Functional properties of a new Wnt11 isoform expressed in colon carcinoma cell line HT29

Colon carcinoma is a common type of neoplastic transformation. The mechanisms of its establishment and progression have been studied for several decades. Aberrant activation of canonical Wnt signaling is frequently observed in colon carcinoma cells. Moreover, expression of “noncanonical” Wnt ligands is also detected in this type of cancer. However, the role of noncanonical Wnt signaling in carcinogenesis and colorectal cancer (CRC) progression is still unclear. To study the characteristics of noncanonical Wnt signaling activation in CRC, expression of “noncanonical” ligand hWnt11 was examined in HT29 human colon carcinoma cells. For the first time it was shown that alternative splicing accompanies hWnt11 expression in CRC. A new hWnt11 isoform (hWnt11sp3) was identified. Unlike hWnt11, the isoform is not secreted and lacks the ability to inhibit canonical Wnt signaling. Different functional properties of the ligand hWnt11 and its isoform may reflect a special role of alternative splicing in carcinogenesis and tumor progression, since aberrant activity of canonical Wnt signaling is observed in many tumor cells. The existence of several Wnt isoforms and the difference in their functional properties should be taken into account when investigating the role of Wnt ligands.     

Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche

Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.     

Multiple mechanisms for Wnt11-mediated repression of the canonical Wnt signaling pathway

The effect of a noncanonical Wnt, Wnt11, on canonical Wnt signaling stimulated by Wnt1 and activated forms of LRP5 (low density lipoprotein receptor-related protein-5), Dishevelled1 (Dvl1), and beta-catenin was examined in NIH3T3 cells and P19 embryonic carcinoma cells. Wnt11 repressed Wnt1-mediated activation of LEF-1 reporter activity in both cell lines. However, Wnt11 was unable to inhibit canonical signaling activated by LRP5, Dvl1, or beta-catenin in NIH3T3 cells, although it could in P19 cells. In addition, Wnt11-mediated inhibition of canonical signaling in NIH3T3 cells is ligand-specific; Wnt11 could effectively repress canonical signaling activated by Wnt1, Wnt3, or Wnt3a but not by Wnt7a or Wnt7b. Co-culture experiments with NIH3T3 cells showed that the co-expression of Wnt11 with Wnt1 was not an essential requirement for the inhibition, suggesting receptor competition as a possible mechanism. Moreover, in both cell types, elevation of intracellular Ca(2+) levels, which can result from Wnt11 treatment, led to the inhibition of canonical signaling. This result suggests that Wnt11 might not be able to signal in NIH3T3. Furthermore, P19 cells were found to express both endogenous canonical Wnts and Wnt11. Knockdown of Wnt11 expression using siRNA resulted in increased LEF-1 reporter activity, thus indicating that Wnt11-mediated suppression of canonical signaling exists in vivo.     

Nuclear beta-catenin localization is not sufficient for canonical Wnt signaling activation in human meianoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called beta-catenin. Mutations promoting beta-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers, but rarely observed in melanomas. Nevertheless, beta-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why, the aim of the investigation was to elucidate the relation between beta-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular beta-catenin localization, and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of beta-catenin does not always correspond to active status canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear beta-catenin canonical Wnt signaling can't be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, the pathway-targeted potential antineoplastic therapy requires the formation of a "molecular pattern of cancer" for localization of the defect in Wnt signaling cascade in the each case.     

When Wnts antagonize Wnts

Secreted Wnt ligands appear to activate a variety of signaling pathways. Two papers in this issue now present genetic evidence that "noncanonical" Wnt signaling inhibits the "canonical" Wnt/beta-catenin pathway. Westfall et al. (2003a) show that zebrafish embryos lacking maternal Wnt-5 function are dorsalized due to ectopic activation of beta-catenin, whereas Topol et al. (2003) report that chondrogenesis in the distal mouse limb bud depends on inhibition of Wnt/beta-catenin signaling by a paralogue of Wnt-5. These studies present the first genetic confirmation of the previous hypothesis that vertebrate Wnt signaling pathways can act in an antagonistic manner.     

Neural crest cells development and neuroblastoma progression: Role of Wnt signaling

Neuroblastoma (NB) is one of the most common heterogeneous extracranial cancers in infancy that arises from neural crest (NC) cells of the sympathetic nervous system. The Wnt signaling pathway, both canonical and noncanonical pathway, is a highly conserved signaling pathway that regulates the development and differentiation of the NC cells during embryogenesis. Reports suggest that aberrant activation of Wnt ligands/receptors in Wnt signaling pathways promote progression and relapse of NB. Wnt signaling pathways regulate NC induction and migration in a similar manner; it regulates proliferation and metastasis of NB. Inhibiting the Wnt signaling pathway or its ligands/receptors induces apoptosis and abrogates proliferation and tumorigenicity in all major types of NB cells. Here, we comprehensively discuss the Wnt signaling pathway and its mechanisms in regulating the development of NC and NB pathogenesis. This review highlights the implications of aberrant Wnt signaling in the context of etiology, progression, and relapse of NB. We have also described emerging strategies for Wnt-based therapies against the progression of NB that will provide new insights into the development of Wnt-based therapeutic strategies for NB.     

Wnts and wing: Wnt signaling in vertebrate limb development and musculoskeletal morphogenesis

In the past twenty years, secreted signaling molecules of the Wnt family have been found to play a central role in controlling embryonic development from hydra to human. In the developing vertebrate limb, Wnt signaling is required for limb bud initiation, early limb patterning (which is governed by several well-characterized signaling centers), and, finally, late limb morphogenesis events. Wnt ligands are unique, in that they can activate several different receptor-mediated signal transduction pathways. The most extensively studied Wnt pathway is the canonical Wnt pathway, which controls gene expression by stabilizing beta-catenin in regulating a diverse array of biological processes. Recently, more attention has been given to the noncanonical Wnt pathway, which is beta-catenin-independent. The noncanonical Wnt pathway signals through activating Ca(2+) flux, JNK activation, and both small and heterotrimeric G proteins, to induce changes in gene expression, cell adhesion, migration, and polarity. Abnormal Wnt signaling leads to developmental defects and human diseases affecting either tissue development or homeostasis. Further understanding of the biological function and signaling mechanism of Wnt signaling is essential for the development of novel preventive and therapeutic approaches of human diseases. This review provides a critical perspective on how Wnt signaling regulates different developmental processes. As Wnt signaling in tumor formation has been reviewed extensively elsewhere, this part is not included in the review of the clinical significance of Wnt signaling.     

The mechanism of endogenous receptor activation functionally distinguishes prototype canonical and noncanonical Wnts

Wnt glycoproteins are developmentally essential signaling molecules, and lesions afflicting Wnt pathways play important roles in human diseases. Some Wnts signal to the canonical pathway by stabilizing beta-catenin, while others lack this activity. Frizzled serpentine receptors mediate distinct signaling pathways by both classes of Wnts. Here, we tandemly linked noncanonical Wnt5a with the C-terminal half of Dickkopf-2 (Dkk2C), a distinct ligand of the Wnt coreceptor LRP5/6. Whereas Wnt5a, Dkk2C, or both together were incapable of stimulating endogenous canonical signaling, the Wnt5a/Dkk2C chimera efficiently activated this pathway in a manner inhibitable by specific antagonists of either frizzled or LRP receptors. Thus, activation of the canonical pathway requires ligand coupling of an endogenous frizzled/LRP coreceptor complex, rather than Wnt triggering each receptor independently. Moreover, fusion of Wnt5a with Dkk2C unmasked its ability to signal to Dishevelled through multiple frizzleds, indicating that the lack of functional interaction with LRP distinguishes noncanonical Wnt5a from canonical Wnts in mammalian cells. These findings provide a novel mechanism by which the same receptor can be switched between distinct signaling pathways depending on the differential recruitment of a coreceptor by members of the same ligand family.     

Nuclear β-catenin localization is not sufficient for canonical Wnt signaling activation in human melanoma cell lines

In most cases, advanced stages of melanoma are practically incurable due to high metastatic potential of tumor cells. Multiple observations support the idea that aberrations in the Wnt signaling pathway play a significant role in melanoma development and progression. Canonical Wnt signaling activation results in stabilization and accumulation of the major effector molecule called & gb-catenin. Mutations promoting & gb-catenin stabilization and, thereby, activation of canonical Wnt signaling pathway are frequently found in different cancers but rarely observed in melanomas. Nevertheless, & gb-catenin nuclear and cytoplasmic accumulation is the feature of many human melanoma cell lines and original tumors. That is why the aim of the investigation was to elucidate the relation between & gb-catenin intracellular localization and activity status of Wnt signaling pathway in human melanoma cell lines. Ten human melanoma cell lines were characterized on the basis of the following parameters: canonical Wnt ligand expression, intracellular & gb-catenin localization and activity status of canonical Wnt signaling pathway. Here, it has been demonstrated that nuclear localization of & gb-catenin does not always correspond to active status of canonical Wnt signaling pathway. Moreover, in the majority of cell lines with nuclear & gb-catenin, canonical Wnt signaling cannot be activated by exogenous expression of an appropriate ligand. Human melanoma cell lines differ in activity of canonical Wnt signaling pathway as well as in mechanisms of its regulation. Therefore, pathway-targeted potential antineoplastic therapy requires the formation of a & ldmolecular pattern of cancer” for localization of the defect in Wnt signaling cascade in each case.     

Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis

The signaling molecule Wnt regulates bone homeostasis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.     

Axis formation--beta-catenin catches a Wnt

In this issue of Cell, the Heasman group implicates Wnt11 as a component of the canonical Wnt signaling pathway that specifies Xenopus laevis axis formation (Tao et al., 2005). This important work not only identifies a long-sought-after dorsalizing factor but also highlights the pivotal role of extracellular cofactors in specifying the activation of either canonical or noncanonical Wnt pathways.     

The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape

Members of the vertebrate Wnt family have been subdivided into two functional classes according to their biological activities. Some Wnts signal through the canonical Wnt-1/wingless pathway by stabilizing cytoplasmic beta-catenin. By contrast other Wnts stimulate intracellular Ca2+ release and activate two kinases, CamKII and PKC, in a G-protein-dependent manner. Moreover, putative Wnt receptors belonging to the Frizzled gene family have been identified that preferentially couple to the two prospective pathways in the absence of ectopic Wnt ligand and that might account for the signaling specificity of the Wnt pathways. As Ca2+ release was the first described feature of the noncanonical pathway, and as Ca2+ probably plays a key role in the activation of CamKII and PKC, we have named this Wnt pathway the Wnt/Ca2+ pathway.     

Daam2 is required for dorsal patterning via modulation of canonical Wnt signaling in the developing spinal cord

The Daam family of proteins consists of Daam1 and Daam2. Although Daam1 participates in noncanonical Wnt signaling during gastrulation, Daam2 function remains completely uncharacterized. Here we describe the role of Daam2 in canonical Wnt signal transduction during spinal cord development. Loss-of-function studies revealed that Daam2 is required for dorsal progenitor identities and canonical Wnt signaling. These phenotypes are rescued by β-catenin, demonstrating that Daam2 functions in dorsal patterning through the canonical Wnt pathway. Complementary gain-of-function studies demonstrate that Daam2 amplifies Wnt signaling by potentiating ligand activation. Biochemical examination found that Daam2 association with Dvl3 is required for Wnt activity and dorsal patterning. Moreover, Daam2 stabilizes Dvl3/Axin2 binding, resulting in enhanced intracellular assembly of Dvl3/Axin2 complexes. These studies demonstrate that Daam2 modulates the formation of Wnt receptor complexes, revealing new insight into the functional diversity of Daam proteins and how canonical Wnt signaling contributes to pattern formation in the developing spinal cord.     

Wnt-4 activates the canonical beta-catenin-mediated Wnt pathway and binds Frizzled-6 CRD: functional implications of Wnt/beta-catenin activity in kidney epithelial cells

The Wnt signaling pathway is central to the development of all animals and to cancer progression, yet largely unknown are the pairings of secreted Wnt ligands to their respective Frizzled transmembrane receptors or, in many cases, the relative contributions of canonical (beta-catenin/LEF/TCF) versus noncanonical Wnt signals. Specifically, in the kidney where Wnt-4 is essential for the mesenchymal to epithelial transition that generates the tissue's collecting tubules, the corresponding Frizzled receptor(s) and downstream signaling mechanism(s) are unclear. In this report, we addressed these issues using Madin-Darby Canine Kidney (MDCK) cells, which are competent to form tubules in vitro. Employing established reporter constructs of canonical Wnt/beta-catenin pathway activity, we have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions, precisely reflecting functional findings from Wnt-4 null kidney mesenchyme ex vivo rescue studies. We have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/beta-catenin pathway using beta-Engrailed and dnTCF-4 constructs that suppress this pathway. We have further found that MDCK cells express the Frizzled-6 receptor and that Wnt-4 forms a biochemical complex with the Frizzled-6 CRD. Since Frizzled-6 did not appear to transduce Wnt-4's canonical signal, data supported recently by Golan et al., there presumably exists another as yet unknown Frizzled receptor(s) mediating Wnt-4 activation of beta-catenin/LEF/TCF. Finally, we report that canonical Wnt/beta-catenin signals cells help maintain cell growth and survival in MDCK cells but do not contribute to standard HGF-induced (nonphysiologic) tubule formation. Our results in combination with work from Xenopus laevis (not shown) lead us to believe that Wnt-4 binds both canonical and noncanonical Frizzled receptors, thereby activating Wnt signaling pathways that may each contribute to kidney tubulogenesis.