Abstract
Wnt signaling plays critical biological roles during normal embryonic development and homeostasis in adults. In the canonical pathway, binding of Wnt ligands to the Frizzled (Fzd) receptor and the low density lipoprotein-related receptor (LRP) 5 or LRP6 coreceptor initiates downstream signaling events leading to gene activation by beta-catenin and the T-cell factor (TCF)-lymphoid enhancer factor (LEF) family transcription factor complex. In this study, we provide several lines of evidence that the mouse Cristin/R-spondin family proteins function as Fzd8 and LRP6 receptor ligands and induce the canonical Wnt/beta-catenin signaling pathway, leading to TCF-dependent gene activation. First, conditioned medium containing Cristin/R-spondin proteins effectively induced reporter activity in a TCF-binding site-dependent manner. Second, stimulation of cells with Cristin/R-spondin was accompanied by stabilization of endogenous beta-catenin proteins and induction of canonical Wnt target genes. Third, Cristin/R-spondin proteins physically interacted with the extracellular domains of the LRP6 and Fzd8 receptors in vivo and in vitro. Interestingly, unlike canonical Wnt ligands, Cristin/R-spondin failed to form a ternary complex with both LRP6 and Fzd8 receptors, suggesting that R-spondin may activate the canonical Wnt signaling pathway by different mechanisms. Furthermore, Cristin/R-spondin proteins possess an intriguing positive modulatory activity on Wnt ligands, possibly through a direct interaction. Our findings expand the repertoire of ligands that induce beta-catenin/TCF-dependent gene activation and implicate the presence of active beta-catenin-dependent gene activation in a Wnt-free biological context.
Highlights
Recent discoveries changed the conventional view of Wnt ligands and Fzd/lipoprotein-related receptor (LRP) receptors (8 –11)
Xenopus R-spondin2 and human R-spondin1 protein activity on canonical Wnt signaling was demonstrated recently [14, 15], consistent with our findings. These results suggest that the function of Cristin/Rspondin family proteins is conserved in vertebrates
Multigene Family of Novel Secreted Mouse Cristin/R-spondin Proteins—We previously demonstrated that mouse mutants lacking the pMesogenin1 gene, which encodes a presomitic mesoderm-specific basic helix-loop-helix transcription factor [20], display severe defects in
Summary
Recent discoveries changed the conventional view of Wnt ligands and Fzd/LRP receptors (8 –11). The RYK, Derailed, and LIN-18 proteins, receptor tyrosine kinases with unknown function, were determined to be novel Wnt receptors in mouse, Drosophila, and Caenorhabditis elegans, respectively (9 –11) These discoveries expanded the repertoire of ligands and receptors involved in Wnt/Fzd/LRP signaling and suggest the possibility of unknown ligands unrelated to the Wnt proteins and receptors unrelated to Fzd and LRP5 or LRP6. We provide evidence strongly suggesting that a multigene family of novel secreted proteins, mouse Cristin/R-spondin proteins, may work at the receptor level to activate downstream -catenin-dependent gene activation and may function as a class of ligands unrelated to Wnt proteins. Xenopus R-spondin and human R-spondin protein activity on canonical Wnt signaling was demonstrated recently [14, 15], consistent with our findings These results suggest that the function of Cristin/Rspondin family proteins is conserved in vertebrates
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