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.     

Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of beta-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including beta-catenin. Here, we have examined the role of CK2 in dorsal axis formation. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2alpha was able to block ectopic axis formation induced by XWnt8 and beta-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis.     

beta-catenin is a target for the ubiquitin-proteasome pathway.

beta-catenin is a central component of the cadherin cell adhesion complex and plays an essential role in the Wingless/Wnt signaling pathway. In the current model of this pathway, the amount of beta-catenin (or its invertebrate homolog Armadillo) is tightly regulated and its steady-state level outside the cadherin-catenin complex is low in the absence of Wingless/Wnt signal. Here we show that the ubiquitin-dependent proteolysis system is involved in the regulation of beta-catenin turnover. beta-catenin, but not E-cadherin, p120(cas) or alpha-catenin, becomes stabilized when proteasome-mediated proteolysis is inhibited and this leads to the accumulation of multi-ubiquitinated forms of beta-catenin. Mutagenesis experiments demonstrate that substitution of the serine residues in the glycogen synthase kinase 3beta (GSK3beta) phosphorylation consensus motif of beta-catenin inhibits ubiquitination and results in stabilization of the protein. This motif in beta-catenin resembles a motif in IkappaB (inhibitor of NFkappaB) which is required for the phosphorylation-dependent degradation of IkappaB via the ubiquitin-proteasome pathway. We show that ubiquitination of beta-catenin is greatly reduced in Wnt-expressing cells, providing the first evidence that the ubiquitin-proteasome degradation pathway may act downstream of GSK3beta in the regulation of beta-catenin.     

Defining the roles of beta-catenin and plakoglobin in cell-cell adhesion: isolation of beta-catenin/plakoglobin-deficient F9 cells

F9 teratocarcinoma cells in which beta-catenin and/or plakoglobin genes are knocked-out were generated and investigated in an effort to define the role of beta-catenin and plakoglobin in cell adhesion. Loss of beta-catenin expression only did not affect cadherin-mediated cell adhesion activity. Loss of both beta-catenin and plakoglobin expression, however, severely affected the strong cell adhesion activity of cadherin. In beta-catenin-deficient cells, the amount of plakoglobin associated with E-cadherin dramatically increased. In beta-catenin/plakoglobin-deficient cells, the level of E-cadherin and alpha-catenin markedly decreased. In these cells, E-cadherin formed large aggregates in cytoplasm and membrane localization of alpha-catenin was barely detected. These data confirmed that beta-catenin or plakoglobin is required for alpha-catenin to form complex with E-cadherin. It was also demonstrated that plakoglobin can compensate for the absence of beta-catenin. Moreover it was suggested that beta-catenin or plakoglobin is required not only for the cell adhesion activity but also for the stable expression and cell surface localization of E-cadherin.     

Proteasome-dependent degradation of alpha-catenin is regulated by interaction with ARMc8alpha

ARMc8 (armadillo-repeat-containing protein 8) is a key component of the CTLH (C-terminal to lissencephaly type-1-like homology motif) complex in mammalian cells. This complex is well conserved in Saccharomyces cerevisiae and has been characterized as a FBPase (fructose-1, 6-bisphosphatase)-degrading complex. The yeast homologue of ARMc8, Gid (glucose-induced degradation) 5p, plays an essential role in the ubiquitin- and proteasome-dependent degradation of FBPase. To elucidate the function of ARMc8, we used a yeast two-hybrid system to screen a human skeletal muscle cDNA library. alpha-Catenin was isolated as a binding protein of ARMc8alpha. This association was confirmed by co-immunoprecipitation assay using MDCK (Madin-Darby canine kidney) cells in which exogenous alpha-catenin and ARMc8alpha were overexpressed. The association was also confirmed by co-immunoprecipitation assay using endogenous proteins in untransfected MDCK cells. We then used immunofluorescence microscopy of MDCK cells and C2C12 cells to investigate the intracellular distribution of ARMc8. Exogenously expressed ARMc8 was co-localized with alpha-catenin and beta-catenin along the cell membrane, suggesting an association between alpha-catenin and ARMc8 in the cells. To compare the binding domain of alpha-catenin with ARMc8alpha with that of beta-catenin, we performed a co-immunoprecipitation assay, again using 5'- and 3'-deletion constructs of alpha-catenin. The N-terminal sequence (amino acids 82-148) of alpha-catenin was sufficient to bind to both ARMc8alpha and beta-catenin. Next, we investigated the proteasome-dependent degradation of alpha-catenin by immunoblotting using proteasome inhibitors. Co-expression of ARMc8alpha with alpha-catenin resulted in rapid degradation of the exogenous alpha-catenin. Furthermore, ARMc8 knockdown inhibited alpha-catenin degradation and prolonged the half-life of alpha-catenin. We conclude that ARMc8alpha associates with alpha-catenin and up-regulates its degradation.     

Wnt/beta-catenin signaling suppresses Rapsyn expression and inhibits acetylcholine receptor clustering at the neuromuscular junction

The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.     

A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

One of the most important issues in stem cell research is to understand the regulatory mechanisms responsible for their differentiation. An extensive understanding of mechanism underlying the process of differentiation is crucial in order to prompt stem cells to perform a particular function after differentiation. To elucidate the molecular mechanisms responsible for the hematopoietic differentiation of embryonic stem cells (ESCs), we investigated murine ES cells for the presence of hematopoietic lineage markers as well as Wnt signaling pathway during treatments with different cytokines alone or in combination with another. Here we report that Wnt/beta-catenin signaling is down-regulated in hematopoietic differentiation of murine ES cells. We also found that differentiation induced by the interleukin-3, interleukin-6, and erythropoietin combinations resulted in high expression of CD3e, CD11b, CD45R/B220, Ly-6G, and TER-119 in differentiated ES cells. A high expression of beta-catenin was observed in two undifferentiated ES cell lines. Gene and protein expression analysis revealed that the members downstream of Wnt in this signaling pathway including beta-catenin, GSK-3beta, Axin, and TCF4 were significantly down-regulated as ES cells differentiated into hematopoietic progenitors. Our results show that the Wnt/beta-catenin signaling pathway plays a role in the hematopoietic differentiation of murine ESCs and also may support beta-catenin as a crucial factor in the maintenance of ES cells in their undifferentiated state.     

A targeted mutation of Nkd1 impairs mouse spermatogenesis

Nkd1 is an antagonist of the canonical Wnt/beta-catenin signaling pathway. The EF-hand motif of Nkd1 is required for its inhibitory function. Early studies suggested that Nkd1 might play important roles in mouse embryonic development and tumorigenesis. We constructed Nkd1(-/-) mice whose Nkd1 protein lacked the EF-hand and was unable to inhibit Wnt/beta-catenin signaling. The homozygotes were viable and grew normally, but their fertility in males was reduced. In wild-type adult testes, Nkd1 mRNA was expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand caused reductions in the testis weight and sperm count by 30 and 60%, respectively. During testis development, Nkd1 mRNA expression started at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increased in the elongating spermatids, suggesting that the mutant Nkd1 failed to inhibit the Wnt/beta-catenin pathway. These results suggest that deletion of the EF-hand from Nkd1 reduces the number of the elongating spermatids at haploid stage. In contrast, the mutant Nkd1 did not affect intestinal polyposis in Apc(Delta716) mice.     

Lithium chloride regulates connexin43 in skeletal myoblasts in vitro: possible involvement in Wnt/beta-catenin signaling

Gap junction channels composed of connexin43 (Cx43) are essential for normal myogenic differentiation and skeletal muscle regeneration. Here, the aim was to study whether lithium chloride (LiCl) could regulate Cx43 expression and gap junction channel function by mimicking the Wnt/beta-catenin pathway in primary myoblasts. Cx43 mRNA expression in myoblasts was up-regulated in response to 5 mM LiCl. The enhanced Cx43 protein expression resulting from treatment with 5 and 10 mM LiCl for 24 h increased gap-junctional coupling in myoblasts. However, no obvious changes were observed with 20 mM LiCl. Furthermore, chronic treatment with 10 mM LiCl decreased Cx43 protein expression compared with untreated cells. The authors showed that LiCl mimicked the active canonical Wnt/beta-catenin signaling by glycogen synthase kinase-3beta (GSK-3beta) inactivation and accumulation of the effector protein beta-catenin into the nucleus. These results suggest that LiCl regulates Cx43 expression in skeletal myoblasts in vitro partly by a Wnt/beta-catenin-dependent pathway.     

Wdr5 is essential for osteoblast differentiation

Wdr5 is developmentally expressed in osteoblasts and accelerates osteoblast differentiation in vitro and in vivo. To address whether Wdr5 is essential for osteoblast differentiation, plasmid-based small interfering RNAs were used to stably suppress endogenous Wdr5 protein levels in MC3T3-E1 cells. Reduction of endogenous Wdr5 levels markedly inhibited osteoblast differentiation, evidenced by a significant decrease in alkaline phosphatase activity, Runx-2 and osteocalcin mRNAs, and absence of mineralized matrix formation. Wdr5 suppression also resulted in a reduction of histone H3 lysine 4 trimethylation, confirming its critical role in this modification. Because Wdr5 overexpression enhances canonical Wnt signaling in osteoblasts in vivo, the effects of Wdr5 silencing on this pathway were examined. The expression of the canonical Wnt target gene, c-myc, was decreased, whereas that of sfrp2, which is repressed by Wnt signaling, was increased with Wdr5 knockdown. Although only a minimal increase in apoptosis was observed, the antiapoptotic effect of Wnt signaling was also impaired with Wdr5 silencing. The expression of canonical Wnts was significantly decreased with Wdr5 knockdown, resulting in a decrease in nuclear beta-catenin protein levels. Activation of the canonical Wnt signaling pathway did not overcome the effects of Wdr5 knockdown on the expression of Wnt target genes. Chromatin immunoprecipitation demonstrated that Wdr5 is present on the Wnt1 promoter and on canonical Wnt response elements of the c-myc and Runx-2 promoters. These studies demonstrate that Wdr5 suppression interferes with the canonical Wnt signaling pathway at multiple stages and that optimal Wdr5 levels are required for induction of the osteoblast phenotype.