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Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling

Nature volume 476, pages 293–297 (2011)Cite this article

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Abstract

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.

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Figure 1: Conditional deletion of Lgr4 and Lgr5.
Figure 2: Wnt target genes are downstream of Lgr4/5.
Figure 3: LGR4 is essential for transmitting RSPO1 signals but dispensable for transmitting WNT3A signals.
Figure 4: Direct physical interaction of RSPO1 with LGR4/5/6 exodomains.
Figure 5: Rescue of Lgr4/5 deletion in cultured crypt organoids by Wnt signals.

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Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the GEO database under accession number GSE28265. Mass spectrometry data sets are available at ProteomeCommons.org Tranche Repository https://proteomecommons.org/tranche/data-downloader.jsp?h52LOW5tCJBOfT%2FpcCAtMrPqCgTTOd247s6poPgSvwu16KiVwCfExWdJ0jifGdI4FraidTHUnl1PYhIoT0nTs1zdwKmKEAAAAAAAACzw%3D%3D.

References

  1. Barker, N. & Clevers, H. Leucine-rich repeat-containing G-protein-coupled receptors as markers of adult stem cells. Gastroenterology 138, 1681–1696 (2010)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  3. Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro . Cell Stem Cell 6, 25–36 (2010)

    Article  CAS  Google Scholar 

  4. Jaks, V. et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nature Genet. 40, 1291–1299 (2008)

    Article  CAS  Google Scholar 

  5. Snippert, H. J. et al. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327, 1385–1389 (2010)

    Article  ADS  CAS  Google Scholar 

  6. Van Schoore, G., Mendive, F., Pochet, R. & Vassart, G. Expression pattern of the orphan receptor LGR4/GPR48 gene in the mouse. Histochem. Cell Biol. 124, 35–50 (2005)

    Article  CAS  Google Scholar 

  7. Mustata, R. C. et al. Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells I. EMBO Reports 12, 558–564 (2011)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Morita, H. et al. Neonatal lethality of LGR5 null mice is associated with ankyloglossia and gastrointestinal distension. Mol. Cell. Biol. 24, 9736–9743 (2004)

    Article  CAS  Google Scholar 

  10. Kato, S. et al. Eye-open at birth phenotype with reduced keratinocyte motility in LGR4 null mice. FEBS Lett. 581, 4685–4690 (2007)

    Article  CAS  Google Scholar 

  11. Ireland, H., Houghton, C., Howard, L. & Winton, D. J. Cellular inheritance of a Cre-activated reporter gene to determine Paneth cell longevity in the murine small intestine. Dev. Dyn. 233, 1332–1336 (2005)

    Article  CAS  Google Scholar 

  12. Garcia, M. I. et al. LGR5 deficiency deregulates Wnt signaling and leads to precocious Paneth cell differentiation in the fetal intestine. Dev. Biol. 331, 58–67 (2009)

    Article  CAS  Google Scholar 

  13. van der Flier, L. G., Haegebarth, A., Stange, D. E., van de Wetering, M. & Clevers, H. OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells. Gastroenterology 137, 15–17 (2009)

    Article  Google Scholar 

  14. Muncan, V. et al. Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc . Mol. Cell. Biol. 26, 8418–8426 (2006)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  16. Pinto, D., Gregorieff, A., Begthel, H. & Clevers, H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 17, 1709–1713 (2003)

    Article  CAS  Google Scholar 

  17. Kuhnert, F. et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc. Natl Acad. Sci. USA 101, 266–271 (2004)

    Article  ADS  CAS  Google Scholar 

  18. van Es, J. H. et al. Notch/γ-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435, 959–963 (2005)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  20. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005)

    Article  ADS  CAS  Google Scholar 

  21. Nakatani, Y. & Ogryzko, V. Immunoaffinity purification of mammalian protein complexes. Methods Enzymol. 370, 430–444 (2003)

    Article  CAS  Google Scholar 

  22. Kazanskaya, O. et al. R-Spondin2 is a secreted activator of Wnt/β-catenin signaling and is required for Xenopus myogenesis. Dev. Cell 7, 525–534 (2004)

    Article  CAS  Google Scholar 

  23. Kim, K. A. et al. R-spondin family members regulate the Wnt pathway by a common mechanism. Mol. Biol. Cell 19, 2588–2596 (2008)

    Article  CAS  Google Scholar 

  24. Kim, K. A. et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259 (2005)

    Article  ADS  CAS  Google Scholar 

  25. Zhao, J. et al. R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice. Gastroenterology 132, 1331–1343 (2007)

    Article  CAS  Google Scholar 

  26. Ohkawara, B., Glinka, A. & Niehrs, C. Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis. Dev. Cell 20, 303–314 (2011)

    Article  CAS  Google Scholar 

  27. Nam, J. S., Turcotte, T. J., Smith, P. F., Choi, S. & Yoon, J. K. Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate β-catenin-dependent gene expression. J. Biol. Chem. 281, 13247–13257 (2006)

    Article  CAS  Google Scholar 

  28. Wei, Q. et al. R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and β-catenin signaling. J. Biol. Chem. 282, 15903–15911 (2007)

    Article  CAS  Google Scholar 

  29. Binnerts, M. E. et al. R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6. Proc. Natl Acad. Sci. USA 104, 14700–14705 (2007)

    Article  ADS  CAS  Google Scholar 

  30. Glinka, A. et al. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391, 357–362 (1998)

    Article  ADS  CAS  Google Scholar 

  31. Korinek, V. et al. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma. Science 275, 1784–1787 (1997)

    Article  CAS  Google Scholar 

  32. Flanagan, C. A. A. GPCR that is not “DRY”. Mol. Pharmacol. 68, 1–3 (2005)

    CAS  PubMed  Google Scholar 

  33. Robine, S., Sahuquillo-Merino, C., Louvard, D. & Pringault, E. Regulatory sequences on the human villin gene trigger the expression of a reporter gene in a differentiating HT29 intestinal cell line. J. Biol. Chem. 268, 11426–11434 (1993)

    CAS  PubMed  Google Scholar 

  34. Klein, P. S. & Melton, D. A. A molecular mechanism for the effect of lithium on development. Proc. Natl Acad. Sci. USA 93, 8455–8459 (1996)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  36. Korinek, V. et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nature Genet. 19, 379–383 (1998)

    Article  CAS  Google Scholar 

  37. Ng, S. S. et al. Phosphatidylinositol 3-kinase signaling does not activate the Wnt cascade. J. Biol. Chem. 284, 35308–35313 (2009)

    Article  CAS  Google Scholar 

  38. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996)

    Article  CAS  Google Scholar 

  39. Raijmakers, R. et al. Automated online sequential isotope labeling for protein quantitation applied to proteasome tissue-specific diversity. Mol. Cell. Proteomics 7, 1755–1762 (2008)

    Article  CAS  Google Scholar 

  40. Frese, C. K. et al. Improved peptide identification by targeted fragmentation using CID, HCD and ETD on a LTQ-Orbitrap Velos. J. Proteome Res. 10, 2377–2388 (2011)

    Article  CAS  Google Scholar 

  41. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnol. 26, 1367–1372 (2008)

    Article  CAS  Google Scholar 

  42. Saeed, A. I. et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34, 374–378 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Vassart for Lgr4-LacZ intestinal tissue, D. Winton for Ah-Cre mice, S. Robine for Villin-CreERT2 mice, A. Moerkamp and C. Verheul for experimental help and H. Farin for figures.

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Author notes

  1. Nick Barker & Andrea Haegebarth

    Present address: Present addresses: Institute of Medical Biology, 06-06 Immunos, Singapore 138648 (N.B.); Bayer Schering Pharma AG, 13353 Berlin, Germany (A.H.).,

  2. Wim de Lau and Nick Barker: These authors contributed equally to this work.

Authors and Affiliations

  1. Hubrecht Institute and University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands ,

    Wim de Lau, Nick Barker, Bon-Kyoung Koo, Vivian S. W. Li, Hans Teunissen, Andrea Haegebarth, Marc van de Wetering, D. E. Stange, J. van Es, Daniele Guardavaccaro & Hans Clevers

  2. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center, Utrecht University, 3584 CH Utrecht, The Netherlands ,

    Teck Y. Low, Shabaz Mohammed & Albert J. R. Heck

  3. Antoni van Leeuwenhoek Hospital Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands ,

    Pekka Kujala & Peter J. Peters

  4. Medical Cell Biophysics, MIRA institute, University of Twente, 7500 AE Enschede, The Netherlands ,

    Richard B. M. Schasfoort

  5. Tohoku University, 981-8555 Sendai, Miyagi, Japan ,

    Yasuaki Mohri & Katsuhiko Nishimori

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Contributions

All Hubrecht Institute authors performed experiments under guidance of H.C.; S.M., A.J.R.H. and T.Y.L. performed mass spectrometry; P.K. and P.J.P. performed electron microscopy analysis; R.B.M.S. performed plasmon surface resonance; and Y.M. and K.N. generated the Lgr4 knockout mouse.

Corresponding author

Correspondence to Hans Clevers.

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The authors declare competing financial interests as inventors on several patents relating to this work.

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de Lau, W., Barker, N., Low, T. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011). https://doi.org/10.1038/nature10337

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