Large Genomic Rearrangements of Desmosomal Genes in Italian Arrhythmogenic Cardiomyopathy Patients.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is an inherited heart muscle disease associated with point mutations in genes encoding for cardiac desmosome proteins. Conventional mutation screening is positive in ≈50% of probands. Copy number variations (CNVs) have recently been linked to AC pointing to the need to determine the prevalence of CNVs in desmosomal genes and to evaluate disease penetrance by cosegregation analysis in family members. METHODS AND RESULTS: A total of 160 AC genotype-negative probands for 5 AC desmosomal genes by conventional mutation screening underwent multiplex ligation-dependent probe amplification. Nine heterozygous CNVs were identified in 11 (6.9%) of the 160 probands. Five carried a deletion of the entire plakophilin-2 (PKP2) gene, 2 a deletion of only PKP2 exon 4, 1 a deletion of the PKP2 exons 6 to 11, 1 a PKP2 duplication of 5' untranslated region till exon 1, 1 the desmocollin-2 (DSC2) duplication of exons 7 to 9, and 1 a large deletion of chromosome 18 comprising both DSC2 and desmoglein-2 genes. All probands were affected by moderate-severe forms of the disease, whereas 10 (32%) of the 31 family members carrying one of these deletions fulfilled the diagnostic criteria. CONCLUSIONS: Genomic rearrangements were detected in ≈7% of AC probands negative for pathogenic point mutations in desmosomal genes, highlighting the potential of CNVs analysis to substantially increase the diagnostic yield of genetic testing. Genotype-phenotype correlation demonstrated the presence of the disease in about one third of family members carrying the CNV, underlying the role of other factors in the development and progression of the disease.

[Change in E-cadherin, alpha-, beta- and gamma-catenin expression after hyperthermia of a human colon carcinoma cell line in vitro].

OBJECTIVE: To investigate changes in E-cadherin, alpha-, beta-, and gamma-catenin expression after hyperthermia of a human colon cancer cell line in vitro. METHODS: E-cadherin and alpha-, beta-, and gamma-catenin expression on a human colon carcinoma cell line HT29 at 7 successive times after hyperthermia at 43 degrees C for 60 min were investigated in vitro by RT-PCR and immunochemistry. RESULTS: In vitro studies revealed that E-cadherin expression had increased significantly on HT29 cells at 24 hours after hyperthermia at 43 degrees C for 60 min. In a time-course analysis, the expression of E-cadherin had major increase at 24 to 72 hours after hyperthermia compared with an unheated control cell. gamma-catenin expression had increased significantly at 8 to 72 hours. After hyperthermia at 43 degrees C for 60 min, beta-catenin expression had decreased gradually at 4 hours to 72 hours. alpha-catenin expression kept unchanged after hyperthermia. CONCLUSION: Hyperthermia at 43C for 60 min upregulated E-cadherin and gamma-catenin expressions but downregulated beta-catenin expression on colon carcinoma cells in vitro. alpha-catenin expression was not regulated by hyperthermia.

Tyrosine phosphorylation of plakoglobin causes contrary effects on its association with desmosomes and adherens junction components and modulates beta-catenin-mediated transcription.

Plakoglobin is a protein closely related to beta-catenin that links desmosomal cadherins to intermediate filaments. Plakoglobin can also substitute for beta-catenin in adherens junctions, providing a connection between E-cadherin and alpha-catenin. Association of beta-catenin with E-cadherin and alpha-catenin is regulated by phosphorylation of specific tyrosine residues; modification of beta-catenin Tyr654 and Tyr142 decreases binding to E-cadherin and alpha-catenin, respectively. We show here that plakoglobin can also be phosphorylated on tyrosine residues, but unlike beta-catenin, this modification is not always associated with disrupted association with junctional components. Protein tyrosine kinases present distinct specificities on beta-catenin and plakoglobin, and phosphorylation of beta-catenin-equivalent Tyr residues of plakoglobin affects its interaction with components of desmosomes or adherens junctions differently. For instance, Src, which mainly phosphorylates Tyr86 in beta-catenin, modifies Tyr643 in plakoglobin, decreasing the interaction with E-cadherin and alpha-catenin and increasing the interaction with the alpha-catenin-equivalent protein in desmosomes, desmoplakin. The tyrosine kinase Fer, which modifies beta-catenin Tyr142, lessening its association with alpha-catenin, phosphorylates plakoglobin Tyr549 and exerts the contrary effect: it raises the binding of plakoglobin to alpha-catenin. These results suggest that tyrosine kinases like Src or Fer modulate desmosomes and adherens junctions differently. Our results also indicate that phosphorylation of Tyr549 and the increased binding of plakoglobin to components of adherens junctions can contribute to the upregulation of the transcriptional activity of the beta-catenin-Tcf-4 complex observed in many epithelial tumor cells.

Cytoskeletal proteins in uterine epithelial cells only partially return to the pre-receptive state after the period of receptivity.

Immunohistochemical staining of 5 cytoskeletal proteins (actin, alpha-actinin, gelsolin, plectin and plakoglobin) was used to investigate changes in distribution patterns of these proteins after the period of uterine receptivity for blastocyst implantation in the rat. Actin was found throughout the cytoplasm but it was concentrated along the apical plasma membrane on day 1 of pregnancy, decreased by day 6 and then increased again at day 9. Alpha-actinin and gelsolin were localized in distinctive bands along the apical plasma membrane at day 6 of pregnancy but became diffusely distributed at day 9. Plectin was localized along the apical and basal plasma membranes at day 6 but in higher amounts apically and at day 9, it was concentrated in apical and basal zones in the cells. Plakoglobin was found along the lateral and basal membranes with increased intensity along the apical third of the lateral plasma membrane from day 6 to day 9 of pregnancy. These results show that all 5 cytoskeletal proteins redistributed after the period of uterine receptivity: some exhibited a similar pattern of labelling to that found during the prereceptive state, whereas others only partially returned to the pre-receptive state. This change in distribution patterns may reflect differences in the epithelial barrier function before and after the period of receptivity.

Activation of AXIN2 expression by beta-catenin-T cell factor. A feedback repressor pathway regulating Wnt signaling.

The Wnt pathway regulates cell fate, proliferation, and apoptosis, and defects in the pathway play a key role in many cancers. Although Wnts act to stabilize beta-catenin levels in the cytosol and nucleus, a multiprotein complex containing adenomatous polyposis coli, glycogen synthase kinase 3beta, and Axin1 or its homolog Axin2/Axil/conductin promotes beta-catenin phosphorylation and subsequent proteasomal degradation. We found that the rat Axil gene was strongly induced upon neoplastic transformation of RK3E cells by mutant beta-catenin or gamma-catenin or after ligand-induced activation of a beta-catenin-estrogen receptor fusion protein. Expression of Wnt1 in murine breast epithelial cells activated the conductin gene, and human cancers with defective beta-catenin regulation had elevated AXIN2 gene and protein expression. Expression of AXIN2/Axil was strongly repressed in cancer cells by restoration of wild type adenomatous polyposis coli function or expression of a dominant negative form of T cell factor (TCF)-4. TCF binding sites in the AXIN2 promoter played a key role in the ability of beta-catenin to activate AXIN2 transcription. In contrast to AXIN2/Axil, expression of human or rat Axin1 homologs was nominally affected by beta-catenin-TCF. Because Axin2 can inhibit beta-catenin abundance and function, the data implicate AXIN2 in a negative feedback pathway regulating Wnt signaling. Additionally, although Axin1 and Axin2 have been thought to have comparable functions, the observation that Wnt pathway activation elevates AXIN2 but not AXIN1 expression suggests that there may be potentially significant functional differences between the two proteins.

Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation.

The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1- and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1- and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5' regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.

Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes.

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.

Induction of mutual stabilization and retardation of tumor growth by coexpression of plakoglobin and E-cadherin in mouse skin spindle carcinoma cells.

The influence of plakoglobin on the phenotype and tumorigenicity of murine spindle carcinoma cells was analyzed by stable transfection of plakoglobin cDNA in the presence or absence of E-cadherin expression. In either situation, overexpression of plakoglobin was unable to modify the fibroblastic phenotype or to completely suppress the tumorigenic behavior of the spindle cells, but a moderate reduction in the growth rate of the tumors was induced by plakoglobin and was further enhanced by E-cadherin. Coexpression of E-cadherin and plakoglobin induced a mutual stabilization, increasing the half-life of both molecules in the double transfectants more than 5- and 30-fold, respectively, with a turnover rate similar to that observed in control keratinocytes. The stabilization of E-cadherin, as well as that of plakoglobin, was maintained in the tumors induced by the double transfectants, in contrast to the unstable expression of E-cadherin observed in tumors induced in plakoglobin-deficient cells. The E-cadherin/catenin complexes present in the double transfectants were functional in calcium-dependent aggregation assays and similar in composition to those of control keratinocytes. However, most of the components of the complexes of the transfectants were solubilized by non-ionic detergents, indicating a weak interaction with the actin cytoskeleton. These results indicated that restoration of E-cadherin/catenin complexes was not sufficient to induce the transition of the fibroblastic cells to an epithelial phenotype or to completely suppress the tumorigenicity of mouse skin spindle carcinoma cells.

The role of cadherin-catenin-cytoskeleton complex in angiogenesis: antisense oligonucleotide of plakoglobin promotes angiogenesis in vitro, and protein kinase C (PKC) enhances angiogenesis through the plakoglobin signaling pathway.

Angiogenesis plays an important role in various diseases and conditions such as malignant tumor, wound healing, and atherosclerosis. Since cell-to-cell adhesion may play a key role in angiogenesis, we investigated the effect of the cadherin-catenin-cytoskeleton complex on angiogenesis in human umbilical vein endothelial cells (HUVECs). Immunofluorescence staining revealed that alpha-catenin, beta-catenin, and plakoglobin were concentrated at cell-cell contacts in HUVECs. Antisense oligonucleotide (AS-oligo), complementary to the region of human plakoglobin was dissolved in saline and applied to the media at 1 mM every 12 h for 4 days, and sense oligonucleotide (S-oligo) was used as control. HUVEC migration from an injury line was enhanced by AS-oligo. Interestingly, HUVECs migrated in line with S-oligo, and in a scattered fashion with AS-oligo. Tube formation on Matrigel occurred earlier with AS-oligo than with S-oligo. These findings indicate that plakoglobin inhibited HUVEC migration and tube formation (angiogenesis) by regulating cell-cell adhesion.

Association of human protein-tyrosine phosphatase kappa with members of the armadillo family.

We have identified a human receptor-like protein-tyrosine phosphatase (PTP) in the mammary carcinoma cell line SK-BR-3, which represents the human homolog of murine PTPkappa (Jiang, Y.-P., Wang, H., D'Eustachio, P., Musacchio, J. M., Schlessinger, J., and Sap, J. (1993) Mol. Cell. Biol. 13, 2942-2951) and was therefore termed hPTPkappa. We show here that hPTPkappa expression is dependent on cell density and find it colocalized with two members of the arm family of proteins, beta-catenin and gamma-catenin/plakoglobin, at adherens junctions. Using both in vitro and in vivo binding assays, we demonstrate specific complex formation between endogenous hPTPkappa and beta- and gamma-catenin/plakoglobin. In addition, we present evidence that suggests that beta-catenin may represent a substrate for the catalytic activity of hPTPkappa. The identification of specific binding partners for this receptor-like PTP provides insight into the mechanisms of its biological action and suggests a role for hPTPkappa in the regulation of processes involving cell contact and adhesion such as growth control, tumor invasion, and metastasis.

Characterization of cadherin-4 and cadherin-5 reveals new aspects of cadherins.

Several properties of cadherin-4 and cadherin-5 were characterized by using the cDNA transfection approach. The proteins of both cadherins had a relative molecular mass of about 130 kDa and were present at the cell periphery, especially at cell-cell contact sites. These cadherins were easily digested with trypsin, and Ca2+ protected cadherin-4, but not cadherin-5, from the digestion. In immunoprecipitation, cadherin-4 co-precipitated with two major proteins of 105 kDa and 95 kDa, respectively. The 105 kDa and the 95 kDa proteins are likely to correspond to alpha- and beta-catenins. Cadherin-5 co-precipitated with only one major protein of 95 kDa, but seems to associate with the 105 kDa protein. On the other hand, plakoglobin or gamma-catenin did not co-precipitate well with either cadherin-4 or cadherin-5 in immunoprecipitation, but plakoglobin also appears to associated weakly with these cadherins. Cadherin-4 transfectants aggregated within 30 minutes in a cell aggregation assay, but cadherin-5 transfectants did not aggregate under the same conditions. Furthermore, the transfectants of chimeric cadherin-4 with cadherin-5 cytoplasmic domain showed cell aggregation activity comparable to that of wild-type cadherin-4 transfectants, whereas the transfectants of chimeric cadherin-5 with cadherin-4 cytoplasmic domain did not show appreciable cell aggregation, suggesting that the extracellular domains of cadherins, in conjunction with their cytoplasmic domains, play an important role in cell aggregation activity. These results show that cadherin-4 is very similar to the classical cadherins, whereas cadherin-5 is functionally as well as structurally distinct from classical cadherins.