Mouse cristin/R-spondin family proteins are novel ligands for the frizzled 8 and LRP6 receptors and activate β-catenin-dependent gene expression

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.