These findings provide a connection between metabolism and the fate determination of ISCs.

中文翻译：

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LKB1通过PDK4和能量代谢抑制ATOH1，并调节肠道干细胞的命运。

背景与目的除Notch和Wnt信号通路外，能量代谢还调节肠道干细胞（ISC）的功能。肿瘤抑制因子和激酶STK11（也称为LKB1）调节干细胞和细胞代谢。我们调查了LKB1的丢失是否改变了小鼠的ISC动态平衡。方法我们使用Lgr5调控的CRE-ERT2（Lkb1Lgr5-KO小鼠）从小鼠的ISC中删除LKB1，并使用CRE依赖性TdTomato报告基因删除了所追踪的谱系。收集肠组织并通过免疫组织化学和免疫荧光分析进行分析。我们使用流式细胞仪纯化了ISC和肠道祖细胞，并进行了RNA测序分析。我们使用来自Lkb1Lgr5-KO小鼠的肠组织测量了类器官形成能力和ISC百分比。我们通过免疫印迹，实时定量聚合酶链反应和海马活细胞代谢分析法分析了具有LKB1或其他蛋白质敲低作用的人Ls174t细胞。结果与对照小鼠的隐窝相比，Lkb1Lgr5-KO小鼠的一些肠道隐窝失去了ISC。但是，与对照小鼠相比，来自Lkb1Lgr5-KO小鼠的大多数隐窝均包含功能性ISC，这些ISC表达Atoh1信使RNA（mRNA）的水平升高，获得了与分泌细胞相关的基因表达特征，并在分泌谱系中产生了更多的细胞。敲除Ls174t细胞中的LKB1会诱导Atoh1 mRNA的表达和粘蛋白产生增加的表型。击倒ATOH1阻止了该表型的诱导。从ISC或Ls174t细胞丢失LKB1后，Atoh1 mRNA的表达增加并不涉及Notch或Wnt信号传导。敲除丙酮酸脱氢酶激酶4（PDK4）或用二氯乙酸抑制可降低Ls174t细胞中LKB1敲除后Atoh1 mRNA的上调。敲低LKB1的细胞的耗氧率降低，通过二氯乙酸盐抑制PDK4可以部分恢复。敲除LKB1的ISC增加PDK4的表达，并改变代谢谱。结论LKB1在ISC中通过PDK4抑制ATOH1的转录，从而限制了它们向分泌谱系的分化。这些发现提供了新陈代谢与ISC命运决定之间的联系。敲除丙酮酸脱氢酶激酶4（PDK4）或用二氯乙酸抑制可降低Ls174t细胞中LKB1敲除后Atoh1 mRNA的上调。敲低LKB1的细胞的耗氧率降低，通过二氯乙酸盐抑制PDK4可以部分恢复。敲除LKB1的ISC增加PDK4的表达，并改变代谢谱。结论LKB1在ISC中通过PDK4抑制ATOH1的转录，从而限制了它们向分泌谱系的分化。这些发现提供了新陈代谢与ISC命运决定之间的联系。敲除丙酮酸脱氢酶激酶4（PDK4）或用二氯乙酸抑制可降低Ls174t细胞中LKB1敲除后Atoh1 mRNA的上调。敲低LKB1的细胞的耗氧率降低，通过二氯乙酸对PDK4的抑制可以部分恢复。敲除LKB1的ISC增加PDK4的表达，并改变代谢谱。结论LKB1在ISC中通过PDK4抑制ATOH1的转录，从而限制了它们向分泌谱系的分化。这些发现提供了新陈代谢与ISC命运决定之间的联系。敲除LKB1的ISC增加PDK4的表达，并改变代谢谱。结论LKB1在ISC中通过PDK4抑制ATOH1的转录，从而限制了它们向分泌谱系的分化。这些发现提供了新陈代谢与ISC命运决定之间的联系。敲除LKB1的ISC增加PDK4的表达，并改变代谢谱。结论LKB1在ISC中通过PDK4抑制ATOH1的转录，从而限制了它们向分泌谱系的分化。这些发现提供了新陈代谢与ISC命运决定之间的联系。