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Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling.

Authors
  • Luca, Vincent C1
  • Miao, Yi1
  • Li, Xingnan2
  • Hollander, Michael J3
  • Kuo, Calvin J2
  • Garcia, K Christopher1
  • 1 Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, and Department of Structural Biology, Stanford University School of Medicine, Stanford, California, United States of America. , (United States)
  • 2 Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, California, United States of America. , (United States)
  • 3 Department of Bioengineering, Stanford University, Stanford, California, United States of America. , (United States)
Type
Published Article
Journal
PLoS ONE
Publisher
Public Library of Science
Publication Date
Jan 01, 2020
Volume
15
Issue
1
Identifiers
DOI: 10.1371/journal.pone.0226928
PMID: 31914456
Source
Medline
Language
English
License
Unknown

Abstract

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.

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