Stem cells 2013 July-5

 

Troy, a tumor necrosis factor receptor family member, interacts with lgr5 to inhibit wnt signaling in intestinal stem cells.

Gastroenterology. 2013 Feb;144(2):381-91.

Fafilek B, Krausova M, Vojtechova M, Pospichalova V, Tumova L, Sloncova E, Huranova M, Stancikova J, Hlavata A, Svec J, Sedlacek R, Luksan O, Oliverius M, Voska L, Jirsa M, Paces J, Kolar M, Krivjanska M, Klimesova K, Tlaskalova-Hogenova H, Korinek V.

Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

Abstract

BACKGROUND & AIMS: The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice.

METHODS: We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIPon-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc+/Min and ApcCKO/CKO/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization.

RESULTS: We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer– derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast cycling intestinal stem cells.

TROY associated with the leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a surface protein that mediates the response of the intestinal stem cell to the Wnt agonist R-spondin (Rspo). The suppressive role of TROY on Wnt signaling was documented using gene knockdowns and the intestinal organoid cultures established from Troy-deficient mice. In both types of experiments Troy-deficiency potentiated cell responsiveness to Wnt3a and/or Rspo stimulation (Figure 1). Immunoblotting analysis revealed that the absence of TROY resulted in increased phosphorylation and cellular level of the Wnt coreceptor LRP6.

CONCLUSIONS: TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells (Figure 2).

 

Vladimir Korinek-11

Figure 1: Stereomicroscopic images of the intestinal organoids propagated at various concentrations of Rspo1. The images were taken for 6 consecutive days as indicated. Original magnification: 20×.

Vladimir Korinek-22

Figure 2: A possible model of TROY action. The absence of TROY increases levels and phosphorylation of LRP at specific residue (indicated by “P” in red circle). This potentiates the outcome of Wnt/Rspo signaling and leads to accumulation of β-catenin.

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