Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (K(D) = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (M(r) 240K) with [(125)I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through beta-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.

Wnt ligands signal through b-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through b-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. We demonstrate here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). We identify Ror2 Ser 864 as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, we provide evidence that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways.[Keywords: Ror1/2; LRP5/6; Wnt3a; Wnt5a; receptor activation; noncanonical Wnt signaling] Supplemental material is available at http://www.genesdev.org.

Autocrine Wnt signaling in the mouse mammary tumor virus model was the first identified mechanism of canonical pathway activation in cancer. In search of this transformation mechanism in human cancer cells, we identified breast and ovarian tumor lines with upregulation of the uncomplexed transcriptionally active form of beta-catenin without mutations afflicting downstream components. Extracellular Wnt antagonists FRP1 and DKK1 caused a dramatic downregulation of beta-catenin levels in these tumor cells associated with alteration of biological properties and increased expression of epithelial differentiation markers. Colorectal carcinoma cells with knockout of the mutant beta-catenin allele retained upregulated beta-catenin levels, which also could be inhibited by these Wnt antagonists. Together, these findings establish the involvement of autocrine Wnt signaling in human cancer cells.

LDL receptor-related protein 6 (LRP6) is a Wnt coreceptor in the canonical signaling pathway, which plays essential roles in embryonic development. We demonstrate here that wild-type LRP6 forms an inactive dimer through interactions mediated by epidermal growth factor repeat regions within the extracellular domain. A truncated LRP6 comprising its transmembrane and cytoplasmic domains is expressed as a constitutively active monomer whose signaling ability is inhibited by forced dimerization. Conversely, Wnts are shown to activate canonical signaling through LRP6 by inducing an intracellular conformational switch which relieves allosteric inhibition imposed on the intracellular domains. Thus, Wnt canonical signaling through LRP6 establishes a novel mechanism for receptor activation which is opposite to the general paradigm of ligand-induced receptor oligomerization.The Wnt family of secreted signaling molecules is essential in embryonic induction, cell polarity generation, and cell fate specification (46). Deregulation of Wnt signaling results in defects in development and growth control. The canonical Wnt pathway involves activation of ␤-catenin-dependent transcription and is evolutionarily conserved from Caenorhabditis elegans to humans. Mutations in components, which constitutively activate canonical signaling, have been identified in several tumor types, including colorectal cancer (34). Wnts bind to two coreceptors, the Frizzled-type seven-transmembrane-domain receptor (5,17,19,49) and the low-density receptor-related protein (LRP) 5/6 (35, 41) or the Drosophila melanogaster ortholog Arrow (44). These interactions cause ␤-catenin stabilization through inhibition of its phosphorylation by glycogen synthase kinase 3␤ (GSK3␤), which is assembled in a large cytoplasmic complex that includes Dishevelled, casein kinase I, Axin, APC, and Frat (36). As a consequence, stabilized cytoplasmic ␤-catenin is translocated to the nucleus and forms a complex with a family of high-mobility group-like transcription factors, including leukocyte enhancer factor-1 (LEF-1) and T-cell factors (TCF), activating transcription of target genes (4).Frizzled family members have been shown to possess various affinities for different Wnt ligands. For example, Drosophila frizzled, Dfz1 and Dfz2, bind to Wingless (Wg), the Drosophila orthologue of Wnt, to activate the canonical pathway. However, Dfz2 has a 10-fold higher affinity for Wg than Dfz1 and plays a predominant role in transducing the Wg canonical signal in vivo (37). Frizzled family members also signal through the planar polarity pathway, which similarly involves Dsh (25) but is mediated through JNK and RhoA rather than ␤-catenin stabilization (7). In Drosophila, Dfz1 but not Dfz2 appears to regulate planar polarity signaling (8, 37). LRP5, LRP6, and arrow receptors specifically function in the canonical pathway. Inactivation of arrow in Drosophila results in a phenotype similar to that of the wingless mutant (41), and mice deficient in LRP6 exhibit developmental defects resembling ...

Genetic evidence indicates that Wnt signaling is critically involved in bone homeostasis. In this study, we investigated the functions of canonical Wnts on differentiation of adult multipotent human mesenchymal stem cells (hMSCs) in vitro and in vivo. We observe differential sensitivities of hMSCs to Wnt inhibition of osteogenesis versus adipogenesis, which favors osteoblastic commitment under binary in vitro differentiation conditions. Wnt inhibition of osteogenesis is associated with decreased expression of osteoblastic transcription factors and inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation, which are involved in osteogenic differentiation. An hMSC subpopulation exhibits high endogenous Wnt signaling, the inhibition of which enhances osteogenic and adipogenic differentiation in vitro. In an in vivo bone formation model, high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However, hMSCs with exogenous expression of Wnt1 but not stabilized β-catenin markedly stimulate bone formation by naive hMSCs, arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo.