ABSTRACT␤-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated ␤-catenin levels in colorectal cancer caused by mutations in ␤-catenin or by the adenomatous polyposis coli molecule, which regulates ␤-catenin degradation, result in the binding of ␤-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the ␤-catenin͞LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of ␤-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by ␤-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased ␤-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.

Transcriptional repression of E-cadherin, characteristic of epithelial to mesenchymal transition, is often found also during tumor cell invasion. At metastases, migratory fibroblasts sometimes revert to an epithelial phenotype, by a process involving regulation of the E-cadherin–β-catenin complex. We investigated the molecular basis of this regulation, using human colon cancer cells with aberrantly activated β-catenin signaling. Sparse cultures mimicked invasive tumor cells, displaying low levels of E-cadherin due to transcriptional repression of E-cadherin by Slug. Slug was induced by β-catenin signaling and, independently, by ERK. Dense cultures resembled a differentiated epithelium with high levels of E-cadherin and β-catenin in adherens junctions. In such cells, β-catenin signaling, ErbB-1/2 levels, and ERK activation were reduced and Slug was undetectable. Disruption of E-cadherin–mediated contacts resulted in nuclear localization and signaling by β-catenin, induction of Slug and inhibition of E-cadherin transcription, without changes in ErbB-1/2 and ERK activation. This autoregulation of E-cadherin by cell–cell adhesion involving Slug, β-catenin and ERK could be important in tumorigenesis.

β-Catenin and plakoglobin are homologous proteins that function in cell adhesion by linking cadherins to the cytoskeleton and in signaling by transactivation together with lymphoid-enhancing binding/T cell (LEF/TCF) transcription factors. Here we compared the nuclear translocation and transactivation abilities of β-catenin and plakoglobin in mammalian cells. Overexpression of each of the two proteins in MDCK cells resulted in nuclear translocation and formation of nuclear aggregates. The β-catenin-containing nuclear structures also contained LEF-1 and vinculin, while plakoglobin was inefficient in recruiting these molecules, suggesting that its interaction with LEF-1 and vinculin is significantly weaker. Moreover, transfection of LEF-1 translocated endogenous β-catenin, but not plakoglobin to the nucleus. Chimeras consisting of Gal4 DNA-binding domain and the transactivation domains of either plakoglobin or β-catenin were equally potent in transactivating a Gal4-responsive reporter, whereas activation of LEF-1– responsive transcription was significantly higher with β-catenin. Overexpression of wild-type plakoglobin or mutant β-catenin lacking the transactivation domain induced accumulation of the endogenous β-catenin in the nucleus and LEF-1–responsive transactivation. It is further shown that the constitutive β-catenin–dependent transactivation in SW480 colon carcinoma cells and its nuclear localization can be inhibited by overexpressing N-cadherin or α-catenin. The results indicate that (a) plakoglobin and β-catenin differ in their nuclear translocation and complexing with LEF-1 and vinculin; (b) LEF-1–dependent transactivation is preferentially driven by β-catenin; and (c) the cytoplasmic partners of β-catenin, cadherin and α-catenin, can sequester it to the cytoplasm and inhibit its transcriptional activity.

We studied the effect of N-cadherin, and its free or membrane-anchored cytoplasmic domain, on the level and localization of ␤-catenin and on its ability to induce lymphocyte enhancer-binding factor 1 (LEF-1)-responsive transactivation. These cadherin derivatives formed complexes with ␤-catenin and protected it from degradation. N-cadherin directed ␤-catenin into adherens junctions, and the chimeric protein induced diffuse distribution of ␤-catenin along the membrane whereas the cytoplasmic domain of N-cadherin colocalized with ␤-catenin in the nucleus. Cotransfection of ␤-catenin and LEF-1 into Chinese hamster ovary cells induced transactivation of a LEF-1 reporter, which was blocked by the N-cadherin-derived molecules. Expression of N-cadherin and an interleukin 2 receptor͞cadherin chimera in SW480 cells relocated ␤-catenin from the nucleus to the plasma membrane and reduced transactivation. The cytoplasmic tails of N-or E-cadherin colocalized with ␤-catenin in the nucleus, and suppressed the constitutive LEF-1-mediated transactivation, by blocking ␤-catenin-LEF-1 interaction. Moreover, the 72 C-terminal amino acids of N-cadherin stabilized ␤-catenin and reduced its transactivation potential. These results indicate that ␤-catenin binding to the cadherin cytoplasmic tail either in the membrane, or in the nucleus, can inhibit ␤-catenin degradation and efficiently block its transactivation capacity.

β-Catenin and plakoglobin (γ-catenin) are closely related molecules of the armadillo family of proteins. They are localized at the submembrane plaques of cell–cell adherens junctions where they form independent complexes with classical cadherins and α-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to their role in junctional assembly, β-catenin has been shown to play an essential role in signal transduction by the Wnt pathway that results in its translocation into the nucleus. To study the relationship between plakoglobin expression and the level of β-catenin, and the localization of these proteins in the same cell, we employed two different tumor cell lines that express N-cadherin, and α- and β-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of β-catenin in each clone. Induction of plakoglobin expression increased the turnover of β-catenin without affecting RNA levels, suggesting posttranslational regulation of β-catenin. In plakoglobin overexpressing cells, both β-catenin and plakoglobin were localized at cell– cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin binding domain, but not the α-catenin binding domain, abolished β-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in β-catenin levels and resulted in accumulation of both β-catenin and plakoglobin in the nucleus. These results suggest that (a) plakoglobin substitutes effectively with β-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional β-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess β-catenin and plakoglobin translocate into the nucleus.

Abstract. Plakoglobin is a major component of the submembranal plaque of adherens junctions and desmosomes in mammalian ceils. It is closely related to the Drosophila segment polarity gene armadillo which has a role in the transduction of transmembrane signals that regulate cell fate. Like its close homologue [3-catenin, plakoglobin can associate with the product of the tumor suppressor gene APC that is linked to human colon cancer. We have studied the effect of plakoglobin overexpression, and the cooperation between plakoglobin and N-cadherin, on the morphology and tumorigenic ability of cells either lacking, or expressing cadherin and eL-and 6-catenin.Overexpression of plakoglobin in SV40-transformed 3T3 (SVT2) cells suppressed the tumorigenicity of the cells in syngeneic mice. Transfection with N-cadherin conferred an epithelial phenotype on the cell culture, but had no significant effect on the tumorigenicity of the cells. Cotransfection of plakoglobin and N-cadherin into SVT2 cells, however, was considerably more effective in tumor suppression than plakoglobin overexpression alone. Finally, transfection of plakoglobin into a human renal carcinoma cell line that expresses neither cadherins nor plakoglobin, or or-and 13-catenin, resulted in a dose-dependent suppression of tumor formation by these cells in nude mice. Plakoglobin, in these cells, did not exhibit junctional localization and was diffusely distributed in the cytoplasm, with a significant amount of the protein also localized in the nucleus. The results suggest that plakoglobin can efficiently suppress the tumorigenicity of cells in the presence of, or independently of the cadherin-catenin complex.

Using a dual pipette assay that measures the force required to separate adherent cell doublets, we have quantitatively compared intercellular adhesiveness mediated by Type I (E-or N-cadherin) or Type II (cadherin-7 or -11) cadherins. At similar cadherin expression levels, cells expressing Type I cadherins adhered much more rapidly and strongly than cells expressing Type II cadherins. Using chimeric cadherins, we found that the extracellular domain exerts by far the dominant effect on cell adhesivity, that of E-cadherin conferring high adhesivity, and that of cadherin-7 conferring low adhesivity. Type I cadherins were incorporated to a greater extent into detergent-insoluble cytoskeletal complexes, and their cytoplasmic tails were much more effective in disrupting strong adherent junctions, suggesting that Type II cadherins form less stable complexes with ␤-catenin. The present study demonstrates compellingly, for the first time, that cadherins are dramatically different in their ability to promote intercellular adhesiveness, a finding that has profound implications for the regulation of tissue morphogenesis.Adhesive interactions, so essential to multicellular life, are mediated by a diversity of cell surface receptors. Prominent among them are the cadherins, calcium-dependent adhesion molecules central to tissue development and morphogenesis (1-3). The growing superfamily of cadherins is subdivided into five families: classical Type I cadherins, atypical Type II cadherins, desmosomal cadherins, protocadherins, and seven-pass transmembrane cadherins (4, 5). Classical Type I and desmosomal cadherins are found primarily in tissues where a high degree of cell cohesion is required for tissue integrity. Other types of cadherins are expressed in situations where cells are more motile, and intercellular interactions are more transitory (6 -9). It is increasingly clear that cadherins contribute to other cellular functions, including cell signaling, proliferation, differentiation, segregation, and migration (10 -17).Predictably, the pattern of cadherin expression during development is complex. For example, development of the neural crest involves epithelial to mesenchymal transitions, cell migration, cell aggregation, and cell differentiation (18), each of which is associated with tightly regulated, differential expression of Type I and II cadherins. Premigratory cells of the avian neural crest express first N-cadherin, and then they down-regulate N-cadherin and express Type II cadherin-6B, but later down-regulate it and induce expression of the Type II cadherin-7 as they migrate throughout the embryo (19,20). Another Type II cadherin, cadherin-11, is similarly induced in migrating neural crest cells of rat and Xenopus embryos (21-23). Cell grafting experiments in vivo verify that expression of cadherin-7 correlates with cell dispersion and migration along migratory pathways, whereas that of N-cadherin fosters strong intercellular cohesivity and failure to migrate (6).Regulation of cellular adhesion can be achieved in a v...

b-Catenin and plakoglobin are closely related armadillo family proteins with shared and distinct properties; Both are associated with cadherins in actin-containing adherens junctions. Plakoglobin is also found in desmosomes where it anchors intermediate ®laments to the desmosomal plaques. b-Catenin, on the other hand, is a component of the Wnt signaling pathway, which is involved in embryonic morphogenesis and tumorigenesis. A key step in the regulation of this pathway involves modulation of b-catenin stability. A multiprotein complex, regulated by Wnt, directs the phosphorylation of bcatenin and its degradation by the ubiquitin-proteasome system. Plakoglobin can also associate with members of this complex, but inhibition of proteasomal degradation has little e ect on its levels while dramatically increasing the levels of b-catenin. b-TrCP, an F-box protein of the SCF E3 ubiquitin ligase complex, was recently shown to play a role in the turnover of b-catenin. To elucidate the basis for the apparent di erences in the turnover of bcatenin and plakoglobin we compared the handling of these two proteins by the ubiquitin-proteasome system. We show here that a deletion mutant of b-TrCP, lacking the F-box, can stabilize the endogenous b-catenin leading to its nuclear translocation and induction of b-catenin/ LEF-1-directed transcription, without a ecting the levels of plakoglobin. However, when plakoglobin was overexpressed, it readily associated with b-TrCP, e ciently competed with b-catenin for binding to b-TrCP and became polyubiquitinated. Fractionation studies revealed that about 85% of plakoglobin in 293 cells, is Triton X-100-insoluble compared to 50% of b-catenin. These results suggest that while both plakoglobin and b-catenin can comparably interact with b-TrCP and the ubiquitination system, the sequestration of plakoglobin by the membrane-cytoskeleton system renders it inaccessible to the proteolytic machinery and stabilizes it.