Oncogene (2004) 23, 964–972 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc Plakoglobin (c-catenin) has TCF/LEF family-dependent transcriptional activity in b-catenin-deficient cell line Osamu Maeda1,2, Noriyasu Usami2,3, Masashi Kondo1,2, Masahide Takahashi4, Hidemi Goto1, Kaoru Shimokata1,2, Kazuo Kusugami1 and Yoshitaka Sekido*,2 1Department of Internal Medicine, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8560, Japan; 2Department of Clinical Preventive Medicine, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8560, Japan; 3Department of Thoracic Surgery, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8560, Japan; 4Department of Pathology, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8560, Japan b-Catenin is an essential element for the transcriptional Introduction activation of target genes in the Wnt signaling cascade and is also a cell adhesion molecule that couples with Plakoglobin (g-catenin) and b-catenin are homologous cadherins. Although plakoglobin (c-catenin), a closely proteins with characteristic structural features contain- related homologue of b-catenin, is also known to be a cell ing a central armadillo repeat domain flanked by the adhesion molecule, its function as a transcriptional factor carboxyl and amino terminal domains, and function in has not been revealed in detail. Using a human malignant cell–cell junctions when coupled with cadherins (Ru- mesothelioma cell line, NCI-H28, in which we have binfeld et al., 1995; Barker and Clevers, 2000). identified a homozygous deletion of the b-catenin gene, Plakoglobin is present in adherens junctions and we studied whether plakoglobin has a T-cell factor/ desmosomes where it interacts with desmoglein and lymphocyte enhancer factor (TCF/LEF) family-depen- desmocollin, whereas b-catenin is present in adherens dent transcriptional activity. Transfection with the wild- junctions, where it links cadherin family proteins via a- type plakoglobin expression vector induced accumulation catenin to the actin cytoskeleton (Ben-Ze’ev and Geiger, of plakoglobin in the nucleus. Immunoprecipitation assay 1998). with cotransfection of plakoglobin and either TCF-4 or b-Catenin is also an essential element in the Wnt LEF-1 detected binding of plakoglobin to TCF-4 or LEF- signaling cascade that plays a critical role in develop- 1. Luciferase reporter assay demonstrated transcriptional ment and tumorigenesis (Morin et al., 1997; Sparks et al., activity of the wild-type plakoglobin when transfected with 1998; Polakis, 2000; Moon et al., 2002). The amino TCF/LEF, although plakoglobin showed less activity than terminal domain of b-catenin contains a highly con- b-catenin. Exogenous plakoglobin was also shown to served consensus sequence for phosphorylation by promote entrance of exogenous b-catenin into the nuclei. glycogen synthase kinase 3b (GSK3b), while the Furthermore, small interfering RNA directed against carboxyl terminal domain is essential for transcriptional plakoglobin suppressed expression of endogenous plako- activity. In the absence of Wnt signals, GSK3b globin and its transcriptional activity, suggesting that phosphorylates b-catenin, and the phosphorylated b- endogenous plakoglobin has a weak transcriptional catenin is ubiquitinated, resulting in the degradation of activity. These results suggest that plakoglobin can b-catenin by proteasomes. Meanwhile, in the presence of activate the Wnt signaling cascade directly without Wnt signals, the cytoplasmic pool of b-catenin increases interaction of b-catenin, and that plakoglobin has multiple and accumulated b-catenin enters into the nucleus. This functions as a transcriptional activator and a cell adhesion results in the formation of complexes with T-cell factor/ molecule like b-catenin. lymphocyte enhancement factor (TCF/LEF) family Oncogene (2004) 23, 964–972. doi:10.1038/sj.onc.1207254 proteins that are capable of transactivating target genes Published online 8 December 2003 including c-MYC (He et al., 1998), cyclin D1 (Shtutman et al., 1999; Tetsu and McCormick, 1999), the Keywords: plakoglobin; b-catenin; malignant mesothe- matrix metalloproteinase matrilysin (Crawford et al., lioma; transcriptional activity; Wnt signaling cascade 1999), gastrin (Koh et al., 2000), and PPARd (He et al., 1999). Several lines of evidence have suggested that plako- globin is also involved in the Wnt signal transduction cascade (Caca et al., 1999; Kolligs et al., 2000; Williams et al., 2000). Plakoglobin has been shown to bind to *Correspondence: Y Sekido; E-mail: [email protected] LEF-1 and, when overexpressed in mammalian cells, to Received 3 March 2002; revised 9 September 2003; accepted 25 enhance LEF-1-directed transcription, although a September 2003 plakoglobin and LEF-1 complex may be very inefficient Transcriptional activity of plakoglobin O Maeda et al 965 in binding DNA or in forming a ternary complex frequently mutated in a variety of human tumors. containing LEF-1 and the LEF-1-binding DNA se- Sequencing analysis of cDNA generated from NCI-H28 quences (Zhurinsky et al., 2000). It was also suggested did not detect a mutation of APC between codons 1201 that overexpression of plakoglobin does not directly and 1527, where the mutation cluster region is located. activate transcription, and that plakoglobin induces Meanwhile, reverse transcriptase-polymerase chain re- transcription indirectly by elevating the level of action (RT–PCR) and sequencing analyses revealed that endogenous b-catenin by blocking degradation of b- NCI-H28 expresses alternative splicing forms and catenin in the cytoplasm (Zhurinsky et al., 2000). nucleotide changes in AXIN1, but they were identical However, all these studies were conducted using cell to several EST sequences that were already deposited in lines with endogenous b-catenin expression, which the NCBI database. In addition, we found no mutation might interfere in the analysis of the Wnt signal of plakoglobin in the NCI-H28 cells. activation that could be regulated by plakoglobin. To study whether plakoglobin has transcriptional Furthermore, since a recent study has shown that activity in the NCI-H28 cells, we synthesized the plakoglobin has a tumor suppressive activity in non- expression vectors with several forms of plakoglobin: a small-cell lung cancer through inhibition of b-catenin/ wild-type form (Plak WT), an activating form (Plak TCF-dependent transcription (Winn et al., 2002), S28A), and a form with a deleted C-terminus, which has clarification of how plakoglobin is involved in the Wnt been reported to be a region critical to transcriptional signaling cascade now seems to be needed. activity (Plak DC) (Figure 1a, b). We also synthesized To evaluate the possible functions of plakoglobin in the same series of expression vectors with a V5 tag (Plak the Wnt signaling cascade, one essential study would be WT-V5, Plak S28A-V5, and Plak DC-V5). After to test its function in b-catenin-deficient cells. Since we transfecting them into the NCI-H28 cells, their expres- have found that a malignant mesothelioma cell line, sion products were confirmed with Western blot analysis NCI-H28, has a homozygous deletion of the b-catenin using anti-plakoglobin and anti-V5 antibodies gene (Shigemitsu et al., 2001), we employed this cell line (Figure 1c). All the constructs were confirmed to with transfection of plakoglobin expression vectors, translate into proteins of the expected size, which were including a putative activating form that was found in a more abundantly expressed than the endogenous gastric cancer cell line (Caca et al., 1999). Immunopre- plakoglobin. Meanwhile, we also transfected these cipitation assay showed that plakoglobin binds to TCF/ constructs into a non-small-cell lung cancer cell line, LEF, and luciferase reporter assay that plakoglobin has NCI-H1299, which expresses wild-type b-catenin and TCF/LEF-dependent transcriptional activity. Plakoglo- plakoglobin, and confirmed that exogenous products bin was also shown to prompt exogenous b-catenin to were all expressed similarly (data not shown). enter the nuclei. Furthermore, small interfering RNA Using b-catenin constructs of wild-type form (b-cat (siRNA) directed against plakoglobin suppressed the WT-V5), an activating mutation form that lacks a serine expression of plakoglobin and decreased the transcrip- residue at the GSK3b phosphorylation site (b-cat S37C- tional activity. These findings suggest that plakoglobin V5), and a deletion form (b-cat DC-V5), we previously has TCF/LEF transcriptional activity independent of b- reported that the activating form was more abundantly catenin, although this activity is lower than that of expressed than the wild type-form (Figure 1c) (Usami b-catenin. et al., 2003). In the present study, however, we did not detect any apparent difference in the expression levels between Plak WT and S28A mutant, which has been proposed to be an analogous mutation of the activated Results b-catenin (Figure 1c). Thus, we suspect that the Expression of exogenous plakoglobin in NCI-H28 mutation of plakoglobin in the GSK3b phosphorylation site in the N-terminal domain may not confer greater We have previously shown with Western blot analysis stability to plakoglobin in this cell line. that a malignant mesothelioma cell line, NCI-H28, has a -catenin homozygous deletion of the b gene, and that Overexpressed plakoglobin accumulates in the nucleus these cells