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ADAM10 regulates Notch function in intestinal stem cells of mice.

ABSTRACT: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a cell surface sheddase that regulates physiologic processes, including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types, but the requirement for ADAM10 signaling in crypt homeostasis is not well defined.We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10(f/f) mice) and conditional (Vil-CreER;Adam10(f/f) and Leucine-rich repeat-containing GPCR5 [Lgr5]-CreER;Adam10(f/f) mice) deletion of ADAM10. We performed cell lineage-tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26(NICD)), or mice with intestine-specific disruption of Notch (Rosa26(DN-MAML)), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance.Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26(NICD) and Rosa26(DN-MAML) mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage-tracing experiments showed that ADAM10 is required for survival of Lgr5(+) crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche.ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance.

SUBMITTER: Tsai YH

PROVIDER: S-EPMC4176890 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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ADAM10 regulates Notch function in intestinal stem cells of mice.

Tsai Yu-Hwai YH VanDussen Kelli L KL Sawey Eric T ET Wade Alex W AW Kasper Chelsea C Rakshit Sabita S Bhatt Riha G RG Stoeck Alex A Maillard Ivan I Crawford Howard C HC Samuelson Linda C LC Dempsey Peter J PJ

Gastroenterology 20140716 4

<h4>Background & aims</h4>A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a cell surface sheddase that regulates physiologic processes, including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types, but the requirement for ADAM10 signaling in crypt homeostasis is not well defined.<h4>Methods</h4>We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10(f/f) mice) and conditional (Vil-CreER;Adam10(f/f) and Leucine-rich repeat- ...[more]

PMID: 25038433

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Project description:BackgroundIn mammalian intestines, Notch signaling plays a critical role in mediating cell fate decisions; it promotes the absorptive (or enterocyte) cell fate, while concomitantly inhibiting the secretory cell fate (i.e. goblet, Paneth and enteroendocrine cells). We recently reported that intestinal-specific Kaiso overexpressing mice (Kaiso Tg ) exhibited chronic intestinal inflammation and had increased numbers of all three secretory cell types, hinting that Kaiso might regulate Notch signaling in the gut. However, Kaiso's precise role in Notch signaling and whether the Kaiso Tg secretory cell fate phenotype was linked to Kaiso-induced inflammation had yet to be elucidated.MethodsIntestines from 3-month old Non-transgenic and Kaiso Tg mice were "Swiss" rolled and analysed for the expression of Notch1, Dll-1, Jagged-1, and secretory cell markers by immunohistochemistry and immunofluorescence. To evaluate inflammation, morphological analyses and myeloperoxidase assays were performed on intestines from 3-month old Kaiso Tg and control mice. Notch1, Dll-1 and Jagged-1 expression were also assessed in stable Kaiso-depleted colon cancer cells and isolated intestinal epithelial cells using real time PCR and western blotting. To assess Kaiso binding to the DLL1, JAG1 and NOTCH1 promoter regions, chromatin immunoprecipitation was performed on three colon cancer cell lines.ResultsHere we demonstrate that Kaiso promotes secretory cell hyperplasia independently of Kaiso-induced inflammation. Moreover, Kaiso regulates several components of the Notch signaling pathway in intestinal cells, namely, Dll-1, Jagged-1 and Notch1. Notably, we found that in Kaiso Tg mice intestines, Notch1 and Dll-1 expression are significantly reduced while Jagged-1 expression is increased. Chromatin immunoprecipitation experiments revealed that Kaiso associates with the DLL1 and JAG1 promoter regions in a methylation-dependent manner in colon carcinoma cell lines, suggesting that these Notch ligands are putative Kaiso target genes.ConclusionHere, we provide evidence that Kaiso's effects on intestinal secretory cell fates precede the development of intestinal inflammation in Kaiso Tg mice. We also demonstrate that Kaiso inhibits the expression of Dll-1, which likely contributes to the secretory cell phenotype observed in our transgenic mice. In contrast, Kaiso promotes Jagged-1 expression, which may have implications in Notch-mediated colon cancer progression.

Project description:Background & aimsFunctions of intestinal stem cells (ISCs) are regulated by diet and metabolic pathways. Hepatocyte nuclear factor 4 (HNF4) family are transcription factors that bind fatty acids. We investigated how HNF4 transcription factors regulate metabolism and their functions in ISCs in mice.MethodsWe performed studies with Villin-CreERT2;Lgr5-EGFP-IRES-CreERT2;Hnf4?f/f;Hnf4?Crispr/Crispr mice, hereafter referred to Hnf4??DKO. Mice were given tamoxifen to induce Cre recombinase. Mice transgenic with only Cre alleles (Villin-CreERT2, Lgr5-EGFP-IRES-CreERT2, Hnf4?+/+, and Hnf4?+/+) or mice given vehicle were used as controls. Crypt and villus cells were isolated, incubated with fluorescently labeled fatty acids or glucose analog, and analyzed by confocal microscopy. Fatty acid oxidation activity and tricarboxylic acid (TCA) cycle metabolites were measured in cells collected from the proximal half of the small intestine of Hnf4??DKO and control mice. We performed chromatin immunoprecipitation and gene expression profiling analyses to identify genes regulated by HNF4 factors. We established organoids from duodenal crypts, incubated them with labeled palmitate or acetate, and measured production of TCA cycle metabolites or fatty acids. Acetate, a precursor of acetyl coenzyme A (CoA) (a product of fatty acid ?-oxidation [FAO]), or dichloroacetate, a compound that promotes pyruvate oxidation and generation of mitochondrial acetyl-CoA, were used for metabolic intervention.ResultsCrypt cells rapidly absorbed labeled fatty acids, and messenger RNA levels of Lgr5+ stem cell markers (Lgr5, Olfm4, Smoc2, Msi1, and Ascl2) were down-regulated in organoids incubated with etomoxir, an inhibitor of FAO, indicating that FAO was required for renewal of ISCs. HNF4A and HNF4G were expressed in ISCs and throughout the intestinal epithelium. Single knockout of either HNF4A or HNF4G did not affect maintenance of ISCs, but double-knockout of HNF4A and HNF4G resulted in ISC loss; stem cells failed to renew. FAO supports ISC renewal, and HNF4 transcription factors directly activate FAO genes, including Acsl5 and Acsf2 (encode regulators of acyl-CoA synthesis), Slc27a2 (encodes a fatty acid transporter), Fabp2 (encodes fatty acid binding protein), and Hadh (encodes hydroxyacyl-CoA dehydrogenase). In the intestinal epithelium of Hnf4??DKO mice, expression levels of FAO genes, FAO activity, and metabolites of TCA cycle were all significantly decreased, but fatty acid synthesis transcripts were increased, compared with control mice. The contribution of labeled palmitate or acetate to the TCA cycle was reduced in organoids derived from Hnf4??DKO mice, compared with control mice. Incubation of organoids derived from double-knockout mice with acetate or dichloroacetate restored stem cells.ConclusionsIn mice, the transcription factors HNF4A and HNF4G regulate the expression of genes required for FAO and are required for renewal of ISCs.

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ADAM10 regulates Notch function in intestinal stem cells of mice.

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ADAM10 regulates Notch function in intestinal stem cells of mice.

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