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.

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.

SUMMARY Wnt canonical signaling is critical for normal development as well as homeostasis of several epithelial tissues, and constitutive activation of this pathway is commonly observed in carcinomas. We show here that 50% of human sarcomas (n=45) and 70% of sarcoma cell lines (n=23) of diverse histological subtypes exhibit upregulated autocrine canonical Wnt signaling. Further, we identify alterations including overexpression or gene amplification of Wnt ligands and/or LRP5/6 co-receptors, epigenetic silencing of different cell surface Wnt antagonists in autocrine and mutations in adenomatous polyposis coli (APC) gene in two non-autocrine Wnt positive sarcoma cell lines. Finally, downregulation of the activated Wnt pathway inhibited sarcoma cell proliferation both in vitro and in vivo by a mechanism involving the downregulation of CDC25A. SIGNIFICANCE Sarcomas comprise the most common malignancy of childhood and afflict adults as well. Canonical Wnt signaling influences the maintenance of mesenchymal stem cells, and our findings indicate that sarcomas commonly select for upregulation of Wnt autocrine signaling, which acts to increase their proliferation through the functions of a TCF/β-catenin target gene, CDC25A, a major regulator of cell cycle progression. The high frequency at which the Wnt pathway is activated in diverse human sarcomas identifies Wnt signaling as a potential target for therapies that could decrease morbidity and mortality from this disease.

The canonical Wnt pathway plays a crucial role in embryonic development, and its deregulation is involved in human diseases. The LRP6 single-span transmembrane coreceptor is essential for transmission of canonical Wnt signaling. However, due to the lack of immunological reagents, our understanding of LRP6 structure and function has relied on studies involving its overexpression, and regulation of the endogenous receptor by the Wnt ligand has remained unexplored. Using a highly sensitive and specific antibody to LRP6, we demonstrate that the endogenous receptor is modified by N-glycosylation and is phosphorylated in response to Wnt stimulation in a sustained yet ligand-dependent manner. Moreover, following triggering by Wnt, endogenous LRP6 is internalized and recycled back to the cellular membrane within hours of the initial stimulus. Finally, we have identified a novel feedback mechanism by which Wnt, acting through ␤-catenin, negatively regulates LRP6 at the mRNA level. Together, these findings contribute significantly to our understanding of LRP6 function and uncover a new level of regulation of Wnt signaling. In light of the direct role that the Wnt pathway plays in human bone diseases and malignancies, our findings may support the development of novel therapeutic approaches that target Wnt signaling through LRP6.The highly conserved canonical Wnt pathway plays a critical role in cell fate determination and tissue development (7, 23). Moreover, aberrant activation of Wnt signaling is causally involved in human cancers (9, 28). Members of this family of secreted glycoproteins interact with two coreceptors, the Frizzled seven-pass transmembrane receptor and the low-density lipoprotein (LDL) receptor-related protein LRP5/6. Wnt-receptor interactions lead to inhibition of ␤-catenin phosphorylation by casein kinase 1-␣ (CK1-␣) and glycogen synthase kinase-␤, which occurs within a protein complex containing axin and the tumor suppressor adenomatous polyposis coli. Inhibition of ␤-catenin phosphorylation impairs its degradation and results in accumulation of the uncomplexed cytosolic molecule, which translocates to the nucleus and interacts with TCF/LEF factors to activate transcription (9,13,24).Frizzled receptors are known to mediate signaling through both the Wnt-␤-catenin "canonical" pathway and other, "noncanonical" ones, such as the planar cell polarity and Wnt/Ca 2ϩ pathways. In contrast, the LRP6 receptor and the family member LRP5 specifically function in the Wnt-␤-catenin pathway (5, 13, 17). In fact, inactivation of the LRP5/6 homologue arrow in Drosophila melanogaster results in a phenotype similar to that of the wingless mutant, and injection of LRP6 mRNA into Xenopus laevis embryos enhances Wnt-induced developmental defects (33, 35). Moreover, mice deficient for LRP6 exhibit defects resembling those caused by the loss of various Wnt proteins (27). There is evidence supporting a dual-receptor model in which independent binding of Wnt to Frizzled and LRP6 recruits these two proteins into a receptor comp...

The loss of the Y chromosome is a frequent numerical chromosomal abnormality observed in human prostate cancer. In cancer, loss of specific genetic material frequently accompanies simultaneous inactivation of tumor suppressor genes. It is not known whether the Y chromosome harbors such genes. To address the role of genes on the Y chromosome in human prostate cancer, we transferred a tagged Y chromosome into PC-3, a human prostate cancer cell line lacking a Y chromosome. A human Y chromosome was tagged with the hisD gene and transferred to PC-3 by microcell-mediated chromosome transfer. Tumorigenicity of these PC-3 hybrids was tested in vivo and in vitro, and the results were compared with those of the polymerase chain reaction analyses conducted on the PC-3 hybrids using Y chromosome-specific markers. Among 60 mice injected with 12 different PC-3 hybrids (five mice per hybrid), tumor growth was apparent in only one mouse, whereas tumors grew in all mice injected with the parental PC-3 cells. An in vitro assay showed that the Y chromosome did not suppress anchorage-independent growth of PC-3 cells. We found that addition of the Y chromosome suppressed tumor formation by PC-3 in athymic nude mice, and that this block of tumorigenesis was independent of the in vitro growth properties of the cells. This observation suggests the presence of a gene important for prostate tumorigenesis on the Y chromosome.