LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals1. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5+ stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.
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We thank A. A. Rolf, I. Kuper, D. V. F. Tauriello, M. van den Born, C. Kroon-Veenboer, H. Begthel, J. Korving and S. van den Brink for technical assistance, L. Lum for providing IWP1 and S. Bartfeld for the schematic drawing. This work was funded in part by grants from the European Research Council, EU/232814-StemCeLLMark and the National Research Foundation of Korea, NRF-2011-357-C00093 (B.-K.K.); EU/Health-F4-2007-200720 (M.v.d.W.); The Centre van Biomedical Genetics (D.E.S.); Ti Pharma/T3-106 (J.H.v.E.); the European Research Council, ERC-StG no.242958 (M.M.M.) and the KNAW/3V-fund.
H.C. is an inventor on several patents involving the culture system in this paper. The other authors declare no competing financial interests.
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WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop
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