Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling.

Abstract

Wnts modulate cell proliferation, differentiation and stem cell self-renewal, by inducing β-catenin dependent signaling through Frizzled (Fzd) and Lrp5/6 to regulate cell fate decisions, and the growth and repair of a multitude of tissues1. The 19 mammalian Wnts interact promiscuously with the 10 Fzds, which has complicated the attribution of specific Fzd/Wnt subtype interactions to distinct biological functions. Furthermore, Wnts are post-translationally modified by palmitoylation, which is essential for Wnt secretion and functions as a critical site of interaction with Fzd 2–4. As a result of their acylation, Wnts are very hydrophobic proteins requiring detergents for purification, which presents major obstacles for the preparation and application of recombinant Wnts. This has hindered the delineation of the molecular mechanisms of Wnt signaling activation, understanding of the functional significance of Fzd subtypes, and the use of Wnts as therapeutics. Here we developed surrogate Wnt agonists, water-soluble Fzd-Lrp5/6 heterodimerizers, consisting of Fzd5/8-specific and broadly Fzd-reactive binding domains, that elicit a characteristic β-catenin signaling response in a Fzd-selective fashion, enhance osteogenic lineage commitment of primary mesenchymal stem cells (MSCs), and support the growth of a broad range of primary human organoid cultures comparably to Wnt3a. Furthermore, we demonstrate that the surrogates can be systemically expressed and exhibit Wnt activity in vivo, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signaling can be activated simply through bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced non-lipidated Wnt surrogate agonists offer a new avenue to facilitate functional studies of Wnt signaling and the exploration of Wnt agonists for translational applications in regenerative medicine.