Abstract

Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues1. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors2,3,4. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD–LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.

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Acknowledgements

We thank the staff of the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory, for support and access to beamline 8.2.2, and P. Chu from the Department of Comparative Medicine Animal Histology Service Center for sample preparation. This work was supported by National Institutes of Health (NIH) R01 GM097015 (to K.C.G), K08DK096048 (to K.S.Y), U01 DK085527 (to C.J.K.), U19 AI116484 (to C.J.K.), U01 CA176299 (to C.J.K.); DFG SFB 944 (to J.P.); Bu,rroughs Wellcome Fund CAMS (to K.S.Y.); the Stinehart/Reed Foundation (to K.C.G.); the Ludwig Foundation (K.C.G., C.J.K.); the Howard Hughes Medical Institute (to K.C.G., D.B.), the European Union’s Horizon 2020 research and innovation program under grant agreement no. 668294 (to H.C.), and the NWO translational Adult Stem Cell Research grant 40-41400-98-1108 (to H.C.).

Author information

Author notes

    • Claudia Y. Janda
    •  & Luke T. Dang

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, and Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Claudia Y. Janda
    • , Dirk Siepe
    •  & K. Christopher Garcia
  2. Department of Biochemistry, Howard Hughes Medical Institute, and the Institute for Protein Design, University of Washington, Seattle, Washington 98195, USA

    • Luke T. Dang
    • , James D. Moody
    •  & David Baker
  3. Division of Biophysics, Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany

    • Changjiang You
    •  & Jacob Piehler
  4. Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Junlei Chang
    • , Kelley S. Yan
    • , Xingnan Li
    •  & Calvin J. Kuo
  5. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences, and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

    • Wim de Lau
    •  & Hans Clevers
  6. Program for Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell Biology, Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, Michigan 49503, USA

    • Zhendong A. Zhong
    •  & Bart O. Williams
  7. Hagey Laboratory for Pediatric Regenerative Medicine and Department of Surgery, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

    • Owen Marecic

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Contributions

C.Y.J. designed experiments, performed biophysical measurements, determined crystal structures, performed in vitro functional assays and prepared the manuscript. D.S. analysed data. D.B., L.T.D. and J.D.M. designed the B12 binding module, and performed affinity maturation. J.P. and C.Y. performed TIRF microscopy, analysed data and contributed to manuscript preparation. Z.A.Z. and B.O.W. performed osteogenesis assays, and analysed data. W.d.L. and H.C. performed organoid culture assays, analysed data and contributed to manuscript preparation. J.C., K.S.Y. and X.L. carried out in vivo experiments in mice and analysed data. O.M. performed parabiosis surgery. C.J.K. designed and supervised in vivo experiments, analysed data and contributed to manuscript preparation. K.C.G. conceived of the project, analysed data, supervised execution of the project, and prepared the manuscript.

Competing interests

K.C.G., C.J.K. and C.Y.J. are founders of Surrozen, Inc.

Corresponding author

Correspondence to K. Christopher Garcia.

Reviewer Information Nature thanks W. DeGrado, Y. Jones and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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  1. 1.

    Supplementary Information

    This file contains Supplementary Tables 1-2 and Supplementary Figure 1, the uncropped western blots.

Videos

  1. 1.

    Co-locomoting Lrp6/Fzd8 measured without treatment

    Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-colour single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell without treatment. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.

  2. 2.

    Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of scFv-DKK1c

    Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of scFV-DKK1c. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of scFV-DKK1c. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.

  3. 3.

    Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of B12-DKK1c

    Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of B12-DKK1c. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.

  4. 4.

    Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of XWnt8

    Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of XWnt8. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.

  5. 5.

    Co-locomoting Lrp6/Fzd8 measured within 30 min after the addition of Wnt3a

    Co-locomoting Lrp6/Fzd8 visualized by time-lapse single molecule imaging. Time-lapse dual-color single molecule imaging of Fzd8 (red) and Lrp6 (blue) in the plasma membrane of HeLa cell within 30 min after the addition of 100 nM of Wnt3a. Co-locomotion/heterodimerization of Fzd8 and Lrp6 are shown in magenta. Playback in real time. Scale bar: 2 µm.

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https://doi.org/10.1038/nature22306

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