LKB1 Represses ATOH1 via PDK4 and Energy Metabolism and Regulates Intestinal Stem Cell Fate

Abstract

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 (Lkb1^Lgr5-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 Lkb1^Lgr5-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 Lkb1^Lgr5-KO mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1^Lgr5-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.

Keywords: DCA; Gene Regulation; Mitochondria; Renewal.