Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors

This article has been updated

Abstract

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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: LGR5+ stem cell genes: Rnf43 and Znrf3.
Figure 2: Strong proliferation of the Rnf43Znrf3 compound mutant intestine is accompanied by Wnt/β-catenin activation as well as stem cell and Paneth cell metaplasia.
Figure 3: RNF43 suppresses the Wnt/β-catenin pathway by reducing surface levels of frizzled receptors.
Figure 4: RNF43 promotes ubiquitin-mediated endocytosis of frizzled receptors.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The data for the microarray analysis have been deposited to the Gene Expression Omnibus under accession number GSE36497.

Change history

  • 29 August 2012

    An addition was made to the Acknowledgements.

References

  1. 1

    Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Cheng, H. & Leblond, C. P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am. J. Anat. 141, 461–479 (1974)

    CAS  Article  Google Scholar 

  3. 3

    Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Yui, S. et al. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nature Med. 18, 618–623 (2012)

    CAS  Article  Google Scholar 

  6. 6

    van der Flier, L. G. et al. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell 136, 903–912 (2009)

    CAS  Article  Google Scholar 

  7. 7

    El Marjou, F. et al. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis 39, 186–193 (2004)

    CAS  Article  Google Scholar 

  8. 8

    Ireland, H. et al. Inducible cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of β-catenin. Gastroenterology 126, 1236–1246 (2004)

    CAS  Article  Google Scholar 

  9. 9

    Sansom, O. J. et al. Myc deletion rescues Apc deficiency in the small intestine. Nature 446, 676–679 (2007)

    ADS  CAS  Article  Google Scholar 

  10. 10

    van Es, J. H. et al. Wnt signalling induces maturation of Paneth cells in intestinal crypts. Nature Cell Biol. 7, 381–386 (2005)

    CAS  Article  Google Scholar 

  11. 11

    Carmon, K. S., Gong, X., Lin, Q., Thomas, A. & Liu, Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc. Natl Acad. Sci. USA 108, 11452–11457 (2011)

    ADS  CAS  Article  Google Scholar 

  12. 12

    de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Kazanskaya, O. et al. The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development. Development 135, 3655–3664 (2008)

    CAS  Article  Google Scholar 

  14. 14

    Chen, B. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nature Chem. Biol. 5, 100–107 (2009)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Koo, B. K. et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods 9, 81–83 (2012)

    CAS  Article  Google Scholar 

  16. 16

    Liu, G., Bafico, A., Harris, V. K. & Aaronson, S. A. A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor. Mol. Cell. Biol. 23, 5825–5835 (2003)

    CAS  Article  Google Scholar 

  17. 17

    Ivanov, I., Lo, K. C., Hawthorn, L., Cowell, J. K. & Ionov, Y. Identifying candidate colon cancer tumor suppressor genes using inhibition of nonsense-mediated mRNA decay in colon cancer cells. Oncogene 26, 2873–2884 (2007)

    CAS  Article  Google Scholar 

  18. 18

    Li, V. S. et al. Wnt signaling through inhibition of β-catenin degradation in an intact Axin1 complex. Cell 149, 1245–1256 (2012)

    CAS  Article  Google Scholar 

  19. 19

    Haglund, K. & Dikic, I. The role of ubiquitylation in receptor endocytosis and endosomal sorting. J. Cell Sci. 125, 265–275 (2012)

    CAS  Article  Google Scholar 

  20. 20

    Mukai, A. et al. Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt. EMBO J. 29, 2114–2125 (2010)

    CAS  Article  Google Scholar 

  21. 21

    Van der Flier, L. G. et al. The intestinal Wnt/TCF signature. Gastroenterology 132, 628–632 (2007)

    CAS  Article  Google Scholar 

  22. 22

    Yagyu, R. et al. A novel oncoprotein RNF43 functions in an autocrine manner in colorectal cancer. Int. J. Oncol. 25, 1343–1348 (2004)

    CAS  PubMed  Google Scholar 

  23. 23

    Hao, H. X. et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485, 195–200 (2012)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Niida, A. et al. DKK1, a negative regulator of Wnt signaling, is a target of the β-catenin/TCF pathway. Oncogene 23, 8520–8526 (2004)

    CAS  Article  Google Scholar 

  25. 25

    Shimomura, Y. et al. APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex. Nature 464, 1043–1047 (2010)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Jho, E. H. et al. Wnt/β-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol. Cell. Biol. 22, 1172–1183 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Berndt, J. D. et al. Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling. J. Cell Biol. 194, 737–750 (2011)

    Article  Google Scholar 

  28. 28

    Wu, J. et al. Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways. Proc. Natl Acad. Sci. USA 108, 21188–21193 (2011)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Ong, C. K. e. t. a. l. Exome sequencing of liver fluke-associated cholangiocarcinoma. Nature Genet. 44, 690–693 (2012)

    CAS  Article  Google Scholar 

  30. 30

    March, H. N. et al. Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis. Nature Genet. 43, 1202–1209 (2011)

    CAS  Article  Google Scholar 

  31. 31

    Tauriello, D. V. et al. Loss of the tumor suppressor CYLD enhances Wnt/β-catenin signaling through K63-linked ubiquitination of Dvl. Mol. Cell 37, 607–619 (2010)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

B.-K.K., M.M.M. and H.C. conceived and designed the experiments. B.-K.K., M.S., I.J., D.E.S., M.v.d.W. and J.H.v.E. performed the experiments. T.Y.L., S.M. and A.J.R.H. performed the mass spectrometry analysis. B.-K.K., M.S., I.J., M.M.M. and H.C. analysed the data. B.-K.K., M.M.M. and H.C. wrote the manuscript.

Corresponding authors

Correspondence to Madelon M. Maurice or Hans Clevers.

Ethics declarations

Competing interests

H.C. is an inventor on several patents involving the culture system in this paper. The other authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-12. (PDF 10932 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Koo, BK., Spit, M., Jordens, I. et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488, 665–669 (2012). https://doi.org/10.1038/nature11308

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing