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.     

Epigenetic alterations of CDH1 and APC genes: relationship with activation of Wnt/beta-catenin pathway in invasive ductal carcinoma of breast

Activation of canonical Wnt/beta-catenin pathway in Invasive Ductal Carcinoma of Breast (IDCs) was recently reported from our laboratory. Herein, we analyzed promoter methylation status of CDH1 and Adenomatous polyposis coli (APC) genes in 50 IDCs and correlated with expression of E-cadherin (E-CD) and APC proteins and with activation of oncogenic Wnt/beta-catenin signaling pathway components, Dvl, beta-catenin and CyclinD1. Further, Wnt/beta-catenin driven epithelial mesenchymal transition (EMT) was investigated by correlating the expression of Dvl, beta-catenin and CyclinD1 with vimentin expression in these IDCs. Promoter hypermethylation was observed in 25/50 (50%) IDCs for CDH1 and in 11/50 (22%) tumors for APC, associated with loss of expression of E-CD and APC proteins; concordant hypermethylation of these genes was observed in paired patients' sera. Further, 57% of tumors harboring CDH1 methylation and 50% tumors harboring the methylated APC gene showed nuclear localization of beta-catenin, suggesting activation of the canonical Wnt/beta-catenin pathway. Our study demonstrates significant association between vimentin expression and nuclear beta-catenin (p=0.001; Odds ratio (OR)=25.6) and Dvl (p=0.023; OR=8.0), suggesting that EMT may be driven by Wnt/beta-catenin activation in IDCs. In conclusion, we demonstrate correlation of CDH1 and APC promoter methylation with loss of E-CD and APC proteins and with activation of Wnt/beta-catenin signaling pathway. Association of nuclear Dvl and beta-catenin with vimentin expression suggests the importance of Wnt/beta-catenin pathway driven EMT in IDCs. The concordance between CDH1 and APC methylation in IDCs and paired circulating DNA underscores the utility of serum DNA as a non-invasive tool for methylation analysis in IDC patients.     

The recruitment of phosphatidylinositol 3-kinase to the E-cadherin-catenin complex at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and human keratinocyte differentiation

Calcium induces epidermal keratinocyte differentiation, but the mechanism is not completely understood. We have previously demonstrated that calcium-induced human keratinocyte differentiation requires an intracellular calcium rise caused by phosphatidylinositol 3-kinase (PI3K)-dependent activation of phospholipase C-gamma1. In this study we sought to identify the upstream signaling pathway necessary for calcium activation of PI3K and its subsequent activation of phospholipase C-gamma1. We found that calcium induces the recruitment of PI3K to the E-cadherin-catenin complex at the plasma membrane of human keratinocytes. Knocking-down E-cadherin, beta-catenin, or p120-catenin expression blocked calcium activation of PI3K and phospholipase C-gamma1 and calcium-induced keratinocyte differentiation. However, knocking-down gamma-catenin expression had no effect. Calcium-induced PI3K recruitment to E-cadherin stabilized by p120-catenin at the plasma membrane requires beta-catenin but not gamma-catenin. These data indicate that the recruitment of PI3K to the E-cadherin/beta-catenin/p120-catenin complex via beta-catenin at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and, ultimately, keratinocyte differentiation.     

Protein-tyrosine phosphatase PCP-2 inhibits beta-catenin signaling and increases E-cadherin-dependent cell adhesion

beta-Catenin is a key molecule involved in both cell adhesion and Wnt signaling pathway. However, the exact relationship between these two roles has not been clearly elucidated. Tyrosine phosphorylation of beta-catenin was shown to decrease its binding to E-cadherin, leading to decreased cell adhesion and increased beta-catenin signaling. We have previously shown that receptor-like protein-tyrosine phosphatase PCP-2 localizes to the adherens junctions and directly binds and dephosphorylates beta-catenin, suggesting that PCP-2 might regulate the balance between signaling and adhesive beta-catenin. Here we demonstrate that PCP-2 can inhibit both the wild-type and constitutively active forms of beta-catenin in activating target genes such as c-myc. The phosphatase activity of PCP-2 is required for this effect since loss of catalytic activity attenuates its inhibitory effect on beta-catenin activation. Expression of PCP-2 in SW480 colon cancer cells can lead to stabilization of cytosolic pools of beta-catenin perhaps, by virtue of their physical interaction. PCP-2 expression also leads to increased membrane-bound E-cadherin and greater stabilization of adherens junctions by dephosphorylation of beta-catenin, which could further sequester cytosolic beta-catenin and thus inhibit beta-catenin mediated nuclear signaling. Furthermore, SW480 cells stably expressing PCP-2 have a reduced ability to proliferate and migrate. Thus, PCP-2 may play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may be an alternative mechanism for activating beta-catenin signaling.     

The Wnt/beta-catenin pathway regulates Gli-mediated Myf5 expression during somitogenesis

Canonical Wnt/beta-catenin signaling regulates the activation of the myogenic determination gene Myf5 at the onset of myogenesis, but the underlying molecular mechanism is unknown. Here, we report that the Wnt signal is transduced in muscle progenitor cells by at least two Frizzled (Fz) receptors (Fz1 and/or Fz6), through the canonical beta-catenin pathway, in the epaxial domain of newly formed somites. We show that Myf5 activation is dramatically reduced by blocking the Wnt/beta-catenin pathway in somite progenitor cells, whereas expression of activated beta-catenin is sufficient to activate Myf5 in somites but not in the presomitic mesoderm. In addition, we identified Tcf/Lef sequences immediately 5' to the Myf5 early epaxial enhancer. These sites determine the correct spatiotemporal expression of Myf5 in the epaxial domain of the somite, mediating the synergistic action of the Wnt/beta-catenin and the Shh/Gli pathways. Taken together, these results demonstrate that Myf5 is a direct target of Wnt/beta-catenin, and that its full activation requires a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells.     

Regulating the balance between peroxisome proliferator-activated receptor gamma and beta-catenin signaling during adipogenesis. A glycogen synthase kinase 3beta phosphorylation-defective mutant of beta-catenin inhibits expression of a subset of adipogenic genes

The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of beta-catenin to the proteasome. In fact, sustained activation of beta-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. In this report, we investigated the mechanisms regulating the balance between beta-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes. Specifically, we show that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of beta-catenin, which depends on glycogen synthase kinase (GSK) 3beta activity. Mutation of serine 37 (a target of GSK3beta) to an alanine renders beta-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3beta phosphorylation-defective S37A-beta-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-beta-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant beta-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-beta-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKbeta is a key regulator of the balance between beta-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes.     

Glucose induces an autocrine activation of the Wnt/beta-catenin pathway in macrophage cell lines

The canonical Wnt signalling pathway acts by slowing the rate of ubiquitin-mediated beta-catenin degradation. This results in the accumulation and subsequent nuclear translocation of beta-catenin, which induces the expression of a number of genes involved in growth, differentiation and metabolism. The mechanisms regulating the Wnt signalling pathway in the physiological context is still not fully understood. In the present study we provide evidence that changes in glucose levels within the physiological range can acutely regulate the levels of beta-catenin in two macrophage cell lines (J774.2 and RAW264.7 cells). In particular we find that glucose induces these effects by promoting an autocrine activation of Wnt signalling that is mediated by the hexosamine pathway and changes in N-linked glycosylation of proteins. These studies reveal that the Wnt/beta-catenin system is a glucose-responsive signalling system and as such is likely to play a role in pathways involved in sensing changes in metabolic status.