Murine Frizzled-1 Behaves as an Antagonist of the Canonical Wnt/β-Catenin Signaling

Abstract

Activation of the Wnt signaling cascade provides key signals during development and in disease. Wnt signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single transmembrane low density lipoprotein receptor-related proteins 5 or 6. In the course of the analysis of genes regulated by bone morphogenetic protein 2 in mesenchymal cells we found a significant induction of murine Frizzled-1 (mFz1) gene expression. Unexpectedly overexpression of mFz1 dramatically repressed the induction of alkaline phosphatase mediated by either bone morphogenetic protein 2 or Wnt3a in these cells. Moreover mFz1 overexpression significantly repressed both ␤-catenin translocation into the nucleus and T cell factor signaling mediated by Wnt3a. Importantly microinjection of mFz1 transcript in Xenopus embryo inhibited the ability of Wnt1 to induce the expression of the Wnt/␤-catenin target gene Siamois in animal cap assay and secondary axis formation in whole embryo. By using chimeric constructs in which N-and Cterminal segments of mFz1 were replaced by the corresponding parts of Xfz3 we demonstrated that the antagonistic activity resides in the cysteine-rich domain of the N-terminal part. The antagonist activity of mFz1 could be prevented by overexpression of G␣ q -(305-359), which specifically uncouples G q -coupled receptors, suggesting that G␣ q signaling contributes to the inhibition of Wnt/␤-catenin pathway by mFz1. This is the first time that a Frizzled receptor has been reported to antagonize Wnt/␤-catenin. The transforming growth factor (TGF)1 -␤ family plays a central role in regulating a broad range of cellular responses including cell growth and differentiation. Among members of this family, bone morphogenetic proteins (BMPs) have been shown to regulate growth and differentiation of chondroblast and osteoblast lineage cells and induce the commitment of mesenchymal cells into osteoblast/chondroblast phenotypes (1-3). In addition, when ectopically implanted, BMPs possess the capacity to induce bone and cartilage formation (4, 5). In a series of murine cell lines including C3H10T1/2, C2C12, and ST2, BMP-2 induces the expression of several osteoblast differentiation markers including alkaline phosphatase (ALP) and osteocalcin (6). In contrast, TGF-␤1 has been shown to repress the expression of osteoblast markers, and it abolishes the increase in osteocalcin induced by calcitriol (7, 8). Moreover we have previously showed that TGF-␤1 is able to antagonize BMP-2 activities in several mesenchymal cell lines (9).Wnts are secreted proteins involved in a wide range of developmental processes such as embryonic axis specification and organogenesis (10). They have been shown to activate distinct pathways both in vertebrates and in invertebrates. The better described pathway is the Wnt/␤-catenin pathway. In this pathway, the interaction between Wnt and Frizzled receptors leads to inactivation of glycogen synthase kinase-3␤. Genetic epistasis experiments suggest that Dishevelled lies upstream and represses the activity of gly...

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