G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
cadherin 2, type 1, N-cadherin (neuronal)
G00000625 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000059940 (Vega human gene)
ENSG00000170558 (Ensembl human gene)
1000 (Entrez Gene)
68 (G2Cdb plasticity & disease)
CDH2 (GeneCards)
114020 (OMIM)
Marker Symbol
HGNC:1759 (HGNC)
Protein Expression
141 (human protein atlas)
Protein Sequence
P19022 (UniProt)

Synonyms (2)

  • CD325
  • CDHN

Literature (117)

Pubmed - other

  • Rac1-induced connective tissue growth factor regulates connexin 43 and N-cadherin expression in atrial fibrillation.

    Adam O, Lavall D, Theobald K, Hohl M, Grube M, Ameling S, Sussman MA, Rosenkranz S, Kroemer HK, Schäfers HJ, Böhm M and Laufs U

    Klinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarland, Homburg/Saar, Germany. oliver.adam@uks.eu

    Objectives: We studied the signal transduction of atrial structural remodeling that contributes to the pathogenesis of atrial fibrillation (AF).

    Background: Fibrosis is a hallmark of arrhythmogenic structural remodeling, but the underlying molecular mechanisms are incompletely understood.

    Methods: We performed transcriptional profiling of left atrial myocardium from patients with AF and sinus rhythm and applied cultured primary cardiac cells and transgenic mice with overexpression of constitutively active V12Rac1 (RacET) in which AF develops at old age to characterize mediators of the signal transduction of atrial remodeling.

    Results: Left atrial myocardium from patients with AF showed a marked up-regulation of connective tissue growth factor (CTGF) expression compared with sinus rhythm patients. This was associated with increased fibrosis, nicotinamide adenine dinucleotide phosphate oxidase, Rac1 and RhoA activity, up-regulation of N-cadherin and connexin 43 (Cx43) expression, and increased angiotensin II tissue concentration. In neonatal rat cardiomyocytes and fibroblasts, a specific small molecule inhibitor of Rac1 or simvastatin completely prevented the angiotensin II-induced up-regulation of CTGF, Cx43, and N-cadherin expression. Transfection with small-inhibiting CTGF ribonucleic acid blocked Cx43 and N-cadherin expression. RacET mice showed up-regulation of CTGF, Cx43, and N-cadherin protein expression. Inhibition of Rac1 by oral statin treatment prevented these effects, identifying Rac1 as a key regulator of CTGF in vivo.

    Conclusions: The data identify CTGF as an important mediator of atrial structural remodeling during AF. Angiotensin II activates CTGF via activation of Rac1 and nicotinamide adenine dinucleotide phosphate oxidase, leading to up-regulation of Cx43, N-cadherin, and interstitial fibrosis and therefore contributing to the signal transduction of atrial structural remodeling.

    Journal of the American College of Cardiology 2010;55;5;469-80

  • Cadherin expression in gastrointestinal tract endometriosis: possible role in deep tissue invasion and development of malignancy.

    Van Patten K, Parkash V and Jain D

    Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023, USA.

    Cadherins are cell surface proteins crucial for cell adhesion and tissue integrity. The mechanism of deep tissue invasion in gastrointestinal endometriosis is unknown and may be related to the altered expression of these cell surface proteins. The goal of this study was to evaluate the expression of N-cadherin, E-cadherin, and beta-catenin in peritoneal endometriotic implants, gastrointestinal endometriosis, and carcinoma arising in gastrointestinal endometriosis. Cases of peritoneal endometriosis, gastrointestinal endometriosis, and carcinoma arising in gastrointestinal endometriosis were identified from our pathology database. Immunohistochemistry was performed using antibodies against N-cadherin, E-cadherin, and beta-catenin on representative tissue sections. Cases of normal proliferative and secretory endometrium and adenomyosis were included in the study for comparison. The intensity and extent of staining for each marker was scored semiquantitatively. Appropriate positive and negative controls were used. A total of 38 cases (peritoneal endometriosis (n=14), gastrointestinal endometriosis (n=21: 11 colon, 8 appendix, 2 small bowel), and 3 cases of endometrioid carcinoma arising in colonic endometriosis (n=3)) were included in the study. Compared with normal proliferative endometrium, N-cadherin expression was decreased in intensity and extent in secretory endometrium. Peritoneal and gastrointestinal endometriosis also showed markedly decreased expression of N-cadherin compared with proliferative endometrium. All three cases of carcinoma arising in colonic endometriosis showed a total loss of N-cadherin in the tumor, but preserved E-cadherin and beta-catenin expression. In these cases, areas of benign endometriotic glands near the tumor showed weak and focal N-cadherin expression that was gradually lost. Moderate-to-strong membranous staining for beta-catenin expression and variable intensity of E-cadherin expression was seen diffusely in normal endometrium and all study cases. These results strongly suggest that alterations of N-cadherin expression in gastrointestinal endometriosis may have an important role in the mechanism that underlies deep tissue invasion, and possibly also in the development of malignancy.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2010;23;1;38-44

  • Lebectin increases N-cadherin-mediated adhesion through PI3K/AKT pathway.

    Sarray S, Siret C, Lehmann M, Marrakchi N, Luis J, El Ayeb M and André F

    Institut Pasteur de Tunis, Tunis, Tunisia.

    Cell adhesion molecules, including cadherins and integrins, play an essential role during tumor progression and represent potential targets for the development of new therapeutic agents. We previously showed that lebectin, a C-type lectin protein (CLP) issued from Macrovipera lebectina snake venom, inhibits integrin-mediated migration of IGR39 melanoma cells. Here we assessed whether lebectin modulates cell-cell adhesion. We demonstrated that lebectin promotes N-cadherin/catenin complex reorganization at cell-cell contacts, inducing a strengthening of intercellular adhesion. This reorganization is associated to phosphorylation of beta-catenin on tyrosine 142 residue. Interestingly, lebectin acts on N-cadherin-mediated cell-cell contacts through PI3K/Akt pathway. This effect could contribute to the blockage of tumor cell migration previously observed.

    Cancer letters 2009;285;2;174-81

  • IGF-1 released by corneal epithelial cells induces up-regulation of N-cadherin in corneal fibroblasts.

    Ko JA, Yanai R and Nishida T

    Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan. jiae0831@yamaguchi-u.ac.jp

    Interactions between epithelial cells and fibroblasts play important roles in tissue homeostasis. With the use of a coculture system in which human corneal fibroblasts and epithelial cells are cultured on opposite sides of a collagen (vitrigel) membrane, we have examined the effects of epithelial cells on expression of the adherens-junction protein N-cadherin in fibroblasts. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that the presence of epithelial cells increased the expression of N-cadherin in fibroblasts at the mRNA and protein levels. This effect of epithelial cells was mimicked by insulin-like growth factor-1 (IGF-1) but not by epidermal growth factor or fibroblast growth factor. Depletion of IGF-1 in epithelial cells by RNA interference abolished the effect of these cells on N-cadherin expression in fibroblasts. The presence of epithelial cells activated the IGF-1 receptor as well as up-regulated expression of the transcriptional regulator ZEB1 in fibroblasts. RNA interference-mediated depletion of IGF-1 in epithelial cells prevented the effect of these cells on ZEB1 expression in fibroblasts. These results suggest that IGF-1 released from corneal epithelial cells up-regulates the expression of N-cadherin in corneal fibroblasts.

    Journal of cellular physiology 2009;221;1;254-61

  • Active matrix metalloproteinase-2 promotes apoptosis of hepatic stellate cells via the cleavage of cellular N-cadherin.

    Hartland SN, Murphy F, Aucott RL, Abergel A, Zhou X, Waung J, Patel N, Bradshaw C, Collins J, Mann D, Benyon RC and Iredale JP

    MRC/University of Edinburgh Centre for Inflammation Research, Edinburgh, UK.

    Hepatic stellate cells (HSC) are known to synthesise excess matrix that characterises liver fibrosis and cirrhosis. Activated HSC express the matrix-degrading matrix metalloproteinase enzymes (MMPs) and their tissue inhibitors (TIMPs). During spontaneous recovery from experimental liver fibrosis, the expression of TIMP-1 declines and hepatic collagenolytic activity increases. This is accompanied by HSC apoptosis. In this study, we examine a potential mechanism whereby MMP activity might induce HSC apoptosis by cleaving N-cadherin at the cell surface.

    Results: N-cadherin expression was upregulated in human HSC during activation in culture. Addition of function-blocking antibodies or a peptide targeting the extracellular domain of N-cadherin, to cultured HSC, promoted apoptosis. During apoptosis, there was cleavage of N-cadherin into 20-100 kDa fragments. MMP-2 became activated early during HSC apoptosis and directly cleaved N-cadherin in vitro. Addition of activated MMP-2 to HSCs in culture resulted in enhanced apoptosis and loss of N-cadherin.

    Conclusions: Together, these studies identify a role for both N-cadherin and MMP-2 in mediating HSC apoptosis, where N-cadherin works to provide a cell survival stimulus and MMP-2 promotes HSC apoptosis concomitant with N-cadherin degradation.

    Funded by: Medical Research Council: G0600033, G116/84, G9900297

    Liver international : official journal of the International Association for the Study of the Liver 2009;29;7;966-78

  • Epstein-Barr virus latent membrane protein-1 effects on junctional plakoglobin and induction of a cadherin switch.

    Shair KH, Schnegg CI and Raab-Traub N

    Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

    Latent membrane protein-1 (LMP1) is considered the major oncoprotein of Epstein-Barr virus and is frequently expressed in nasopharyngeal carcinoma (NPC). LMP1 promotes growth and migration of epithelial cells, and the loss of plakoglobin has been identified as a contributing factor to LMP1-induced migration. Plakoglobin is a junctional protein that can also serve as a transcription factor in Tcf/Lef signaling. To determine the effects of LMP1 on the molecular and functional properties of plakoglobin, LMP1 was overexpressed in the NPC cell line C666-1. LMP1 did not affect plakoglobin stability but did decrease plakoglobin transcription. The resultant decreased levels of nuclear plakoglobin did not affect Tcf/Lef activity or the amount of plakoglobin bound to Tcf4. Although LMP1 induced and stabilized beta-catenin, a protein with common binding partners to plakoglobin, the loss of plakoglobin did not affect its association with Tcf4. However, LMP1 did induce a cadherin switch from E- to N-cadherin, a process involved in cancer progression, and enhanced the association of junctional beta-catenin with N-cadherin. LMP1 decreased overall levels of junctional plakoglobin but the remaining junctional plakoglobin was found associated with the induced N-cadherin. This increased association of junctional plakoglobin with N-cadherin was a distinguishing feature of LMP1-expressing cells that have reduced migration due to restoration of plakoglobin. Low levels of plakoglobin were also detected in human NPC tissues. These findings reveal that the effects of LMP1 on junctional plakoglobin and the initiation of a cadherin switch likely contribute to metastasis of NPC.

    Funded by: NCI NIH HHS: CA 32979, CA103634, R01 CA032979, R01 CA032979-25, R01 CA103634, R01 CA103634-05

    Cancer research 2009;69;14;5734-42

  • Genetic associations of 115 polymorphisms with cancers of the upper aerodigestive tract across 10 European countries: the ARCAGE project.

    Canova C, Hashibe M, Simonato L, Nelis M, Metspalu A, Lagiou P, Trichopoulos D, Ahrens W, Pigeot I, Merletti F, Richiardi L, Talamini R, Barzan L, Macfarlane GJ, Macfarlane TV, Holcátová I, Bencko V, Benhamou S, Bouchardy C, Kjaerheim K, Lowry R, Agudo A, Castellsagué X, Conway DI, McKinney PA, Znaor A, McCartan BE, Healy CM, Marron M and Brennan P

    Department of Environmental Medicine and Public Health, University of Padova, Padova, Italy.

    Cancers of the upper aerodigestive tract (UADT) include malignant tumors of the oral cavity, pharynx, larynx, and esophagus and account for 6.4% of all new cancers in Europe. In the context of a multicenter case-control study conducted in 14 centers within 10 European countries and comprising 1,511 cases and 1,457 controls (ARCAGE study), 115 single nucleotide polymorphisms (SNP) from 62 a priori-selected genes were studied in relation to UADT cancer. We found 11 SNPs that were statistically associated with UADT cancers overall (5.75 expected). Considering the possibility of false-positive results, we focused on SNPs in CYP2A6, MDM2, tumor necrosis factor (TNF), and gene amplified in squamous cell carcinoma 1 (GASC1), for which low P values for trend (P trend<0.01) were observed in the main effects analyses of UADT cancer overall or by subsite. The rare variant of CYP2A6 -47A>C (rs28399433), a phase I metabolism gene, was associated with reduced UADT cancer risk (P trend=0.01). Three SNPs in the MDM2 gene, involved in cell cycle control, were associated with UADT cancer. MDM2 IVS5+1285A>G (rs3730536) showed a strong codominant effect (P trend=0.007). The rare variants of two SNPs in the TNF gene were associated with a decreased risk; for TNF IVS1+123G>A (rs1800610), the P trend was 0.007. Variants in two SNPs of GASC1 were found to be strongly associated with increased UADT cancer risk (for both, P trend=0.008). This study is the largest genetic epidemiologic study on UADT cancers in Europe. Our analysis points to potentially relevant genes in various pathways.

    Cancer research 2009;69;7;2956-65

  • The expression of three genes in primary non-small cell lung cancer is associated with metastatic spread to the brain.

    Grinberg-Rashi H, Ofek E, Perelman M, Skarda J, Yaron P, Hajdúch M, Jacob-Hirsch J, Amariglio N, Krupsky M, Simansky DA, Ram Z, Pfeffer R, Galernter I, Steinberg DM, Ben-Dov I, Rechavi G and Izraeli S

    Cancer Research Center and Pathology Department, Sheba Medical Center, Ramat Gan, Israel.

    Purpose: Brain metastases affect 25% of patients with non-small cell lung cancer (NSCLC). We hypothesized that the expression of genes in primary NSCLC tumors could predict brain metastasis and be used for identification of high-risk patients, who may benefit from prophylactic therapy.

    The expression of 12 genes was measured by real-time quantitative reverse transcriptase PCR in 142 frozen NSCLC tissue samples. Univariate and multivariate Cox regression analysis was used to analyze the correlation between gene expression and the occurrence of brain metastasis. Immunohistochemistry on independent samples was used to verify the findings.

    Results: A score based on the expression levels of three genes, CDH2 (N-cadherin), KIFC1, and FALZ, was highly predictive of brain metastasis in early and advanced lung cancer. The probability of remaining brain metastasis-free at 2 years after diagnosis was 90.0+/-9.5% for patients with stage I/stage II tumors and low score compared with 62.7+/-12% for patients with high score (P<0.01). In patients with more advanced lung cancer, the brain metastasis-free survival at 24 months was 89% for patients with low score compared with only 37% in patients with high score (P<0.02). These results were confirmed by immunohistochemical detection of N-cadherin in independent cohort of primary NSCLC.

    Conclusions: The expression levels of three genes in primary NSCLC tumors may be used to identify patients at high risk for brain metastasis who may benefit from prophylactic therapy to the central nervous system.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15;5;1755-61

  • N-cadherin knock-down decreases invasiveness of esophageal squamous cell carcinoma in vitro.

    Li K, He W, Lin N, Wang X and Fan QX

    Department of Oncology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China.

    Aim: To examine the expressions of N-cadherin and E-cadherin in specimens of 62 normal esophageal epithela, 31 adjacent atypical hyperplastic epithelia and 62 esophageal squamous cell carcinomas (ESCCs), and to investigate the roles of N-cadherin in the invasiveness of ESCC cell line EC9706 transfected by N-cadherin shRNA.

    Methods: PV immunohistochemistry was used to detect the expression pattern of N-cadherin and E-cadherin in specimens of 62 normal esophageal epithelia, 31 adjacent atypical hyperplastic epithelia and 62 ESCCs. The invasiveness of ESCC line EC9706 was determined by transwell assay after EC9706 was transfected by N-cadherin shRNA.

    Results: The positive rates of N-cadherin decreased in the carcinoma, adjacent atypical hyperplastic and normal esophageal tissues (75.8%, 61.3% and 29.0%, P < 0.05), respectively, while those of E-cadherin increased (40.3%, 71.0% and 95.2%, P < 0.05). The increased expression of N-cadherin and decreased expression of E-cadherin were related to invasion, differentiation, and lymph node metastasis (P < 0.05). The expression level of N-cadherin decreased in the N-cadherin knocked down cells, and the invasiveness of those cells decreased significantly as well. The number of cells which crossed the basement membrane filter decreased from 123.40 +/- 8.23 to 49.60 +/- 6.80 (P < 0.05).

    Conclusion: E-cadherin and N-cadherin expression is correlated with the invasion and aggravation of ESCC. The down-regulation of N-cadherin lowers the invasiveness of EC9706 cell line.

    World journal of gastroenterology : WJG 2009;15;6;697-704

  • Protein phosphatase Dusp26 associates with KIF3 motor and promotes N-cadherin-mediated cell-cell adhesion.

    Tanuma N, Nomura M, Ikeda M, Kasugai I, Tsubaki Y, Takagaki K, Kawamura T, Yamashita Y, Sato I, Sato M, Katakura R, Kikuchi K and Shima H

    Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, Natori, Japan.

    Recent studies have demonstrated essential functions for KIF3, a microtubule-directed protein motor, in subcellular transport of several cancer-related proteins, including the beta-catenin-cadherin(s) complex. In this study, we report identification of the protein-phosphatase Dusp26 as a novel regulator of the KIF3 motor. Here we undertake yeast two-hybrid screening and identify Kif3a, a motor subunit of the KIF3 heterotrimeric complex, as a novel Dusp26-binding protein. Co-immunoprecipitation and colocalization experiments revealed that Dusp26 associates not only with Kif3a, but also with Kap3, another subunit of the KIF3 complex. Dephosphorylation experiments in vitro and analysis using mutant forms of Dusp26 in intact cells strongly suggested that Dusp26 is recruited to the KIF3 motor mainly by interaction with Kif3a, and thereby dephosphorylates Kap3. Forced expression of Dusp26, but not its catalytically inactive mutant, promoted distribution of beta-catenin/N-cadherin, an established KIF3 cargo, to cell-cell junction sites, resulting in increased cell-cell adhesiveness. We also showed that Dusp26 mRNA expression was downregulated in human glioblastoma samples. These results suggest previously unidentified functions of Dusp26 in intracellular transport and cell-cell adhesion. Downregulation of Dusp26 may contribute to malignant phenotypes of glioma.

    Oncogene 2009;28;5;752-61

  • beta-catenin is involved in N-cadherin-dependent adhesion, but not in canonical Wnt signaling in E2A-PBX1-positive B acute lymphoblastic leukemia cells.

    Nygren MK, Døsen-Dahl G, Stubberud H, Wälchli S, Munthe E and Rian E

    Department of Immunology, Institute for Cancer Research, Norwegian Radium Hospital, Rikshospitalet University Hospital, Montebello, Oslo, Norway.

    Objective: The t(1;19)(q23;13) translocation, resulting in the production of the E2A-PBX1 chimeric protein, is a common nonrandom translocation in pediatric B-lineage acute lymphoblastic leukemia (B-ALL). The E2A-PBX1 chimeric protein activates expression of several genes, including Wnt16. In the present study, we explored the role of Wnt16 and beta-catenin in t(1;19) B-ALL cells.

    Canonical Wnt signaling was measured by TOPflash activity. Localization of beta-catenin in the cell membrane and its involvement in leukemia-stroma interaction were studied by confocal microscopy. Adhesion to N-cadherin was analyzed by adding (3)H-thymidin-labeled cells to N-cadherin-coated wells.

    Results: In contrast to previous reports, we detected no effects on cell viability or proliferation upon modulation of the Wnt16 levels. Moreover, despite high levels of Wnt16 and beta-catenin, the cells had very low levels of canonical Wnt signaling. Instead, beta-catenin was located in the cell membrane along with N-cadherin. E2A-PBX1-positive leukemia cells adhered strongly to bone marrow stroma cells, and we showed that adherence junctions stained strongly for both proteins. Moreover, knockdown of beta-catenin reduced the adhesion of E2A-PBX1-positive leukemia cells to N-cadherin, suggesting that beta-catenin and N-cadherin play a central role in homotypic cell-to-cell adhesion and in leukemia-stroma adhesion. Interestingly, knockdown of Wnt16 by small interfering RNA reduced the level of N-cadherin.

    Conclusion: Wnt16 does not activate canonical Wnt signaling in E2A-PBX1-positive cells. Instead, beta-catenin is involved in N-cadherin-dependent adherence junctions, suggesting for the first time that leukemia-stroma interactions may be mediated via an N-cadherin-dependent mechanism.

    Experimental hematology 2009;37;2;225-33

  • N-cadherin serves as diagnostic biomarker in intrahepatic and perihilar cholangiocarcinomas.

    Mosnier JF, Kandel C, Cazals-Hatem D, Bou-Hanna C, Gournay J, Jarry A and Laboisse CL

    Université de Nantes, Faculté de Médecine de Nantes, EA Biometadys, Nantes, France. jfmosnier@chu-nantes.fr

    As a definite immunoprofile of this tumor is missing, the histopathologic diagnosis of intrahepatic cholangiocarcinoma is difficult. The aim of this study was to explore E- and N-cadherin expressions in intrahepatic bile duct tumors, and to determine their potential interest in differential diagnosis. Normal liver tissue, 5 cirrhosis with ductular reaction, 5 focal nodular hyperplasia, 5 bile duct hamartomas, 5 bile duct adenomas, and 45 intrahepatic cholangiocarcinomas from Caucasian patients were studied. Tissue-microarrays including 20 esophageal, 86 gastric, 8 small bowel, 64 colonic, 18 pancreatic, 6 gallbladder, and 7 extrahepatic biliary tract adenocarcinomas, 22 hepatocellular carcinomas, and normal tissues were constructed. Immunohistochemistry was performed using E-cadherin, N-cadherin, NCAM, Hep Par1, and cytokeratins 7, 19 and 20. Immunoblot analysis of frozen liver tissues was performed to control the specificity of E- and N-cadherin antibodies used. In normal liver, epithelial cells of intrahepatic bile ducts, whatever their caliber, as well as hepatocytes, coexpressed E- and N-cadherins at their plasma membranes. In cirrhosis, ductular reactions completely expressed E- and N-cadherins. All the benign lesions and 30 of the 45 intrahepatic cholangiocarcinomas (23/29 peripheral and 7/16 hilar) also expressed N-cadherin. E-cadherin was detected in all the lesions. The expression of N-cadherin at the plasma membrane of tumor cells was significantly more frequent in peripheral than in hilar intrahepatic cholangiocarcinomas (P=0.003). Among noncholangiocarcinomas, only 1% gastric and 66% gallbladder adenocarcinomas and all the hepatocellular carcinomas expressed N-cadherin at the membrane of tumor cells. Finally, for the diagnosis of intrahepatic cholangiocarcinomas, the specificity value of membranous expression of N-cadherin was 88%, whereas that of the combination cytokeratin 7/membranous N-cadherin was 98%. In the gastrointestinal and liver tract, membranous N-cadherin is restricted to the hepatocytes and intrahepatic biliary cells. In combination with cytokeratin 7 and Hep Par1, N-cadherin is a reliable tool for the histopathological diagnosis of primary hepatic tumors.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2009;22;2;182-90

  • Large-scale structural analysis of the classical human protein tyrosine phosphatome.

    Barr AJ, Ugochukwu E, Lee WH, King ON, Filippakopoulos P, Alfano I, Savitsky P, Burgess-Brown NA, Müller S and Knapp S

    University of Oxford, Structural Genomics Consortium, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford, OX3 7DQ, UK. alastair.barr@sgc.ox.ac.uk

    Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a "head-to-toe" dimerization model for RPTPgamma/zeta that is distinct from the "inhibitory wedge" model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.

    Funded by: Wellcome Trust

    Cell 2009;136;2;352-63

  • Expression of N-cadherin in esophageal squamous cell carcinoma and silencing expression of N-cadherin using RNA interference on invasiveness of EC9706 cells.

    Li K, Wang X, He W, Lin N and Fan QX

    Department of Oncology, First Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, PR China.

    E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasion. It has been suggested that, unlike E-cadherin, N-cadherin may promote invasion and metastasis of carcinoma. This study was to explore the correlation of E-cadherin and N-cadherin expression to clinicopathologic features of esophageal squamous cell carcinoma (ESCC), and to investigate the effect of silencing N-cadherin expression by RNA interference (RNAi) on the invasiveness of EC9706 cells.

    Methods: PV immunohistochemistry was used to detect the expression of E-cadherin and N-cadherin in 62 specimens of normal esophageal epithelium, 31 specimens of adjacent atypical hyperplasia epithelium and 62 specimens of ESCC. N-cadherin siRNA was transfected into EC9706 cells, and the effect of RNAi was assessed by RT-PCR and Western blot. The invasiveness of EC9706 cells was determined by Transwell chamber assay.

    Results: The positive rates of E-cadherin and N-cadherin were 95.2% and 29.0% in normal esophageal epithelium, 71.0% and 61.3% in adjacent atypical hyperplasia epithelium, 40.3% and 75.8% in ESCC. The negativity of E-cadherin and positivity of N-cadherin were correlated to invasion, differentiation, and lymph node metastasis of ESCC (p < 0.05). E-cadherin expression was negatively correlated to N-cadherin expression in ESCC (gamma = -0. 534, p < 0.05). N-cadherin RNAi significantly inhibited N-cadherin expression in EC9706 cells, and decreased the number of EC9706 cells that invaded through the basement membrane from (123.40 +/- 8.23) to (49.60 +/- 6.80) (p < 0.05).

    Conclusions: Down-regulation of E-cadherin and up-regulation of N-cadherin may be involved in the genesis of ESCC. Silencing N-cadherin using RNA interference could inhibit the invasiveness of EC9706 cells in vitro.

    Ai zheng = Aizheng = Chinese journal of cancer 2009;28;1;8-13

  • Analysis of APC, alpha-, beta-catenins, and N-cadherin protein expression in aggressive fibromatosis (desmoid tumor).

    Ferenc T, Wroński JW, Kopczyński J, Kulig A, Sidor M, Stalińska L, Dziki A and Sygut J

    Department of Biology and Genetics, Medical University, Pl. Hallera 1, 90-647 Lodz, Poland. biolgen@achilles.wam.lodz.pl

    The aims of this study were to analyze the cadherin/catenin adhesion complex in cells from abdominal and extra-abdominal aggressive fibromatosis tumors, and to estimate the correlation between the expression of the tested proteins and the clinical data of the desmoid patients. Immunohistochemistry was used to examine the expression of the cadherin/catenin adhesion complex: APC protein, alpha-, beta-catenin, and N-cadherin in archival material derived from 15 cases of extra-abdominal desmoid tumor (E-AD) and 20 cases of abdominal (AD) desmoid tumor. The tested proteins demonstrated cytoplasmic (c) staining. Furthermore, nuclear (n) or cytoplasmic and nuclear (c+n) staining was observed for beta-catenin. The mean values of the percentage of positive cells for the tested proteins between E-AD vs. AD did not demonstrate any statistically significant difference except for alpha-catenin. In the E-AD group, in both cases of recurrent tumors, no alpha-catenin expression was observed but the expression of this protein was detected in primary tumors. In the groups investigated, no statistically significant correlation was found between alpha-catenin, beta-catenin (c), (n) and (c+n) expression, and tumor size (p>0.1). The results regarding beta-catenin expression obtained in our study confirm the previous findings that nuclear accumulation of this protein plays a crucial role in the pathogenesis of aggressive fibromatosis.

    Pathology, research and practice 2009;205;5;311-24

  • EGFR-dependent migration of glial cells is mediated by reorganisation of N-cadherin.

    Rappl A, Piontek G and Schlegel J

    Division of Neuropathology, Institute of Pathology, Technische Universität München, Ismaninger Str. 22, 81675 München, Germany.

    Receptor tyrosine kinases of the EGFR family exert their various effects on cellular function through the formation of different dimeric receptor complexes. To investigate the functional impact of EGFR-HER2 heterodimers on migration of glial tumour cells, we stably transfected different HER2 constructs, including a constitutively active (HER2VE) and a dominant-negative (HER2VEKA) receptor, in the EGFR-overexpressing human glioma cell line LN18. Interference of EGFR activation through HER2VEKA inhibited cellular migration, whereas EGFR activation through HER2VE increased migration. These results were corroborated by inhibition of EGFR-HER2 signalling with tyrosine kinase inhibitors, because only the blocking of both receptors in HER2VE-cells with the bi-specific inhibitor AEE788 downregulated migration to levels comparable with those in HER2VEKA cells. The non-migratory phenotype was mediated through upregulation of N-cadherin and its recruitment to the cell membrane in HER2VEKA cells; downregulation of N-cadherin by RNAi restored migration in HER2VEKA cells and N-cadherin was also downregulated in migrating HER2VE-cells. Downregulation of N-cadherin levels in the plasma membrane was accompanied by a direct interaction of the EGFR-HER2 and N-cadherin-beta-catenin complexes, leading to tyrosine phosphorylation of beta-catenin. These results indicate that HER2 affects glial-cell migration by modulating EGFR-HER2 signal transduction, and that this effect is mediated by N-cadherin.

    Journal of cell science 2008;121;Pt 24;4089-97

  • N-cadherin levels in endothelial cells are regulated by monolayer maturity and p120 availability.

    Ferreri DM, Minnear FL, Yin T, Kowalczyk AP and Vincent PA

    Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA.

    Endothelial cells (ECs) express VE-cadherin and N-cadherin, and recent data suggest that VE-cadherin levels are dependent on N-cadherin expression. While investigating changes in N-cadherin levels during endothelial monolayer maturation, the authors found that VE-cadherin levels are maintained in ECs despite a decrease in N-cadherin, suggesting that VE-cadherin levels may not depend on N-cadherin. Knockdown of N-cadherin did not affect VE-cadherin levels in ECs with low endogenous N-cadherin expression. Surprisingly, however, knockdown of N-cadherin in ECs with high endogenous N-cadherin expression increased VE-cadherin levels, suggesting an inverse relationship between the two. This was further supported by a decrease in VE-cadherin following overexpression of N-cadherin. Experiments in which p120, a catenin that binds N- and VE-cadherin, was knocked down or overexpressed indicate that these two cadherins compete for p120. These data demonstrate that VE-cadherin levels are not directly related to N-cadherin levels but may be inversely related due to competition for p120.

    Funded by: NHLBI NIH HHS: HL-68079, HL-77870, R01 HL068079, R01 HL077870, R01 HL077870-03; NIAMS NIH HHS: AR-050501, R01 AR050501

    Cell communication & adhesion 2008;15;4;333-49

  • Expression of E- and N-cadherin and clinicopathology in hepatocellular carcinoma.

    Cho SB, Lee KH, Lee JH, Park SY, Lee WS, Park CH, Kim HS, Choi SK and Rew JS

    Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea.

    Loss or reduced E-cadherin expression and aberrant expression of N-cadherin have been associated with invasiveness of human carcinoma cells and poor prognosis. The role of E- and N-cadherin, however, in hepatocellular carcinoma (HCC) has not yet been elucidated. The aim of the present study was to investigate the expression pattern of E- and N-cadherin in surgically resected HCC specimens according to their relationship with clinicopathological features. The expression patterns of E- and N-cadherin were evaluated on immunohistochemistry in 68 specimens of HCC and adjacent non-tumor tissue. The most different expression pattern between HCC and non-tumor tissue was the decreased staining intensity of E-cadherin (n = 37, 54%) and the dot-like discontinuous staining of N-cadherin (n = 35, 55%). Decreased intensity of E-cadherin and discontinuous staining of N-cadherin in HCC was correlated with advanced stage. The risk factors for expression patterns related to recurrence were loss of E-cadherin expression (odds ratio (OR) = 3.6; 95% confidence interval (CI): 1.1-12.4) and discontinuous staining of N-cadherin (OR = 1.6; 95% CI: 0.8-3.2). In conclusion, discontinuous staining of N-cadherin and loss of E-cadherin expression in HCC predicts a high risk of recurrence after surgical treatment.

    Pathology international 2008;58;10;635-42

  • Candidate gene/loci studies in cleft lip/palate and dental anomalies finds novel susceptibility genes for clefts.

    Vieira AR, McHenry TG, Daack-Hirsch S, Murray JC and Marazita ML

    Department of 1Oral Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA. arv11@dental.pitt.edu

    Purpose: We revisited 42 families with two or more cleft-affected siblings who participated in previous studies. Complete dental information was collected to test the hypothesis that dental anomalies are part of the cleft phenotype spectrum, and can provide new opportunities for identification of cleft susceptibility genes.

    Methods: Genotypes from 1489 single nucleotide polymorphism markers located in 150 candidate genes/loci were reanalyzed. Two sets of association analyses were carried out. First, we ran the analysis solely on the cleft status. Second, we assigned affection to any cleft or dental anomaly (tooth agenesis, supernumerary teeth, and microdontia) and repeated the analysis.

    Results: Significant over-transmission was seen for a single nucleotide polymorphism in ankyrin repeat and sterile alpha motif domain containing 6 (rs4742741, 9q22.33; P = 0.0004) when a dental anomaly phenotype was included in the analysis. Significant over-transmission was also seen for a single nucleotide polymorphism in ERBB2 (rs1810132, 17q21.1; P = 0.0006). In the clefts only data, the most significant result was also for ERBB2 (P = 0.0006). Other markers with suggestive P values included interferon regulatory factor 6 and 6q21-q23 loci. In contrast to the above results, suggestive over-transmission of markers in GART, DPF3, and neurexin 3 were seen only when the dental anomaly phenotype was included in the analysis.

    Conclusions: These findings support the hypothesis that some loci may contribute to both clefts and congenital dental anomalies. Thus, including dental anomalies information in the genetics analysis of cleft lip and palate will provide new opportunities to map susceptibility loci for clefts.

    Funded by: NHGRI NIH HHS: N01HG65403; NIDCR NIH HHS: P50 DE016215, P50 DE016215-04, P50 DE016215-05, P50-DE016215, R01 DE016148, R01-DE016148, R21 DE016718, R21-DE016718, R37 DE008559, R37 DE008559-18, R37 DE008559-19, R37-DE08559

    Genetics in medicine : official journal of the American College of Medical Genetics 2008;10;9;668-74

  • Potential role of N-cadherin in hepatocyte growth factor (HGF) mediated improvement of the cardiac function of dilated cardiomyopathy mice.

    Tang Q, Shen D, Huang Z, Xiong R, Wu H, Huang J, Feng S, Niu P, Yang L and Bian Z

    Objective: To study the potential mechanisms of hepatocyte growth factor (HGF) mediating improvement of the cardiac function of dilated cardiomyopathy mice (DCM).

    We established experimental model of dilated cardiomyopathy by repetitive coxsickievirus B3 (CVB3) infection in Balb/c mice and half of the experimental group mice were injected 0.2 microg recombinated human HGF through tail vein every 2 days starting from the 5th month. At the 5th month, dilated cardiomyopathy occurred in experimental group mice. The HGF level and N-cadherin expression in myocardium of experimental group was downregulated. At the 7th month, after HGF supplement, the HGF level and N-cadherin expression in the myocardium of the mice were increased, the fibrosis of myocardial and cells apoptosis were ameliorated and cardiac function was improved. In vitro experiment, we found that N-cadherin expression was increased in cultured myocardial cells treated with rHGF.

    Conclusion: Increasing in N-cadherin expression may be one of the potential mechanisms of HGF mediating Improvement of cardiac function of dilated cardiomyopathy mice.

    International journal of cardiology 2008;127;3;442-3

  • Neural cadherin overexpression is a predictive marker for early postoperative recurrence in hepatocellular carcinoma patients.

    Seo DD, Lee HC, Kim HJ, Min HJ, Kim KM, Lim YS, Chung YH, Lee YS, Suh DJ, Yu E and Chun SY

    Department of Internal Medicine, University of Inje College of Medicine, Sanggye Paik Hospital, University of Ulsan College of Medicine, Seoul, Korea.

    Recent studies have disclosed alterations in neural and/or epithelial cadherin (E-cadherin) expression in several epithelial tumors. However, the clinical relevance of these phenomena in hepatocellular carcinoma (HCC) remains to be established. In this study, we investigated the expression patterns of neural and epithelial cadherins and their clinical implications in HCC.

    Methods: Immunohistochemical staining for neural and epithelial cadherins was performed on tumor and adjacent non-tumor tissue sections of 52 HCC patients subjected to curative surgical resection. Clinical, radiological, and histopathological characteristics were analyzed, relative to the degree of neural and E-cadherin expression.

    Results: The neural cadherin (N-cadherin) expression was upregulated in 67% of HCC tissues, compared to adjacent non-tumoral liver tissues. Patients expressing high levels of N-cadherin experienced more frequent tumor recurrences within 2 years (50% vs 12.5%; P = 0.01) after surgical resection. Consequently, the cumulative overall survival rate tended to be lower in patients overexpressing N-cadherin (P = 0.08). The N-cadherin overexpression was the only independent predictive factor for postoperative recurrence within 2 years in both univariate and multivariate analyses (odds ratio: 8.5; 95% confidence interval: 1.625-44.46; P = 0.011). No correlation was evident between E-cadherin expression patterns and clinicopathological parameters.

    Conclusion: The overexpression of N-cadherin is significantly related to postoperative recurrence within 2 years after surgical resection in HCC. Therefore, immunohistochemical staining for N-cadherin may be effectively applied as a predictive marker for early postoperative recurrence in HCC.

    Journal of gastroenterology and hepatology 2008;23;7 Pt 1;1112-8

  • Is N-cadherin expression important in ductal carcinoma?

    King JA, Shevde LA, Ofori-Acquah S, Watkins G and Jiang WG

    Department of Pharmacology, Center for Lung Biology, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36688, USA. jking@usouthal.edu

    Objectives: Neural (N)-cadherin is a calcium-dependent cell adhesion molecule that is associated with invasive tumors in breast cancer, but no association with grade or nodal status has been shown in previous studies. The present study examined the expression of N-cadherin in human breast cancer and assessed its prognostic value in long-term patient follow-up (10 years).

    Methods: Using real-time polymerase chain reaction, the number of N-cadherin transcripts in normal breasts (n = 32) and infiltrating ductal carcinomas (n = 90) was assessed. The results were then analyzed in relation to grade, nodal involvement, distant metastasis, TNM stage, Nottingham Prognostic Index, and survival over 10 years.

    Results: The levels of N-cadherin transcripts (normalized to glyceraldehyde 3-phosphate dehydrogenase) in primary tumors were lower in patients with metastases (P = 0.05), with local recurrence (P = 0.037), and those who died as a result of breast cancer (P = 0.038). There was no significant association with grade, nodal status, TNM stage, or Nottingham Prognostic Index.

    Conclusion: In ductal carcinomas, decreased levels of N-cadherin (normalized to glyceraldehyde 3-phosphate dehydrogenase) in primary tumors correlate with local recurrence and death in long-term follow-up of patients.

    Southern medical journal 2008;101;5;470-5

  • Collagen I-mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1.

    Shintani Y, Fukumoto Y, Chaika N, Svoboda R, Wheelock MJ and Johnson KR

    Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA.

    Tumor cells undergo epithelial-to-mesenchymal transition (EMT) to convert from a benign to a malignant phenotype. Our recent focus has been signaling pathways that promote EMT in response to collagen. We have shown that human pancreatic cancer cells respond to collagen by up-regulating N-cadherin, which promotes tumor growth, invasion, and metastasis. Initial characterization showed that knocking down c-Jun NH2-terminal kinase prevented N-cadherin up-regulation and limited tumor growth and invasion in a mouse model for pancreatic cancer. The current study was designed to understand the pathway from collagen to N-cadherin up-regulation. Initiation of the signal requires two collagen receptors, alpha2beta1 integrin and discoidin domain receptor (DDR) 1. Each receptor propagates signals through separate pathways that converge to up-regulate N-cadherin. Focal adhesion kinase (FAK)-related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of alpha2beta1 integrin. Both receptor complexes rely on the p130 Crk-associated substrate scaffold. Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

    Funded by: NCI NIH HHS: P30 CA036727, P30 CA36727; NIDCR NIH HHS: R01 DE012308, R01-DE12308; NIGMS NIH HHS: R01 GM051188, R01-GM51188

    The Journal of cell biology 2008;180;6;1277-89

  • Compartmentalization in membrane rafts defines a pool of N-cadherin associated with catenins and not engaged in cell-cell junctions in melanoma cells.

    Rossier-Pansier L, Baruthio F, Rüegg C and Mariotti A

    Division of Experimental Oncology, Centre Pluridisciplinaire d'Oncologie, Lausanne Cancer Center and Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges s/Lausanne, Switzerland.

    Melanoma progression is associated with changes in adhesion receptor expression, in particular upregulation of N-cadherin which promotes melanoma cell survival and invasion. Plasma membrane lipid rafts contribute to the compartmentalization of signaling complexes thereby regulating their function, but how they may affect the properties of adhesion molecules remains elusive. In this study, we addressed the question whether lipid rafts in melanoma cells may contribute to the compartmentalization of N-cadherin. We show that a fraction of N-cadherin in a complex with catenins is associated with cholesterol/sphingolipid-rich membrane microdomains in aggressive melanoma cells in vitro and experimental melanomas in vivo. Partitioning of N-cadherin in membrane rafts is not modulated by growth factors and signaling pathways relevant to melanoma progression, is not necessary for cell-cell junctions' establishment or maintenance, and is not affected by cell-cell junctions' and actin cytoskeleton disruption. These results reveal that two independent pools of N-cadherin exist on melanoma cell surface: one pool is independent of lipid rafts and is engaged in cell-cell junctions, while a second pool is localized in membrane rafts and does not participate in cell-cell adhesions. Targeting to membrane rafts may represent a previously unrecognized mechanism regulating N-cadherin function in melanoma cells.

    Journal of cellular biochemistry 2008;103;3;957-71

  • Differential expression of neural-cadherin in pulmonary epithelial tumours.

    Zynger DL, Dimov ND, Ho LC, Laskin WB and Yeldandi AV

    Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA. d-zynger@md.northwestern.edu

    Aims: Neural (N)-cadherin belongs to a group of transmembrane molecules with a crucial role in tissue morphogenesis and maintenance of an epithelioid phenotype and increased N-cadherin expression is implicated in tumour progression and dedifferentiation. The aim was to determine whether evaluation of N-cadherin in pulmonary tumours might assist in identifying lesions with more aggressive potential.

    One hundred and fifty-five pulmonary lesions were analysed for N-cadherin expression using immunohistochemistry, including neuroendocrine hyperplasia (n = 3), typical carcinoid (n = 59), atypical carcinoid (n = 12), small cell lung carcinoma (n = 11), large cell neuroendocrine carcinoma (n = 12), adenocarcinoma (n = 35) and squamous cell carcinoma (n = 23). Lymph node status was correlated with immunohistochemical expression. N-cadherin expression was demonstrated in all cases of neuroendocrine hyperplasia, 96% of typical carcinoids, 83% of atypical carcinoids, 63% of the small cell lung carcinomas and 32% of large cell neuroendocrine carcinomas. Over 90% of the adenocarcinomas and 100% of the squamous cell carcinomas were negative. Increased N-cadherin expression in typical carcinoids was associated with negative lymph node status (P < 0.001).

    Discussion: N-cadherin is differentially expressed in pulmonary tumours and is predominantly observed in neuroendocrine lung lesions with high expression in typical and atypical pulmonary carcinoids. The level of expression of N-cadherin between types of lung tumours does not appear to indicate malignant potential or aggressive behaviour.

    Histopathology 2008;52;3;348-54

  • Modulation of human Kv1.5 channel kinetics by N-cadherin.

    Koutsouki E, Lam RS, Seebohm G, Ureche ON, Ureche L, Baltaev R and Lang F

    Department of Physiology, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany.

    Kv1.5 is expressed in multiple tissues including heart, brain, macrophages, as well as vascular, airway, and intestinal smooth muscle cells. Kv1.5 currents contribute to cardiac repolarization. In cardiac myocytes Kv1.5 colocalizes with N-cadherin. As Kv1.5 expression increases following establishment of cell-cell contacts and N-cadherin influences the activity of other ion channels, we explored whether N-cadherin participates in the regulation of Kv1.5 activity. To this end, we expressed Kv1.5 in Xenopus oocytes with or without additional expression of N-cadherin. Coexpression of N-cadherin was followed by a approximately 2- to 3-fold increase of Kv1.5 induced current. The effect of N-cadherin was not paralleled by significant alterations of Kv1.5 channel abundance within the oocyte cell membrane but resulted primarily from accelerated recovery from inactivation. In conclusion, N-cadherin modifies Kv1.5 channel activity and is thus a novel candidate signaling molecule participating in the regulation of a variety of functions including cardiac action potential and vascular tone.

    Biochemical and biophysical research communications 2007;363;1;18-23

  • N-cadherin engagement provides a dominant stop signal for the migration of MDA-MB-468 breast carcinoma cells.

    Potthoff S, Entschladen F, Niggemann B, Zaenker KS and Lang K

    Institute for Immunology, Witten/Herdecke University, Stockumer Str. 10, Witten , 58448, Germany.

    The development of a primary tumor as such is not the main cause of death, but is rather the spreading of metastases, which causes over 90% of deaths in cancer patients. This largely depends on the ability of tumor cells to migrate away from the tumor and relocate at other areas of the body. Cell migration is known to be regulated by various extracellular signal substances such as neurotransmitters. However, before single tumor cells can start to invade into distant tissue, they have to dissociate from the primary tumor. This requires the disruption of cell-cell contacts, which are provided by a plethora of adhesion molecules like the family of cadherins. Using our well, established three-dimensional collagen-based cell migration assay, we show that engagement of N-cadherin results in a significant decrease of the spontaneous and the norepinephrine-induced migration of MDA-MB-468 breast carcinoma cells, which was due to an increase in the average break length. Moreover, this N-cadherin driven influence on the migratory activity is intracellularly integrated via multiple signaling pathways. Our results show that the impact of N-cadherin on the locomotion of MDA cells involves the activation of the adenylyl cyclase and the phosphatidylinositol-3-kinase (PI3K), but is independent of the protein kinase C (PKC) alpha. In summary, we provide evidence that the engagement of N-cadherin provides a stop signal for breast carcinoma cell migration, and accordingly the use of anti-N-cadherin antibodies or soluble ligands might be a tool to inhibit metastasis formation in E-cadherin negative but N-cadherin positive tumors.

    Breast cancer research and treatment 2007;105;3;287-95

  • The green tea compound, (-)-epigallocatechin-3-gallate downregulates N-cadherin and suppresses migration of bladder carcinoma cells.

    Rieger-Christ KM, Hanley R, Lodowsky C, Bernier T, Vemulapalli P, Roth M, Kim J, Yee AS, Le SM, Marie PJ, Libertino JA and Summerhayes IC

    Cell and Molecular Biology Laboratory, Robert E. Wise Research and Education Institute, Lahey Clinic Medical Center, 31 Mall Road, Burlington, Massachusetts 01805, USA.

    Green tea has been reported as potential dietary protection against numerous cancers and has been shown to have activity in bladder tumor inhibition in different animal models. The goal of this study was to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on growth inhibition and behavior of human bladder carcinoma cells and to identify the altered signaling pathway(s) underlying the response to EGCG exposure. EGCG inhibited the in vitro growth of invasive bladder carcinoma cells with an IC(50) range of 70-87 microM. At a concentration of 20 microM, EGCG decreased the migratory potential of bladder carcinoma cells with concomitant activation of p42/44 MAPK and STAT3 and inactivation of Akt. Using biochemical inhibitors of MAPK/ERK, and siRNA to knockdown STAT3 and Akt, inhibition of migration was recorded associated with Akt but not MAPK/ERK or STAT3 signaling in bladder cells. In addition, EGCG downregulated N-cadherin in a dose-dependent manner where reduction in N-cadherin expression paralleled declining migratory potential. Continuous feeding of EGCG to mice prior to and during the establishment of bladder carcinoma xenografts in vivo revealed >50% reduction in mean final tumor volume (P </= 0.05) with no detectable toxicity. EGCG inhibited bladder carcinoma cell growth and suppressed the in vitro migration capacity of cells via downregulation of N-cadherin and inactivation of Akt signaling. Continuous administration of EGCG to mice revealed significant inhibition of tumor growth in vivo indicating a possible preventative role for green tea in bladder cancer.

    Funded by: NCI NIH HHS: CA-104236, CA-94187; NIDDK NIH HHS: 1DK59400

    Journal of cellular biochemistry 2007;102;2;377-88

  • Genome-wide association with bone mass and geometry in the Framingham Heart Study.

    Kiel DP, Demissie S, Dupuis J, Lunetta KL, Murabito JM and Karasik D

    Hebrew SeniorLife Institute for Aging Research and Harvard Medical School, Boston, MA, USA. kiel@hrca.harvard.edu

    Background: Osteoporosis is characterized by low bone mass and compromised bone structure, heritable traits that contribute to fracture risk. There have been no genome-wide association and linkage studies for these traits using high-density genotyping platforms.

    Methods: We used the Affymetrix 100K SNP GeneChip marker set in the Framingham Heart Study (FHS) to examine genetic associations with ten primary quantitative traits: bone mineral density (BMD), calcaneal ultrasound, and geometric indices of the hip. To test associations with multivariable-adjusted residual trait values, we used additive generalized estimating equation (GEE) and family-based association tests (FBAT) models within each sex as well as sexes combined. We evaluated 70,987 autosomal SNPs with genotypic call rates > or =80%, HWE p > or = 0.001, and MAF > or =10% in up to 1141 phenotyped individuals (495 men and 646 women, mean age 62.5 yrs). Variance component linkage analysis was performed using 11,200 markers.

    Results: Heritability estimates for all bone phenotypes were 30-66%. LOD scores > or =3.0 were found on chromosomes 15 (1.5 LOD confidence interval: 51,336,679-58,934,236 bp) and 22 (35,890,398-48,603,847 bp) for femoral shaft section modulus. The ten primary phenotypes had 12 associations with 100K SNPs in GEE models at p < 0.000001 and 2 associations in FBAT models at p < 0.000001. The 25 most significant p-values for GEE and FBAT were all less than 3.5 x 10(-6) and 2.5 x 10(-5), respectively. Of the 40 top SNPs with the greatest numbers of significantly associated BMD traits (including femoral neck, trochanter, and lumbar spine), one half to two-thirds were in or near genes that have not previously been studied for osteoporosis. Notably, pleiotropic associations between BMD and bone geometric traits were uncommon. Evidence for association (FBAT or GEE p < 0.05) was observed for several SNPs in candidate genes for osteoporosis, such as rs1801133 in MTHFR; rs1884052 and rs3778099 in ESR1; rs4988300 in LRP5; rs2189480 in VDR; rs2075555 in COLIA1; rs10519297 and rs2008691 in CYP19, as well as SNPs in PPARG (rs10510418 and rs2938392) and ANKH (rs2454873 and rs379016). All GEE, FBAT and linkage results are provided as an open-access results resource at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007 webcite.

    Conclusion: The FHS 100K SNP project offers an unbiased genome-wide strategy to identify new candidate loci and to replicate previously suggested candidate genes for osteoporosis.

    Funded by: NCRR NIH HHS: 1S10RR163736-01A1; NHLBI NIH HHS: N01-HC-25195, N01HC25195; NIA NIH HHS: R01 AR/AG 41398; NIAMS NIH HHS: R01 AR041398, R01 AR041398-15, R01 AR050066

    BMC medical genetics 2007;8 Suppl 1;S14

  • Cadherins are regulated by Ep-CAM via phosphaditylinositol-3 kinase.

    Winter MJ, Cirulli V, Briaire-de Bruijn IH and Litvinov SV

    Department of Pathology, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. winterm1@wyeth.com

    The cross-signaling between (cell) adhesion molecules is nowadays a well-accepted phenomenon and includes orchestrated cellular changes and changes in the microenvironment. For example, Ep-CAM is an epithelial adhesion molecule that prevails in active proliferating tissue and is suppressed in a more differentiated state of the cell. E-cadherin adhesion complexes are typical for the advanced and terminal differentiated cell status. During normal proliferation, E-cadherin is not suppressed. We have demonstrated the effect of overexpression of Ep-CAM on E-cadherin, which probably affects the connection of cadherins and F-actin. Phosphatidylinositol 3-kinase (Pi3K) participates in various regulating mechanisms, for example in signaling to nuclei, vesicle transport, and cytoskeletal rearrangements. The effect of Ep-CAM on E-cadherin mediated junctions as well as the involvement of Pi3K in regulating adherens junctions, led us to investigate the potential interaction between Pi3K and Ep-CAM. Introduction of Ep-CAM in the epithelial cells caused abrogation of N-cadherin mediated cell-cell adhesion, which could be inhibited by Pi3K inhibitor LY294002. Moreover, the Pi3K subunit p85 was precipitated with Ep-CAM from cell lysates, and this complex showed kinase activity. The Pi3K activity shuttled from N-cadherin to Ep-CAM. From our results, we conclude that Ep-CAM cross signaling with N-cadherin involves Pi3K, resulting in the abrogation of the cadherin adhesion complexes in epithelial cells.

    Funded by: NIDDK NIH HHS: DK55183, DK63443

    Molecular and cellular biochemistry 2007;302;1-2;19-26

  • Up-regulation of gastric cancer cell invasion by Twist is accompanied by N-cadherin and fibronectin expression.

    Yang Z, Zhang X, Gang H, Li X, Li Z, Wang T, Han J, Luo T, Wen F and Wu X

    Department of General Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan, PR China.

    Twist, a newly found EMT-inducer, has been reported to be up-regulated in those of diffuse-type gastric carcinomas with high N-cadherin level. We show here MKN45, a cell line derived from undifferentiated carcinomas cells, expresses high levels of Twist. Down-regulation of Twist, using an antisense Twist vector in MKN45 cells, inhibits cell migration and invasion, companied with a morphologic changes associated with MET. Suppression of Twist also decreases the expressions of N-cadherin and fibronectin, but not of E-cadherin in MKN45. In contrast, overexpression of Twist in MKN28, a cell line derived from moderate differentiated carcinomas, results in up-regulation of N-cadherin and fibronectin, companied with down-regulation of E-cadherin. Taken together, our results suggest that Twist regulates cell motility and invasion in gastric cancer cell lines, probably through the N-cadherin and fibronectin production.

    Biochemical and biophysical research communications 2007;358;3;925-30

  • GSK3beta activity modifies the localization and function of presenilin 1.

    Uemura K, Kuzuya A, Shimozono Y, Aoyagi N, Ando K, Shimohama S and Kinoshita A

    Horizontal Medical Research Organization, Kyoto University Graduate School of Medicine, Kyoto, Japan.

    Presenilin 1, a causative gene product of familial Alzheimer disease, has been reported to be localized mainly in the endoplasmic reticulum and Golgi membranes. However, endogenous Presenilin 1 also localizes at the plasma membrane as a biologically active molecule. Presenilin 1 interacts with N-cadherin/beta-catenin to form a trimeric complex at the synaptic site through its loop domain, whose serine residues (serine 353 and 357) can be phosphorylated by glycogen synthase kinase 3beta. Here, we demonstrate that cell-surface expression of Presenilin 1/gamma-secretase is enhanced by N-cadherin-based cell-cell contact. Physical interaction between Presenilin 1 and N-cadherin/beta-catenin plays an important role in this process. Glycogen synthase kinase 3beta-mediated phosphorylation of Presenilin 1 reduces its binding to N-cadherin, thereby down-regulating its cell-surface expression. Moreover, reduction of the Presenilin 1.N-cadherin.beta-catenin complex formation leads to an impaired activation of contact-mediated phosphatidylinositol 3-kinase/Akt cell survival signaling. Furthermore, phosphorylation of Presenilin 1 hinders epsilon-cleavage of N-cadherin, whereas epsilon-cleavage of APP remained unchanged. This is the first report that clarifies the regulatory mechanism of Presenilin 1/gamma-secretase with respect to its subcellular distribution and its differential substrate cleavage. Because the cleavage of various membrane proteins by Presenilin 1/gamma-cleavage is involved in cellular signaling, glycogen synthase kinase 3beta-mediated phosphorylation of Presenilin 1 should be deeply associated with signaling functions. Our findings indicate that the abnormal activation of glycogen synthase kinase 3beta can reduce neuronal viability and synaptic plasticity via modulating Presenilin 1/N-cadherin/beta-catenin interaction and thus have important implications in the pathophysiology of Alzheimer disease.

    The Journal of biological chemistry 2007;282;21;15823-32

  • NHERF links the N-cadherin/catenin complex to the platelet-derived growth factor receptor to modulate the actin cytoskeleton and regulate cell motility.

    Theisen CS, Wahl JK, Johnson KR and Wheelock MJ

    Department of Biochemistry and Molecular Biology, Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-7696, USA.

    Using phage display, we identified Na+/H+ exchanger regulatory factor (NHERF)-2 as a novel binding partner for the cadherin-associated protein, beta-catenin. We showed that the second of two PSD-95/Dlg/ZO-1 (PDZ) domains of NHERF interacts with a PDZ-binding motif at the very carboxy terminus of beta-catenin. N-cadherin expression has been shown to induce motility in a number of cell types. The first PDZ domain of NHERF is known to bind platelet-derived growth factor-receptor beta (PDGF-Rbeta), and the interaction of PDGF-Rbeta with NHERF leads to enhanced cell spreading and motility. Here we show that beta-catenin and N-cadherin are in a complex with NHERF and PDGF-Rbeta at membrane ruffles in the highly invasive fibrosarcoma cell line HT1080. Using a stable short hairpin RNA system, we showed that HT1080 cells knocked down for either N-cadherin or NHERF had impaired ability to migrate into the wounded area in a scratch assay, similar to cells treated with a PDGF-R kinase inhibitor. Cells expressing a mutant NHERF that is unable to associate with beta-catenin had increased stress fibers, reduced lamellipodia, and impaired cell migration. Using HeLa cells, which express little to no PDGF-R, we introduced PDGF-Rbeta and showed that it coimmunoprecipitates with N-cadherin and that PDGF-dependent cell migration was reduced in these cells when we knocked-down expression of N-cadherin or NHERF. These studies implicate N-cadherin and beta-catenin in cell migration via PDGF-R-mediated signaling through the scaffolding molecule NHERF.

    Funded by: NCI NIH HHS: T32 CA009476, T32CA09476-14; NIDCR NIH HHS: DE-12308, R01 DE012308; NIGMS NIH HHS: GM-51188, R01 GM051188

    Molecular biology of the cell 2007;18;4;1220-32

  • IQ-domain GTPase-activating protein 1 regulates beta-catenin at membrane ruffles and its role in macropinocytosis of N-cadherin and adenomatous polyposis coli.

    Sharma M and Henderson BR

    Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia.

    Beta-catenin is an integral component of E-cadherin dependent cell-cell junctions. Here we show that beta-catenin co-localizes with IQ-domain GTPase-activating protein 1 (IQGAP1), adenomatous polyposis coli (APC), and N-cadherin at actin-positive membrane ruffles in NIH 3T3 fibroblasts. We used deletion mapping to identify the membrane ruffle-targeting region of beta-catenin, localizing it to amino acids 47-217, which overlap the IQGAP1 binding site. Knockdown by small interference RNA (siRNA) revealed IQGAP1-dependent membrane targeting of beta-catenin, APC, and N-cadherin. Transient overexpression of IQGAP1 or N-cadherin increased beta-catenin at membrane ruffles. IQGAP1/APC regulates cell migration, and using a wound healing assay we demonstrate that siRNA-mediated loss of beta-catenin also caused a modest reduction in the rate of cell migration. More significantly, we discovered that beta-catenin is internalized by Arf6-dependent macropinocytosis near sites of membrane ruffling. The beta-catenin macropinosomes co-stained for APC, N-cadherin, and to a lesser extent IQGAP1, and internalization of each binding partner was abrogated by siRNA-dependent knockdown of beta-catenin. In addition, beta-catenin macropinosomes co-localized with the lysosomal marker, lysosome associated membrane protein 1, consistent with their recycling by the late endosomal machinery. Our findings expand on current knowledge of beta-catenin function. We propose that in motile cells beta-catenin is recruited by IQGAP1 and N-cadherin to active membrane ruffles, wherein beta-catenin mediates the internalization and possible recycling of the membrane-associated proteins N-cadherin and APC.

    The Journal of biological chemistry 2007;282;11;8545-56

  • N-Cadherin is expressed by putative stem/progenitor cells and melanocytes in the human limbal epithelial stem cell niche.

    Hayashi R, Yamato M, Sugiyama H, Sumide T, Yang J, Okano T, Tano Y and Nishida K

    Department of Ophthalmology, Tohoku University School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai 980-8574, Japan.

    Corneal epithelial stem cells are known to be localized to the basal layer of the limbal epithelium, providing a model system for epithelial stem cell biology; however, the mechanisms regarding the maintenance of these stem cells in their specialized niche remain poorly understood. N-cadherin is a member of the classic cadherin family and has previously been demonstrated to be expressed by hematopoietic stem cells. In the present study, we demonstrate that N-cadherin is expressed by putative stem/progenitor cells, as well as melanocytes, in the human limbal epithelial stem cell niche. In addition, we demonstrate that upon in vitro culture using 3T3 feeder layers, loss of N-cadherin expression occurs with cell proliferation. These results indicate that N-cadherin may be a critical cell-to-cell adhesion molecule between corneal epithelial stem/progenitor cells and their corresponding niche cells in the limbal epithelium.

    Stem cells (Dayton, Ohio) 2007;25;2;289-96

  • Collagen I promotes metastasis in pancreatic cancer by activating c-Jun NH(2)-terminal kinase 1 and up-regulating N-cadherin expression.

    Shintani Y, Hollingsworth MA, Wheelock MJ and Johnson KR

    Department of Oral Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.

    We have previously shown that N-cadherin expression is associated with tumor invasion, and that some cancer cells respond to specific extracellular matrix molecules by up-regulating N-cadherin. Pancreatic cancer is characterized by excessive deposition of type I collagen. Here, we show that human pancreatic cancer cells respond to collagen I, but not other matrices, by increasing motility and up-regulating mesenchymal markers, including N-cadherin. Both collagen I-mediated motility and metastasis in a mouse model for pancreatic cancer were inhibited by N-cadherin knockdown. Furthermore, inhibiting c-Jun NH(2)-terminal kinase (JNK) with chemical inhibitors or short hairpin RNA abrogated all collagen I-induced changes. We show that JNK1 is activated in response to collagen I, which increases tumorigenesis by up-regulating N-cadherin expression and by increasing motility.

    Funded by: NCI NIH HHS: P30-CA36727; NIDCR NIH HHS: R01-DE12308; NIGMS NIH HHS: R01-GM51188

    Cancer research 2006;66;24;11745-53

  • A distinct PAR complex associates physically with VE-cadherin in vertebrate endothelial cells.

    Iden S, Rehder D, August B, Suzuki A, Wolburg-Buchholz K, Wolburg H, Ohno S, Behrens J, Vestweber D and Ebnet K

    Institute of Medical Biochemistry, ZMBE, University of Münster, Von-Esmarch-Str. 56, D-48149 Münster, Germany.

    A cell polarity complex consisting of partitioning defective 3 (PAR-3), atypical protein kinase C (aPKC) and PAR-6 has a central role in the development of cell polarity in epithelial cells. In vertebrate epithelial cells, this complex localizes to tight junctions. Here, we provide evidence for the existence of a distinct PAR protein complex in endothelial cells. Both PAR-3 and PAR-6 associate directly with the adherens junction protein vascular endothelial cadherin (VE-cadherin). This association is direct and mediated through non-overlapping domains in VE-cadherin. PAR-3 and PAR-6 are recruited independently to cell-cell contacts. Surprisingly, the VE-cadherin-associated PAR protein complex lacks aPKC. Ectopic expression of VE-cadherin in epithelial cells affects tight junction formation. Our findings suggest that in endothelial cells, another PAR protein complex exists that localizes to adherens junctions and does not promote cellular polarization through aPKC activity. They also point to a direct role of a cadherin in the regulation of cell polarity in vertebrates.

    EMBO reports 2006;7;12;1239-46

  • A specific cadherin phenotype may characterise the disseminating yet non-metastatic behaviour of pseudomyxoma peritonei.

    Bibi R, Pranesh N, Saunders MP, Wilson MS, O'dwyer ST, Stern PL and Renehan AG

    Cancer Research UK Immunology Group, Paterson Institute for Cancer Research, Manchester, UK.

    Pseudomyxoma peritonei (PMP) is a rare neoplasm of mainly appendiceal origin, characterised by excess intra-abdominal mucin production leading to high morbidity and mortality. While histological features are frequently indolent, this tumour disseminates aggressively throughout the abdominal cavity, yet seldom metastasises. This study determined the expression of several markers of colorectal differentiation (carcinoembryonic antigen (CEA), cytokeratins (CK20 and CK7), epithelial membrane antigen), mucin production (MUC-2, interleukin-9 (IL-9), IL-9 receptor (IL-9Ralpha)), and cell adhesion (N- and E-cadherin, vimentin) in PMP tissue (n=26) compared with expressions in normal colonic mucosa (n=19) and colorectal adenocarcinoma (n=26). Expressions of CEA and cytokeratins were similar for PMP as those in colorectal adenocarcinomas with the exception that the CK20-/CK7- pattern was rare in PMP (Fisher's exact test: P=0.001). Similarly, expressions of mucin-related proteins were comparable for adenocarcinoma and PMP, with the exception that IL-9 expression was uncommon in adenocarcinoma (P=0.009). Pseudomyxoma peritonei demonstrated a specific pattern of adhesion-related protein expressions of increased N-cadherin, reduced E-cadherin, and increased vimentin (P=0.004), a phenotype suggesting a possible epithelial-mesenchymal transition state. Primary PMP cell cultures were successfully maintained and demonstrated marker expressions similar to those seen in in vivo tissues. These early characterisation studies demonstrate similarities between PMP and colorectal adenocarcinoma, but also reveal a specific cadherin phenotype that may characterise the distinct non-metastasising behaviour of PMP, and form the basis for future mechanistic and therapy-targeting research.

    British journal of cancer 2006;95;9;1258-64

  • VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin.

    Gavard J and Gutkind JS

    Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, DHHS, Bethesda, MD 20892-4340, USA.

    How vascular endothelial growth factor (VEGF) induces vascular permeability, its first described function, remains poorly understood. Here, we provide evidence of a novel signalling pathway by which VEGF stimulation promotes the rapid endocytosis of a key endothelial cell adhesion molecule, VE-cadherin, thereby disrupting the endothelial barrier function. This process is initiated by the activation of the small GTPase Rac by VEGFR-2 through the Src-dependent phosphorylation of Vav2, a guanine nucleotide-exchange factor. Rac activation, in turn, promotes the p21-activated kinase (PAK)-mediated phosphorylation of a highly conserved motif within the intracellular tail of VE-cadherin. Surprisingly, this results in the recruitment of beta-arrestin2 to serine-phosphorylated VE-cadherin, thereby promoting its internalization into clathrin-coated vesicles and the consequent disassembly of intercellular junctions. Ultimately, this novel biochemical route by which VEGF promotes endothelial permeability through the beta-arrestin2-dependent endocytosis of VE-cadherin may help identify new therapeutic targets for the treatment of many human diseases that are characterized by vascular leakage.

    Funded by: Intramural NIH HHS

    Nature cell biology 2006;8;11;1223-34

  • The role of N-cadherin, MCAM and beta3 integrin in melanoma progression, proliferation, migration and invasion.

    Watson-Hurst K and Becker D

    Department of Cellular and Molecular Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213-1863, USA.

    Melanoma, the most aggressive form of skin cancer, which accounts for 75% of all skin cancer-related deaths, continues to rise at an alarming rate worldwide. Despite a favorable cure rate when surgically removed at an early stage, the response rate of patients with metastatic disease to single agent chemotherapy is less than 15%, and biologic therapies are only marginally effective. Given this bleak picture, there is a great need to identify and characterize genes that play an important role in the advanced stages of melanoma and thus, may represent valuable targets for clinical therapy. The cell adhesion molecules N-cadherin, MCAM and beta3 integrin have been suggested to represent melanoma progression markers; yet, little is known as to whether they may constitute therapeutic targets for the disease. To provide information regarding this aspect, we determined by way of whole-genome and tissue microarray analysis, their level of expression concordant with melanoma progression, and via RNA interference and antisense technology, their role in melanoma cell proliferation, migration, and invasion. The results of these studies demonstrate that N-cadherin and beta3 integrin expression correlates with progression to advanced-stage melanoma, whereas expression of MCAM does not. On the other hand, MCAM and beta3 integrin are the two adhesion molecules that play a pivotal role in melanoma cell migration and invasion, and for this reason, may represent valuable targets for melanoma therapy.

    Cancer biology & therapy 2006;5;10;1375-82

  • Activity-dependent regulation of beta-catenin via epsilon-cleavage of N-cadherin.

    Uemura K, Kihara T, Kuzuya A, Okawa K, Nishimoto T, Bito H, Ninomiya H, Sugimoto H, Kinoshita A and Shimohama S

    Horizontal Medical Research Organization, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan.

    N-cadherin is essential for excitatory synaptic contact in the hippocampus. Presenilin 1 (PS1) is located at sites of synaptic contact, forming a complex with N-cadherin and beta-catenin. Here, we report that human N-cadherin is cleaved by PS1/gamma-secretase in response to physiological concentration of glutamate (Glu) stimulation, yielding a fragment Ncad/CTF2. The expression of Ncad/CTF2 in neuronal cells led to its translocation to the nucleus, and caused a prominent enhancement of cytoplasmic and nuclear beta-catenin levels in a cell-cell contact dependent manner, via following mechanisms: 1, inhibition of beta-catenin phosphorylation; 2, transactivation of beta-catenin; and 3, inhibition of N-cadherin transcription, and finally enhanced beta-catenin nuclear signaling. Since the regulation of cellular beta-catenin level is essential for synaptic function, disruption in the cleavage of N-cadherin may be causally linked to the synaptic dysfunction associated with Alzheimer's disease (AD).

    Biochemical and biophysical research communications 2006;345;3;951-8

  • Differential expression of N-cadherin and E-cadherin in normal human tissues.

    Tsuchiya B, Sato Y, Kameya T, Okayasu I and Mukai K

    Department of Molecular Diagnostics, Kitasato University School of Allied Health Sciences, Sagamihara, Kanagawa, Japan. benio@med.kitasato-u.ac.jp

    E-cadherin, which expressed in various epithelial tissues, is important for the maintenance of normal epithelial phenotypes. However, the distribution of N-cadherin in normal human tissues has not been defined systemically. In the present study, we employed a sensitive, reliable immunohistochemical detection system for N-cadherin on formalin-fixed, paraffin-embedded tissue sections, and succeeded in demonstrating N- and E-cadherin protein expressions and their distribution in normal human tissues. E-cadherin immunoreactivity was detected in all the epithelial tissues examined, except for the adrenal cortical cells and granulosa cells. N-cadherin was selectively expressed on epithelial cells of the thymus, pituitary, pancreas, liver, adrenal, endometrium of the uterus, ovary, and stomach as well as in neuronal tissues. Double immunostaining revealed that N-cadherin expression was closely associated with the hormone-producing ability of cells in the pancreas and pituitary. Thus, this study indicated the possibility that N-cadherin is selectively expressed in relation to hormonal regulation in some organs and plays different functions in different situations. The method presented here should prove useful for the further investigation of the N-cadherin expression and function in several disease conditions on formalin-fixed, paraffin-embedded archival tissues.

    Archives of histology and cytology 2006;69;2;135-45

  • N-cadherin as a novel prognostic marker of progression in superficial urothelial tumors.

    Lascombe I, Clairotte A, Fauconnet S, Bernardini S, Wallerand H, Kantelip B and Bittard H

    Tissue and Cell Biology Engineering, Besançon, France. isabelle.lascombe@viola.fr

    Purpose: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion and spreading. In bladder cancer, loss or reduced E-cadherin expression has been associated with poor survival, and aberrant expression of N-cadherin has been associated with the invasive phenotype of bladder carcinoma cells. The purpose of this study was to investigate whether N-cadherin expression was associated with the bladder tumor progression.

    E-cadherin and N-cadherin expression was evaluated by immunohistochemistry in 101 tumors (pT1 and pT2-T3) and by reverse transcription-PCR analysis and immunohistochemistry in 28 other fresh frozen tumors (pT(a), pT1, and pT2-T3).

    Results: N-cadherin expression was absent in normal urothelium, appeared in stage pT1, and increased in pT2-pT3 tumors. In most cases, increased N-cadherin expression in invasive tumors was associated with loss of E-cadherin expression. Progression-free survival and multivariate analyses revealed that N-cadherin expression is an independent prognostic marker for pT1 tumor progression. Analysis of the 28 frozen tumors by immunohistochemistry and reverse transcription-PCR showed a good correlation between protein and gene expression in pT1 and pT2-T3 tumors. Interestingly, in pT(a) tumors, N-cadherin was not immunodetected, whereas mRNA was present in 50% of cases.

    Conclusion: Regulatory defects in the N-cadherin promoter, abnormalities at the translational, or protein processing levels could explain the discrepancies between protein and mRNA expression. Most importantly, this study identified N-cadherin as a novel prognostic marker of progression in superficial urothelial tumors. Clearly, N-cadherin acts in an invasive mode in bladder cancer, but whether it has a primary role in urothelial neoplastic progression has yet to be investigated.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2006;12;9;2780-7

  • Anti-apoptotic N-cadherin signaling and its prognostic implication in human hepatocellular carcinomas.

    Gwak GY, Yoon JH, Yu SJ, Park SC, Jang JJ, Lee KB, Lee SH, Lee SM, Shin CS, Suh KS and Lee HS

    Department of Internal Medicine, Seoul National University College of Medicine, Chongno-gu, Seoul, Republic of Korea.

    N-cadherin signaling has recently been implicated in the progression of certain epithelial tumors by promoting invasion and dissemination of cancer cells. N-cadherin has also been reported to exert an anti-apoptotic effect. In this study, we attempted to evaluate the participation of this adhesion molecule in the progression of human hepatocellular carcinomas (HCCs) by analyzing its anti-apoptotic signaling as well as its prognostic implication in HCC patients. N-cadherin was found to be expressed in human HCCs. We established a stable human HCC cell line expressing a truncated N-cadherin, NCaddeltaC, with a dominant-negative action. NCaddeltaC-expressing cells were more susceptible to bile acid-induced apoptosis than control cells. N-cadherin was found to complex with procaspase-8, and this association was diminished in NCaddeltaC-expressing cells, leading to enhanced procaspase-8 recruitment to death-inducing signaling complex following bile acid treatment. A clinicopathological analysis in patients who had undergone surgical resection for HCC revealed that tumoral N-cadherin up-regulation was significantly related to poor recurrence-free and overall survival. Our findings implicate N-cadherin signaling as contributing to HCC progression by exerting anti-apoptotic effects. Thus, we suggest that the selective interruption of this signaling may have therapeutic potential.

    Oncology reports 2006;15;5;1117-23

  • Involvement of Src family kinases in N-cadherin phosphorylation and beta-catenin dissociation during transendothelial migration of melanoma cells.

    Qi J, Wang J, Romanyuk O and Siu CH

    Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.

    N-cadherin is recruited to the heterotypic contact during transendothelial migration of melanoma cells in a coculture system with tumor cells seeded on top of a monolayer of endothelial cells. However, beta-catenin dissociates from N-cadherin and redistributes to the nucleus of transmigrating melanoma cells to activate gene transcription. In this report, we demonstrate that Src becomes activated at the heterotypic contact between the transmigrating melanoma cell and neighboring endothelial cells. Src activation shows close temporal correlation with tyrosine phosphorylation of N-cadherin. Expression of a dominant-negative Src in melanoma cells blocks N-cadherin phosphorylation, beta-catenin dissociation, and nuclear translocation in transmigrating cells, consistent with the involvement of Src family kinases. In in vitro binding assays, Src-mediated phosphorylation of the N-cadherin cytoplasmic domain results in a significant reduction in beta-catenin binding. Although five phospho-tyrosine residues can be identified on the N-cadherin cytoplasmic domain by mass spectrometry, site-specific mutagenesis indicates that Tyr-860 is the critical amino acid involved in beta-catenin binding. Overexpression of N-cadherin carrying the Y860F mutation inhibits the transmigration of transfected cells across the endothelium. Together, the data suggest a novel role for tyrosine phosphorylation of N-cadherin by Src family kinases in the regulation of beta-catenin association during transendothelial migration of melanoma cells.

    Molecular biology of the cell 2006;17;3;1261-72

  • Differential use of functional domains by coiled-coil coactivator in its synergistic coactivator function with beta-catenin or GRIP1.

    Yang CK, Kim JH, Li H and Stallcup MR

    Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California 90089, USA.

    beta-Catenin, a pivotal component of the Wnt-signaling pathway, binds to and serves as a transcriptional coactivator for the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcriptional activator proteins and for the androgen receptor (AR), a nuclear receptor. Three components of the p160 nuclear receptor coactivator complex, including CARM1, p300/CBP, and GRIP1 (one of the p160 coactivators), bind to and cooperate with beta-catenin to enhance transcriptional activation by TCF/LEF and AR. Here we report that another component of the p160 nuclear receptor coactivator complex, the coiled-coil coactivator (CoCoA), directly binds to and cooperates synergistically with beta-catenin as a coactivator for AR and TCF/LEF. CoCoA uses different domains to bind GRIP1 and beta-catenin, and it uses different domains to transmit the activating signal to the transcription machinery, depending on whether it is bound to GRIP1 or beta-catenin. CoCoA associated specifically with the promoters of transiently transfected and endogenous target genes of TCF/LEF, and reduction of the endogenous CoCoA level decreased the ability of TCF/LEF and beta-catenin to activate transcription of transient and endogenous target genes. Thus, CoCoA uses different combinations of functional domains to serve as a physiologically relevant component of the Wnt/beta-catenin signaling pathway and the androgen signaling pathway.

    Funded by: NIDDK NIH HHS: DK43093, R01 DK043093

    The Journal of biological chemistry 2006;281;6;3389-97

  • N-cadherin switching occurs in high Gleason grade prostate cancer.

    Jaggi M, Nazemi T, Abrahams NA, Baker JJ, Galich A, Smith LM and Balaji KC

    Urological Surgery, University of Nebraska Medical Center, Omaha, Nebraska 68198-2360, USA. mjaggi@unmc.edu

    Background: The inappropriate expression of non-epithelial N-(neural) cadherin by epithelial cells, called cadherin switching, has been suggested to play a role in prostate cancer (PC) progression. We explored the role of N-cadherin as a biomarker in PC by correlating the expression with clinical parameters.

    Methods: Two pathologists blinded to patients' history independently reviewed and scored the intensity and extent of staining of N-cadherin expression in 44 randomly selected radical prostatectomy specimens. The expression was correlated with total Gleason grade, individual Gleason patterns, tumor stage, and preoperative serum prostate specific antigen (PSA) levels and P-values < 0.05 were considered statistically significant.

    Results: Of the 44 PC specimens, 14 (32%), 23 (52%), 7 (16%) consisted of Gleason grade 5-6, 7, and 8-10, respectively and 20/44 (45%) demonstrated N-cadherin expression. N-cadherin was expressed in 1/14 (7%) of Gleason 5-6 compared to 15/23 (65%) of Gleason grade 7, and 4/7 (57%) of Gleason grade 8-10, demonstrating a significant correlation between N-cadherin switching and higher Gleason grade (P = 0.001). While only about a third of primary or secondary Gleason pattern 3 demonstrated N-cadherin expression, a majority of Gleason patterns of > or = 4 expressed N-cadherin (P > 0.05), further suggesting that N-cadherin switching occurs with higher Gleason pattern. However, N-cadherin expression did not significantly correlate with preoperative serum PSA levels or tumor stage in our study cohort.

    Conclusions: We have demonstrated for the first time that N-cadherin switching occurs in higher grade PC and correlates significantly with increasing Gleason patterns. N-cadherin may be as a useful biomarker of aggressive PC.

    The Prostate 2006;66;2;193-9

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Genistein-induced neuronal differentiation is associated with activation of extracellular signal-regulated kinases and upregulation of p21 and N-cadherin.

    Hung SP, Hsu JR, Lo CP, Huang HJ, Wang JP and Chen ST

    Department of Life Sciences, National Cheng Kung University, Tainan City 701, Taiwan.

    Neuronal differentiation in the mammalian CNS is driven by multiple events. When treated with retinoic acid (RA), hNTera-2 (NT-2) cells undergo postmitotic neuronal differentiation. Here, we show that a prolonged exposure of NT-2 cells with non-cytotoxic doses of genistein, a protein tyrosine kinase (PTK) inhibitor, induced differentiation of NT-2 cells. Additionally, genistein enhanced RA-induced neuronal differentiation by increasing the activation of extracellular signal-related kinase 1/2 (ERK1/2) via phosphorylation at Thr183 and Tyr185 in 3-7 days. Meanwhile, genistein also upregulated N-cadherin and p21 (a Cdk inhibitor), but downregulated proliferating cell nuclear antigen protein (PCNA). MEK1/2 inhibitors, such as PD98059 and U0126, reduced RA-induced ERK1/2 activity, but could not block the genistein effects. Our observations indicate that genistein-induced neuronal differentiation is not dependent of the MEK-ERK signaling cascade. Instead, genistein-upregulated ERK activation is likely due to this chemical's direct effect on chromosome and gene transcription, rather than its inhibition on tyrosine kinases. Failure of inhibition of ERK1/2 activation by the MEK1/2 inhibitors PD98059 and U0126 suggests presence of an unknown activator for ERK1/2 in neuronal cells.

    Journal of cellular biochemistry 2005;96;5;1061-70

  • N-cadherin expression in breast cancer: correlation with an aggressive histologic variant--invasive micropapillary carcinoma.

    Nagi C, Guttman M, Jaffer S, Qiao R, Keren R, Triana A, Li M, Godbold J, Bleiweiss IJ and Hazan RB

    Department of Pathology, Mount Sinai School of Medicine, New York, NY, USA.

    Upregulation of N-cadherin in epithelial tumor cells has been shown to contribute to the invasive/metastatic phenotype. It remains however to be determined whether N-cadherin is increased in human breast cancers with enhanced malignant potential. We examined a large number of invasive breast cancer specimens (n = 114) for N- and E-cadherin. These specimens compared invasive duct carcinomas (IDCs) of varying histologic grades with an aggressive subtype, invasive micropapillary carcinoma of the breast (MPAP), which has a high propensity for lymphatic invasion and lymph node metastasis. Staining scores for N- and E-cadherin were compared between non-MPAP and MPAP IDCs, and between the invasive and ductal carcinoma in situ (DCIS) of each IDC using statistical analysis. We found that N-cadherin was expressed in 76% of MPAP and 52% of non-MPAP carcinomas, and E-cadherin in 57% of MPAP and 36% of non-MPAP tumors. More MPAP (25%) compared to non-MPAP (5%) tumors expressed both cadherins. Of the two cadherins, N-cadherin was significantly associated with MPAP tumors (p = 0.033) compared to E-cad (p = 0.171). Moreover, in the majority of tumors that were positive for N-cadherin, the staining scores were increased in the IDC relative to intraductal components, and this effect was more dramatic in the MPAP carcinomas. This difference for N-cadherin was greater than the corresponding difference for E-cadherin in the MPAP group (p = 0.005), whereas such changes were not significant in the non-MPAP group (p = 0.10). Thus, N-cadherin is associated with tumor aggressiveness and metastatic potential and may contribute to tumor progression.

    Funded by: NCI NIH HHS: 5 R24 CA 88282-04, R01 CA90872

    Breast cancer research and treatment 2005;94;3;225-35

  • Modulating the strength of cadherin adhesion: evidence for a novel adhesion complex.

    Kim YJ, Sauer C, Testa K, Wahl JK, Svoboda RA, Johnson KR, Wheelock MJ and Knudsen KA

    University of Nebraska Medical Center, College of Dentistry, 769605 Nebraska Medical Center, Omaha, NE 68583, USA.

    Adherens junctions and desmosomes are critical for embryogenesis and the integrity of adult tissues. To form these junctions, classical cadherins interact via alpha- and beta-catenin with the actin cytoskeleton, whereas desmosomal cadherins interact with the intermediate filament system. Here, we used a hormone-activated mutant N-cadherin expressed in fibroblasts to show the existence of a novel classical cadherin adhesion system. N-cadherin was fused at its C-terminus to a modified estrogen receptor ligand-binding domain (NcadER) that binds 4-hydroxytamoxifen (4OHT) and expressed in L cells, which lack an endogenous cadherin. Cells with the mutant cadherin (LNER cells) aggregated in the absence of 4OHT, but only in its presence formed tightly compacted aggregates like those formed by L cells expressing wild-type N-cadherin (LN cells). Compaction of LNER cells treated with 4OHT was accompanied by elevated levels of p120ctn in NcadER immunoprecipitates, compared to immunoprecipitates of non-treated cells, but without changes in alpha- and beta-catenin, or actin. Compaction induced by 4OHT was also accompanied by increased interaction of the NcadER with the cytoskeleton and increased vimentin organization. Vimentin co-immunoprecipitated with the NcadER/catenin complex, suggesting an interaction between cadherin and vimentin. The mechanism by which vimentin interacts with the cadherin appears to involve p120ctn as it co-immunoprecipitates and colocalizes with vimentin in the parent L cells, which lack a cadherin and alpha- and beta-catenins. Disrupting the actin cytoskeleton with cytochalasin B inhibited aggregation, whereas knocking down vimentin with specific siRNAs inhibited compaction. Based on our results we propose that a vimentin-based classical cadherin complex functions together with the actin-based complex to promote strong cell-cell adhesion in fibroblasts.

    Funded by: NCRR NIH HHS: P20 RR018759-03, RR18759; NIDCR NIH HHS: DE12308, R01 DE012308-09; NIGMS NIH HHS: GM61702, R01 GM051188-13, R01 GM061702-04

    Journal of cell science 2005;118;Pt 17;3883-94

  • Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC).

    Amanchy R, Kalume DE, Iwahori A, Zhong J and Pandey A

    McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry and Oncology, Johns Hopkins University, 733 N. Broadway, Baltimore, MD 21205, USA.

    Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes.

    Funded by: NCI NIH HHS: CA 88843; NHLBI NIH HHS: HV 28180

    Journal of proteome research 2005;4;5;1661-71

  • Transendothelial migration of melanoma cells involves N-cadherin-mediated adhesion and activation of the beta-catenin signaling pathway.

    Qi J, Chen N, Wang J and Siu CH

    Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.

    Cancer metastasis is a multistep process involving many types of cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of cancer cells. Transendothelial migration of cancer cells was investigated using an in vitro assay, in which melanoma cells were seeded on top of a monolayer of endothelial cells. Attachment of melanoma cells on the endothelium induced a twofold increase in N-cadherin expression in melanoma cells and the redistribution of N-cadherin to the heterotypic contacts. Transendothelial migration was inhibited when N-cadherin expression was repressed by antisense RNA, indicating a key role played by N-cadherin. Whereas N-cadherin and beta-catenin colocalized in the contact regions between melanoma cells and endothelial cells during the initial stages of attachment, beta-catenin disappeared from the heterotypic contacts during transmigration of melanoma cells. Immunolocalization and immunoprecipitation studies indicate that N-cadherin became tyrosine-phosphorylated, resulting in the dissociation of beta-catenin from these contact regions. Concomitantly, an increase in the nuclear level of beta-catenin occurred in melanoma cells, together with a sixfold increase in beta-catenin-dependent transcription. Transendothelial migration was compromised in cells expressing a dominant-negative form of beta-catenin, thus supporting a regulatory role of beta-catenin signaling in this process.

    Molecular biology of the cell 2005;16;9;4386-97

  • Adhesion signaling by a novel mitotic substrate of src kinases.

    Bhatt AS, Erdjument-Bromage H, Tempst P, Craik CS and Moasser MM

    School of Medicine, University of California, San Francisco, CA 94143, USA.

    Src kinases are activated and relocalize to the cytoplasm during mitosis, but their mitotic function has remained elusive. We describe here a novel mitotic substrate of src kinases. Trask (transmembrane and associated with src kinases) is a 140 kDa type I transmembrane glycoprotein unrelated to currently known protein families. Src kinases phosphorylate Trask in vitro and mediate its mitotic hyperphosphorylation in vivo. Trask associates with both yes and src, is localized to the cell membrane during interphase, and undergoes cytoplasmic relocalization during mitosis. Overexpression of Trask leads to cell rounding and a loss of adhesion phenotype. Consistent with a function in cell adhesion, Trask interacts with a number of adhesion and matrix proteins including cadherins, syndecans, and the membrane-type serine protease 1 (MT-SP1), and is proteolytically cleaved by MT-SP1. Trask is unique among cell adhesion molecules in that it is under cell cycle regulation and thus links src kinases with the mitotic regulation of cell adhesion. This suggests a potential pathway by which hyperactive src kinases in tumors can deregulate adhesion signaling and mediate the metastatic phenotype.

    Funded by: NCI NIH HHS: CA 72006, R01 CA113952-01, R01 CA113952-02

    Oncogene 2005;24;34;5333-43

  • N-cadherin-dependent cell-cell contacts promote human saphenous vein smooth muscle cell survival.

    Koutsouki E, Beeching CA, Slater SC, Blaschuk OW, Sala-Newby GB and George SJ

    Bristol Heart Institute, Bristol Royal Infirmary, Bristol, UK.

    Objective: Vascular smooth muscle cell (VSMC) apoptosis is thought to contribute to atherosclerotic plaque instability. Cadherin mediates calcium-dependent homophilic cell-cell contact. We studied the role of N-cadherin in VSMC apoptosis.

    Human saphenous vein VSMCs were grown in agarose-coated wells to allow cadherin-mediated aggregate formation. Cell death and apoptosis were determined after disruption of cadherins using several approaches (n> or =3 per approach). Calcium removal from culture medium or addition of nonspecific cadherin antagonist peptides significantly decreased aggregate formation and increased cell death by apoptosis (34+/-6% versus 75+/-1% and 19+/-1% versus 40+/-5%, respectively; P<0.05). Specific inhibition of N-cadherin using antagonists and neutralizing antibodies similarly increased apoptosis. Supporting this, overexpression of full-length N-cadherin significantly reduced VSMC apoptosis from 44+/-10% to 20+/-3% (P<0.05), whereas abolishing N-cadherin expression by overexpression of a dominant-negative N-cadherin significantly, even in the presence of cell-matrix contacts, increased apoptosis from 9+/-2% to 50+/-1% (P<0.05). Interestingly, cell-cell contacts provided a similar degree of protection from apoptosis to cell-matrix contacts. Finally, N-cadherin-mediated cell-cell contacts initiated anti-apoptotic signaling by increasing Akt and Bad phosphorylation.

    Conclusions: Our results indicate that VSMC survival is dependent on N-cadherin-mediated cell-cell contacts, which could be important in the context of plaque instability.

    Arteriosclerosis, thrombosis, and vascular biology 2005;25;5;982-8

  • Up-regulated expression of zonula occludens protein-1 in human melanoma associates with N-cadherin and contributes to invasion and adhesion.

    Smalley KS, Brafford P, Haass NK, Brandner JM, Brown E and Herlyn M

    Wistar Institute, 3601 Spruce St., Philadelphia, Pennsylvania 19104, USA.

    During the process of malignant transformation, nascent melanoma cells escape keratinocyte control through down-regulation of E-cadherin and instead communicate among themselves and with fibroblasts via N-cadherin-based cell-cell contacts. The zonula occludens (ZO) protein-1 is a membrane-associated component of both the tight and adherens junctions found at sites of cell-cell contact. In most cancers, levels of ZO-1 are typically down-regulated, leading to increased motility. Here we report the novel observation that ZO-1 expression is up-regulated in melanoma cells and is located at adherens junctions between melanoma cells and fibroblasts. Immunofluorescence and co-immunoprecipitation studies showed co-localization of ZO-1 with N-cadherin. Down-regulation of ZO-1 in melanoma cells through RNA interference produced marked changes in cell morphology--leading to a less-dendritic, more rounded phenotype. Consistent with a role in N-cadherin-based adhesion, RNAi-treated melanoma cells were less adherent and invasive when grown in a collagen gel. These data provide the first evidence that increased ZO-1 expression in melanoma contributes to the oncogenic behavior of this tumor and further illustrate that protein products of genes, such as ZO-1, can function in either a pro- or anti-oncogenic manner when expressed in different cellular contexts.

    Funded by: NCI NIH HHS: CA 10815, CA 25874, CA 76674, CA 93372, P01 CA025874, P30 CA010815, P50 CA093372, R01 CA076674

    The American journal of pathology 2005;166;5;1541-54

  • Gonadotropins regulate N-cadherin-mediated human ovarian surface epithelial cell survival at both post-translational and transcriptional levels through a cyclic AMP/protein kinase A pathway.

    Pon YL, Auersperg N and Wong AS

    Department of Zoology, University of Hong Kong, Hong Kong.

    Gonadotropins are the major regulators of ovarian function and may be involved in the etiology of ovarian cancer. In this study, we report a new mechanism whereby gonadotropins regulate the survival of human ovarian surface epithelium (OSE), the tissue of origin of epithelial ovarian carcinomas. Our results indicate that disruption of N-cadherin-mediated cell-cell adhesion is an important molecular event in the apoptosis of human OSE. Treatment with surge serum concentrations of gonadotropins reduced the amount of N-cadherin with a concomitant induction of apoptosis, and this effect was mediated by a cAMP/protein kinase A pathway but not the ERK1/2 and protein kinase C cascades. We further demonstrated that activation of the gonadotropins/cAMP signaling pathway in human OSE led to a rapid down-regulation of N-cadherin protein level followed by a reduction at the level of N-cadherin mRNA, indicating that expression of N-cadherin was regulated by post-translational and transcriptional mechanisms. The former mechanism was mediated by increased turnover of N-cadherin protein and could be reversed by inhibition of proteasomal or matrix metalloproteinase (MMP-2) activity. On the other hand, at the transcriptional level, the addition of actinomycin D abolished the cAMP-mediated decrease in N-cadherin mRNA but did not change its stability. Inhibition of protein kinase A or expressing a dominant negative mutant of cAMP-response element-binding protein blocked this decrease of N-cadherin mRNA. Together, the combined operation of post-translational and transcriptional mechanisms suggests that regulation of N-cadherin is a crucial event and emphasizes the important role that N-cadherin has in controlling the survival capability of human OSE.

    The Journal of biological chemistry 2005;280;15;15438-48

  • Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition.

    Maeda M, Johnson KR and Wheelock MJ

    Department of Oral Biology (College of Dentistry), University of Nebraska Medical Center, Omaha, NE 68198-7696, USA.

    Epithelium-to-mesenchyme transitions (EMTs) are characterized by morphological and behavioral changes in cells. During an EMT, E-cadherin is downregulated while N-cadherin is upregulated. The goal of this study was to understand the role cadherin switching plays in EMT using a classical model system: transforming growth factor beta1 (TGF-beta1)-mediated EMT in mammary epithelial cells. We showed that stress fibers and focal adhesions are increased, and cell-cell junctions are decreased in response to TGF-beta1. Moreover, these changes were reversible upon removal of TGF-beta1. Downregulation of E-cadherin and upregulation of N-cadherin were both transcriptional. Neither experimental knockdown nor experimental overexpression of N-cadherin interfered with the morphological changes. In addition, the morphological changes associated with EMT preceded the downregulation of E-cadherin. Interestingly, TGF-beta1-induced motility in N-cadherin-knockdown cells was significantly reduced. Together, these data suggest that cadherin switching is necessary for increased motility but is not required for the morphological changes that accompany EMT.

    Funded by: NCRR NIH HHS: P20-RR018759; NIDCR NIH HHS: R01-DE12308; NIGMS NIH HHS: R01-GM51188

    Journal of cell science 2005;118;Pt 5;873-87

  • Gingipains from Porphyromonas gingivalis W83 induce cell adhesion molecule cleavage and apoptosis in endothelial cells.

    Sheets SM, Potempa J, Travis J, Casiano CA and Fletcher HM

    Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA. ssheets04b@som.llu.edu

    The presence of Porphyromonas gingivalis in the periodontal pocket and the high levels of gingipain activity detected in gingival crevicular fluid could implicate a role for gingipains in the destruction of the highly vascular periodontal tissue. To explore the effects of these proteases on endothelial cells, we exposed bovine coronary artery endothelial cells and human microvascular endothelial cells to gingipain-active extracellular protein preparations and/or purified gingipains from P. gingivalis. Treated cells exhibited a rapid loss of cell adhesion properties that was followed by apoptotic cell death. Cleavage of N- and VE-cadherin and integrin beta1 was observed in immunoblots of cell lysates. There was a direct correlation between the kinetics of cleavage of N- and VE-cadherin and loss of cell adhesion properties. Loss of cell adhesion, as well as N- and VE-cadherin and integrin beta1 cleavage, could be inhibited or significantly delayed by preincubation of P. gingivalis W83 gingipain-active extracellular extracts with the cysteine protease inhibitor Nalpha-p-tosyl-l-lysine chloromethylketone. Furthermore, purified gingipains also induced endothelial cell detachment and apoptosis. Apoptosis-associated events, including annexin V positivity, caspase-3 activation, and cleavage of the caspase substrates poly(ADP-ribose) polymerase and topoisomerase I (Topo I), were observed in endothelial cells after detachment. All of the effects observed were correlated with the different levels of cysteine-dependent proteolytic activity of the extracts tested. Taken together, these results indicate that gingipains from P. gingivalis can alter cell adhesion molecules and induce endothelial cell death, which could have implications for the pathogenicity of this organism.

    Funded by: NIAID NIH HHS: AI44088; NIDCR NIH HHS: DE09761, DE13664, DE13664-S1, R01 DE009761, R01 DE013664, R56 DE013664

    Infection and immunity 2005;73;3;1543-52

  • Sp1/Sp3 and the myeloid zinc finger gene MZF1 regulate the human N-cadherin promoter in osteoblasts.

    Le Mée S, Fromigué O and Marie PJ

    Laboratory of Osteoblast Biology and Pathology, INSERM U606, Lariboisière Hospital, 75475 Cedex 10 Paris, France.

    To determine the molecular mechanisms by which N-cadherin transcription is regulated, we cloned and sequenced a 3681-bp of the 5'-flanking region of the human N-cadherin gene. Deletion analysis of the proximal region identified a minimal 318-bp region with strong promoter activity in human osteoblasts. The cryptic promoter is characterized by high GC content and a GA-rich binding core that may bind zing finger transcription factors. Electrophoretic mobility shift assays (EMSA), competition and supershift EMSA revealed that an Sp1/Sp3 binding site acts as a basal regulatory element of the promoter in osteoblasts. Incubation of osteoblast nuclear extracts with -163/-131 wild-type probe containing the GA-rich binding core revealed another specific complex, which was not formed with a -163/-131 probe mutated in the GA repeat. EMSA identified the nuclear factor involved as myeloid zinc finger-1 (MZF1). Mutation analysis showed that Sp1/Sp3 and MZF1 binding sites contribute to basal promoter activity. Cotransfection analyses showed that Sp1 and MZF1 overexpression increases whereas Sp3 antagonizes Sp1-induced N-cadherin promoter activity in osteoblasts. RT-PCR analysis showed that human osteoblastic cells express MZF1 and that Sp1/MZF1 overexpression increased N-cadherin expression. These results indicate that Sp1/Sp3 and MZF1 are important transcription factors regulating N-cadherin promoter activity and expression in osteoblasts.

    Experimental cell research 2005;302;1;129-42

  • N-cadherin as an invasion promoter: a novel target for antitumor therapy?

    Cavallaro U

    IFOM-The FIRC Institute of Molecular Oncology, Via Adamello 16, 1-20139 Milan, Italy. ugo.cavallaro@ifom-ieo-campus.it

    Cell adhesion molecules of the cadherin superfamily are frequently altered during tumor progression. The loss of E-cadherin, in particular, has long been described in various epithelial cancers where it correlates with malignancy and metastasis. In addition, a subset of tumors show de novo expression or upregulation of N-cadherin, a cadherin that promotes cell motility and invasion. In spite of the accumulating evidence supporting the relationship between N-cadherin levels and cancer progression, the causal role of N-cadherin in tumor development has not yet been clearly demonstrated. This review will address this issue at three different levels: (i) the expression pattern of N-cadherin in cancer; (ii) the cellular events that are modulated by N-cadherin and could account for its pro-invasive role in tumorigenesis; and (iii) the signaling properties of N-cadherin, with particular emphasis on its cross-talk with cell surface partners such as fibroblast growth factor receptor and with intracellular cascades such as the antiapoptotic machinery. Taken together, the topics discussed in this review point to N-cadherin as an important player in tumor development and, therefore, a potential target for novel therapeutic approaches.

    Current opinion in investigational drugs (London, England : 2000) 2004;5;12;1274-8

  • Correlation of N-cadherin expression in high grade gliomas with tissue invasion.

    Asano K, Duntsch CD, Zhou Q, Weimar JD, Bordelon D, Robertson JH and Pourmotabbed T

    Department of Neurosurgery, The University of Tennessee Health Science Center, Memphis, TN, USA. asanoken@cc.hirosaki-u.ac.jp

    Cadherins are Ca2+-dependent cell adhesion molecules that play an important role in tissue construction and morphogenesis in multicellular organisms. Over the last few years, reports have emerged in the literature describing the involvement of cadherins in tumor invasion and metastasis. Cadherins typically demonstrate up and down-regulation according to the biological needs of the tissue. Additionally, up-regulation of N-cadherin is thought to be important for tumor formation in early stages of tumor development. We studied N-cadherin in surgical specimens of patients with primary glioblastoma by microarray analysis and found that N-cadherin mRNA expression is up-regulated compared to normal brain. To study the effects of N-cadherin expression on invasion and metastasis in vitro and in vivo, we overexpressed N-cadherin in the rat C6 glioma cell line which normally has low levels of N-cadherin. We found that up-regulation of N-cadherin resulted in a slight decreased adhesion to type IV collagen, fibronectin, and laminin, but statistically significant decreased adhesion to type I collagen. Furthermore, increased expression of N-cadherin correlated with a dramatic decrease in invasive behavior in extracellular matrix invasion assays. We then proceeded to study these cell lines in vivo in a rat intracranial glioma model, and found that N-cadherin expression inversely correlated with invasion into surrounding tissues, irregular margins, and extracranial invasion. In summary, these data collectively demonstrate that N-cadherin levels are important in the malignant behavior of gliomas, and may serve as a prognostic indicator for patients with high-grade gliomas.

    Journal of neuro-oncology 2004;70;1;3-15

  • Neoexpression of N-cadherin in E-cadherin positive colon cancers.

    Rosivatz E, Becker I, Bamba M, Schott C, Diebold J, Mayr D, Höfler H and Becker KF

    Institut für Pathologie der TU, München, Germany.

    In our study, we aimed to investigate the expression of N-cadherin and E-cadherin and their dependency on epithelial-mesenchymal transition regulators SNAI1, SIP1 and TWIST in human colon cancer. Expression of E-cadherin and N-cadherin was examined by immunohistochemistry in 80 colon carcinomas by using paraffin embedded and formalin fixed tissues. Those cases were partly analyzed for mRNA expression of N-cadherin (42 cases), TWIST (18 cases), SNAI1 (25 cases) and SIP1 (25 cases) by real-time quantitative RT-PCR. Additionally, colon carcinomas that showed amplification of 20q13, the localization of the human SNAI1 gene, were examined. We found cytoplasmic and/or membrane-associated immunoreactivity of N-cadherin in 35/80 (44%) of the cases. However, there was no correlation to upregulated TWIST mRNA levels, as we have shown previously for diffuse-type gastric cancers with abnormal N-cadherin expression. Reduced and/or cytoplasmic E-cadherin immunoreactivity was detected in 19% (15/80) of the cases. Expression of SNAI1 or SIP1 mRNA was not seen in any of the 25 cases analyzed. There was no correlation between amplification of 20q13 and SNAI1 mRNA expression. Remarkably, N-cadherin was almost exclusively expressed in those cases showing normal E-cadherin immunoreactivity, suggesting a mutual exclusion between abnormal E-cadherin reduction and upregulation of N-cadherin. For the first time, we postulate a role for N-cadherin in primary colon cancer progression, which may be similar to the effect discovered by others in breast cancer cell lines, where coexpressed N-cadherin can exert a dominant function over E-cadherin's adhesive function and thus promote tumor invasiveness.

    International journal of cancer. Journal international du cancer 2004;111;5;711-9

  • Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-beta or wounding.

    De Wever O, Westbroek W, Verloes A, Bloemen N, Bracke M, Gespach C, Bruyneel E and Mareel M

    Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Gent, Belgium.

    Invasion of stromal host cells, such as myofibroblasts, into the epithelial cancer compartment may precede epithelial cancer invasion into the stroma. We investigated how colon cancer-derived myofibroblasts invade extracellular matrices in vitro in the presence of colon cancer cells. Myofibroblast spheroids invade collagen type I in a stellate pattern to form a dendritic network of extensions upon co-culture with HCT-8/E11 colon cancer cells. Single myofibroblasts also invade Matrigel trade mark when stimulated by HCT-8/E11 colon cancer cells. The confrontation of cancer cells with extracellular matrices and myofibroblasts, showed that cancer-cell-derived transforming growth factor-beta (TGF-beta) is required and sufficient for invasion of myofibroblasts. In myofibroblasts, N-cadherin expressed at the tips of filopodia is upregulated by TGF-beta. Functional N-cadherin activity is implicated in TGF-beta stimulated invasion as evidenced by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb), and specific N-cadherin knock-down by short interference RNA (siRNA). TGF-beta1 stimulates Jun N-terminal kinase (also known as stress-activated protein kinase) (JNK) activity in myofibroblasts. Pharmacological inhibition of JNK alleviates TGF-beta stimulated invasion, N-cadherin expression and wound healing migration. Neutralization of N-cadherin activity by the GC-4 or by a 10-mer N-cadherin peptide or by siRNA reduces directional migration, filopodia formation, polarization and Golgi-complex reorientation during wound healing. Taken together, our study identifies a new mechanism in which cancer cells contribute to the coordination of invasion of stromal myofibroblasts.

    Journal of cell science 2004;117;Pt 20;4691-703

  • N-cadherin is regulated by activin A and associated with tumor aggressiveness in esophageal carcinoma.

    Yoshinaga K, Inoue H, Utsunomiya T, Sonoda H, Masuda T, Mimori K, Tanaka Y and Mori M

    Department of Molecular and Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan.

    Purpose: Activin A is a member of the transforming growth factor beta superfamily and plays an important role in the differentiation of embryonic stem cells. We have reported previously that the expression of activin A is associated with lymph node metastasis in esophageal cancer, and our purpose in the current work is to clarify the molecular mechanism of the aggressive behavior of tumors that have high activin A expression.

    We have compared the gene expression profiles of human esophageal carcinoma cell lines that were stably transfected with activin beta A, which is a subunit of activin A, with those of control human esophageal carcinoma cell lines, using a cDNA microarray.

    Results: We found that the expression level of neuronal cadherin (N-cadherin) was higher in the transfectants than in the control cells. N-cadherin was located on the cell surface of the transfectants, irrespective of the expression of epithelial cadherin (E-cadherin), and the expression of N-cadherin mRNA was significantly associated with that of activin beta A mRNA in clinical samples of esophageal carcinoma (n = 51; r = 0.855). A clinicopathologic analysis suggested that expression of N-cadherin mRNA was associated with the depth of tumor wall invasion, and a group of patients with high expression of N-cadherin mRNA showed a significantly poorer prognosis than a group of patients with low N-cadherin expression (P = 0.046).

    Conclusions: These results indicate that activin A might mediate the expression of N-cadherin and that this may be associated with depth of invasion and poor prognosis.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10;17;5702-7

  • Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion.

    Yano H, Mazaki Y, Kurokawa K, Hanks SK, Matsuda M and Sabe H

    Department of Molecular Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan.

    Integrins can intercommunicate with cadherins. Here, we examined their possible relationship by use of small interfering RNA-mediated protein knockdown in HeLa cells. We found that a subset of integrin signaling molecules, namely Fak and paxillin, but not p130 Crk-associated substrate or proline-rich tyrosine kinase 2, participate in processes regulating N-cadherin-based cell-cell adhesion. Paxillin was found to be required primarily for the recruitment of Fak to robust focal adhesions. Our results suggest that at least some signals involving Fak are linked to a mechanism down-regulating Rac1 activity at the cell periphery, which appears to be important for the formation of N-cadherin-based adhesions in motile cells. Our analyses simultaneously exemplified the essential role of Fak in the maintenance of cell-cell adhesions in collective cell migration, a type of migration occurring in embryonic development and carcinoma invasion.

    The Journal of cell biology 2004;166;2;283-95

  • PS1 activates PI3K thus inhibiting GSK-3 activity and tau overphosphorylation: effects of FAD mutations.

    Baki L, Shioi J, Wen P, Shao Z, Schwarzman A, Gama-Sosa M, Neve R and Robakis NK

    Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY 10029, USA.

    Phosphatidylinositol 3-kinase (PI3K) promotes cell survival and communication by activating its downstream effector Akt kinase. Here we show that PS1, a protein involved in familial Alzheimer's disease (FAD), promotes cell survival by activating the PI3K/Akt cell survival signaling. This function of PS1 is unaffected by gamma-secretase inhibitors. Pharmacological and genetic evidence indicates that PS1 acts upstream of Akt, at or before PI3K kinase. PS1 forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association. Furthermore, conditions that inhibit this association prevent the PS1-induced PI3K/Akt activation, indicating that PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. By activating PI3K/Akt signaling, PS1 promotes phosphorylation/inactivation of glycogen synthase kinase-3 (GSK-3), suppresses GSK-3-dependent phosphorylation of tau at residues overphosphorylated in AD and prevents apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1-dependent PI3K/Akt activation, thus promoting GSK-3 activity and tau overphosphorylation at AD-related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway.

    Funded by: NIA NIH HHS: AG-05138, AG-08200, AG-17926, P50 AG005138, R01 AG008200, R01 AG017926, R37 AG017926; NINDS NIH HHS: NS-47229, R01 NS047229

    The EMBO journal 2004;23;13;2586-96

  • N-cadherin expression and epithelial-mesenchymal transition in pancreatic carcinoma.

    Nakajima S, Doi R, Toyoda E, Tsuji S, Wada M, Koizumi M, Tulachan SS, Ito D, Kami K, Mori T, Kawaguchi Y, Fujimoto K, Hosotani R and Imamura M

    Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan. sana@kuhp.kyoto-u.ac.jp

    Purpose: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors.

    We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)beta, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines.

    Results: N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change.

    Conclusion: This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10;12 Pt 1;4125-33

  • Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation.

    Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk GJ, Li Y, Xu C, Fang R, Guegler K, Rao MS, Mandalam R, Lebkowski J and Stanton LW

    Geron Corporation, Menlo Park, California 94025, USA. rbrandenberger@geron.com

    Human embryonic stem (hES) cells hold promise for generating an unlimited supply of cells for replacement therapies. To characterize hES cells at the molecular level, we obtained 148,453 expressed sequence tags (ESTs) from undifferentiated hES cells and three differentiated derivative subpopulations. Over 32,000 different transcripts expressed in hES cells were identified, of which more than 16,000 do not match closely any gene in the UniGene public database. Queries to this EST database revealed 532 significantly upregulated and 140 significantly downregulated genes in undifferentiated hES cells. These data highlight changes in the transcriptional network that occur when hES cells differentiate. Among the differentially regulated genes are several components of signaling pathways and transcriptional regulators that likely play key roles in hES cell growth and differentiation. The genomic data presented here may facilitate the derivation of clinically useful cell types from hES cells.

    Nature biotechnology 2004;22;6;707-16

  • Cadherin switch in tumor progression.

    Hazan RB, Qiao R, Keren R, Badano I and Suyama K

    Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. rhazan@aecom.yu.edu

    The loss of E-cadherin expression or function in epithelial carcinomas has long been thought as a primary reason for disruption of tight epithelial cell-cell contacts and release of invasive tumor cells from the primary tumor. Indeed, E-cadherin serves as a widely acting suppressor of invasion and growth of epithelial cancers, and its functional elimination represents a key step in the acquisition of the invasive phenotype for many tumors. Recent evidence indicates, however, that in addition to the loss of the "invasion-suppressor" E-cadherin, another adhesion molecule, N-cadherin, becomes upregulated in invasive tumor cell lines. N-cadherin was shown to be present in the most invasive and dedifferentiated breast cancer cell lines, and its exogenous expression in tumor cells induces a scattered morphology and heightened motility, invasion, and metastasis. N-cadherin cooperates with the FGF receptor, resulting in signals that lead to the up-modulation of MMP-9 and, hence, cellular invasion. In addition to a signaling function in metastasis, N-cadherin probably also supports the systemic dissemination of tumor cells by enabling circulating tumor cells to associate with the stroma and the endothelium at distant sites. Here, we summarize the various aspects of the E- to N-cadherin switching in epithelial carcinomas and its potential impact on metastatic progression.

    Funded by: NCI NIH HHS: R01 CA90872

    Annals of the New York Academy of Sciences 2004;1014;155-63

  • Cadherin expression pattern in melanocytic tumors more likely depends on the melanocyte environment than on tumor cell progression.

    Krengel S, Grotelüschen F, Bartsch S and Tronnier M

    Department of Dermatology, Medical University Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. svenkrengel@gmx.net

    Background: Adhesion molecules have been assigned an important role in melanocytic tumor progression. By the loss of E-cadherin, melanocytes might escape the control of neighbouring keratinocytes. Although in vitro data support this hypothesis, there are yet no conclusive immunohistochemical results on cadherin expression in melanocytic tumors.

    Objective: To gain detailed insight in the expression of cadherins and their cytoplasmic binding partners, the catenins, in various types of benign and malignant melanocytic neoplasms.

    Methods: Immunohistochemical analysis of the expression of E-, P-, and N-cadherin and alpha-, beta-, and gamma-catenin in compound and dermal nevi, Spitz nevi, blue nevi, ultraviolet B (UVB)-irradiated nevi, and malignant melanomas of various tumor thickness.

    Results: In both nevi and melanomas, E-cadherin expression in melanocytic cells decreased, following a gradient from junctional to deeper dermal localization. The pattern of E-cadherin expression was more heterogeneous in melanomas than in nevi. In some melanomas, E-cadherin was only weakly positive in the epidermal tumor cells. P-cadherin expression was similar to that of E-cadherin. N-cadherin expression in melanocytic lesions was a rare finding, however, a small percentage of melanomas showed expression in some cell nests. Some Spitz nevi exhibited strong N-cadherin immunoreactivity. Most melanocytic cells were alpha- and beta-catenin-positive and gamma-catenin-negative. UVB irradiation did not influence the expression of cadherins and catenins in melanocytic nevi in vivo.

    Conclusions: It is presumed that the gradual loss of E-cadherin expression represents a reaction of melanocytic cells to altered conditions in the dermal environment, e.g. lack of contact to keratinocytes, or new contact with dermal extracellular matrix molecules, respectively. Melanoma cells apparently are less dependent on these environmental factors and, therefore, show a more heterogeneous expression pattern. This might be of importance for the adaptation of the tumor cells to local requirements. However, in view of our results, a causative role of (loss of ) E-cadherin or (gain of ) N-cadherin for melanocytic tumor progression still remains to be proven.

    Journal of cutaneous pathology 2004;31;1;1-7

  • Two regions of cadherin cytoplasmic domains are involved in suppressing motility of a mammary carcinoma cell line.

    Fedor-Chaiken M, Meigs TE, Kaplan DD and Brackenbury R

    Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521, USA. mary.chaiken@uc.edu

    E-cadherin has been termed an "invasion suppressor," yet the mechanism of this suppression is not known. In contrast, several reports indicate N-cadherin does not suppress but, rather, promotes cell motility and invasion. Here, by characterizing a series of chimeric cadherins we defined a previously uncharacterized region consisting of the transmembrane domain and an adjacent portion of the cytoplasmic segment that is responsible for the difference in ability of E- and N-cadherin to suppress movement of mammary carcinoma cells, as quantified from time-lapse video recordings. A mutation in this region enabled N-cadherin to suppress motility, indicating that both E- and N-cadherin can suppress, but the activity of N-cadherin is latent, presumably repressed by binding of a specific inhibitor. To define regions common to E- and N-cadherin that are required for suppression, we analyzed a series of deletion mutants. We found that suppression of movement requires E-cadherin amino acids 699-710. Strikingly, beta-catenin binding is not sufficient for and p120ctn is not involved in suppression of these mammary carcinoma cells. Furthermore, the comparable region of N-cadherin can substitute for this required region in E-cadherin and is required for suppression by the mutant form of N-cadherin that is capable of suppressing. Variations in expression of factors that bind to the two regions we have identified may explain previously observed differences in response of tumor cells to cadherins.

    Funded by: NCI NIH HHS: CA91159; NIAMS NIH HHS: AR44713

    The Journal of biological chemistry 2003;278;52;52371-8

  • A novel cell-cell junction system: the cortex adhaerens mosaic of lens fiber cells.

    Straub BK, Boda J, Kuhn C, Schnoelzer M, Korf U, Kempf T, Spring H, Hatzfeld M and Franke WW

    Division of Cell Biology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

    The anucleate prismoid fiber cells of the eye lens are densely packed to form a tissue in which the plasma membranes and their associated cytoplasmic coat form a single giant cell-cell adhesive complex, the cortex adhaerens. Using biochemical and immunoprecipitation methods in various species (cow, pig, rat), in combination with immunolocalization microscopy, we have identified two different major kinds of cortical complex. In one, the transmembrane glycoproteins N-cadherin and cadherin-11 [which also occur in heterotypic ('mixed') complexes] are associated with alpha- and beta-catenin, plakoglobin (proportions variable among species), p120ctn and vinculin. The other complex contains ezrin, periplakin, periaxin and desmoyokin (and so is called the EPPD complex), usually together with moesin, spectrin(s) and plectin. In sections through lens fiber tissue, the short sides of the lens fiber hexagons appear to be enriched in the cadherin-based complexes, whereas the EPPD complexes also occur on the long sides. Moreover, high resolution double-label fluorescence microscopy has revealed, on the short sides, a finer, almost regular mosaicism of blocks comprising the cadherin-based, catenin-containing complexes, alternating with patches formed by the EPPD complexes. The latter, a new type of junctional plaque ensemble of proteins hitherto known only from certain other cell types, must be added to the list of major lens cortex proteins. We here discuss its possible functional importance for the maintenance of lens structure and functions, notably clear and sharp vision.

    Journal of cell science 2003;116;Pt 24;4985-95

  • Presenilin 1 is involved in maturation and trafficking of N-cadherin to the plasma membrane.

    Uemura K, Kitagawa N, Kohno R, Kuzuya A, Kageyama T, Chonabayashi K, Shibasaki H and Shimohama S

    Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

    One pathological characteristic of Alzheimer's disease (AD) is extensive synapse loss. Presenilin 1 (PS1) is linked to the pathogenesis of early onset familial Alzheimer's disease (FAD) and is localized at the synapse, where it binds N-cadherin and modulates its adhesive activity. To elucidate the role of the PS1/N-cadherin interaction in synaptic contact, we established SH-SY5Y cells stably expressing wild-type (wt) PS1 and dominant-negative (D385A) PS1. We show that the formation of cadherin-based cell-cell contact among SH-SY5Y cells stably expressing D385A PS1 was suppressed. Conversely, wt PS1 cells exhibited enhanced cell-cell contact and colony formation. Suppression of cell-cell contact in D385A cells was accompanied by an alteration in N-cadherin subcellular localization; N-cadherin was retained mainly in the endoplasmic reticulum (ER) and cell surface expression was reduced. We conclude that PS1 is essential for efficient trafficking of N-cadherin from the ER to the plasma membrane. PS1-mediated delivery of N-cadherin to the plasma membrane is important for N-cadherin to exert its physiological function, and it may control the state of cell-cell contact.

    Journal of neuroscience research 2003;74;2;184-91

  • A CBP binding transcriptional repressor produced by the PS1/epsilon-cleavage of N-cadherin is inhibited by PS1 FAD mutations.

    Marambaud P, Wen PH, Dutt A, Shioi J, Takashima A, Siman R and Robakis NK

    Department of Psychiatry and Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY 10029, USA.

    Presenilin1 (PS1), a protein implicated in Alzheimer's disease (AD), forms complexes with N-cadherin, a transmembrane protein with important neuronal and synaptic functions. Here, we show that a PS1-dependent gamma-secretase protease activity promotes an epsilon-like cleavage of N-cadherin to produce its intracellular domain peptide, N-Cad/CTF2. NMDA receptor agonists stimulate N-Cad/CTF2 production suggesting that this receptor regulates the epsilon-cleavage of N-cadherin. N-Cad/CTF2 binds the transcription factor CBP and promotes its proteasomal degradation, inhibiting CRE-dependent transactivation. Thus, the PS1-dependent epsilon-cleavage product N-Cad/CTF2 functions as a potent repressor of CBP/CREB-mediated transcription. Importantly, PS1 mutations associated with familial AD (FAD) and a gamma-secretase dominant-negative mutation inhibit N-Cad/CTF2 production and upregulate CREB-mediated transcription indicating that FAD mutations cause a gain of transcriptional function by inhibiting production of transcriptional repressor N-Cad/CTF2. These data raise the possibility that FAD mutation-induced transcriptional abnormalities maybe causally related to the dementia associated with FAD.

    Funded by: NIA NIH HHS: AG-05138, AG-08200, AG-17926, P50 AG005138-190025, R01 AG008200-18, R01 AG017926-05

    Cell 2003;114;5;635-45

  • N-cadherin-catenin complexes form prior to cleavage of the proregion and transport to the plasma membrane.

    Wahl JK, Kim YJ, Cullen JM, Johnson KR and Wheelock MJ

    University of Nebraska Medical Center, College of Dentistry and Eppley Cancer Center, Omaha, Nebraska 68198-7696, USA. Jwahl@unmc.edu

    Cadherins are calcium-dependent glycoproteins that function as cell-cell adhesion molecules and are linked to the actin cytoskeleton via catenins. Newly synthesized cadherins contain a prosequence that must be proteolytically removed to generate a functional adhesion molecule. The goal of this study was to examine the proteolytic processing of N-cadherin and the assembly of the cadherin-catenin complex in cells that express endogenous N-cadherin. A monoclonal antibody specific for the proregion of human N-cadherin was generated and used to examine N-cadherin processing. Our data show that newly synthesized proN-cadherin is phosphorylated and proteolytically processed prior to transport to the plasma membrane. In addition, we show that beta-catenin and plakoglobin associate only with phosphorylated proN-cadherin, whereas p120(ctn) can associate with both phosphorylated and non-phosphorylated proN-cadherin. Immunoprecipitations using anti-proN-cadherin showed that cadherin-catenin complexes are assembled prior to localization at the plasma membrane. These data suggest that a core N-cadherin-catenin complex assembles in the endoplasmic reticulum or Golgi compartment and is transported to the plasma membrane where linkage to the actin cytoskeleton can be established.

    Funded by: NIDCR NIH HHS: DE12308; NIGMS NIH HHS: GM51188

    The Journal of biological chemistry 2003;278;19;17269-76

  • Promyogenic members of the Ig and cadherin families associate to positively regulate differentiation.

    Kang JS, Feinleib JL, Knox S, Ketteringham MA and Krauss RS

    Department of Molecular, Cell, and Developmental Biology, Box 1020, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.

    Determination and differentiation of cells in the skeletal muscle lineage is positively regulated by cell-cell contact. Cell-surface proteins proposed to mediate this effect include both classical cadherins and Ig superfamily members; potential interactions between the promyogenic activities of these classes of protein, however, are unknown. We show here that CDO and BOC, two promyogenic Ig superfamily members that bind to each other in a cis fashion, form complexes with N- and M-cadherin. These complexes contain beta-catenin and are enriched at sites of cell-cell contact between myoblasts. In transient expression assays, the ectodomains and intracellular regions of CDO, BOC, and N-cadherin each interact independently, suggesting that the interactions occur in a cis fashion; consistent with this conclusion, cadherin-mediated cell adhesion is not required for them to occur. Stable expression in myoblasts of a CDO deletion mutant deficient in its ability to associate with N-cadherin interferes with differentiation as assessed by biochemical, morphological, and reporter gene assays, suggesting that this interaction is functionally important in myogenesis. Thus, some of the cell-cell contact-mediated activities that are required for myogenesis seem to be based on interdependent activities of promyogenic classical cadherins and Ig superfamily members.

    Funded by: NCI NIH HHS: 1R24CA095823-01, CA59474, R24 CA095823; NCRR NIH HHS: 1S10RR09145-01; NIAMS NIH HHS: AR46207, R01 AR046207

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;7;3989-94

  • Targeting of p0071 to desmosomes and adherens junctions is mediated by different protein domains.

    Hatzfeld M, Green KJ and Sauter H

    Institute of Physiological Chemistry, Medical Faculty of the University of Halle, 06097 Halle/Saale, Germany. mechthild.hatzfeld@medizin.uni-halle.de

    p0071, a member of the armadillo protein family, is most closely related to p120(ctn) and the plakophilins 1-3. Whereas plakophilins are desmosomal plaque proteins, p120(ctn) localizes to adherens junctions and interacts with classical cadherins. In contrast, p0071 has been described as a protein with dual localization in adherens junctions and desmosomes depending on the cell type examined. Here we have analyzed the localization of p0071 and its domains in detail. Although by sequence analysis, p0071 is more closely related to the adherens junction proteins p120(ctn), ARVCF and delta-catenin, endogenous p0071 associated preferentially with desmosomes in MCF-7 epithelial cells. Overexpressed p0071 localized along cell borders and overlapped only partially with desmosomal markers but colocalized with non-desmosomal cadherins and recruited cadherins to the membrane. The head domain of p0071 was sufficient for desmosomal targeting, whereas the arm repeat domain associated with adherens junctions and enhanced membrane association of classical cadherins. The tail domain localized preferentially to the nucleus and associated with desmosomes. To examine the mechanism underlying this dual localization more closely we determined binding partners of p0071 by using yeast-two-hybrid and mom-targeting assays. These approaches show that the head domain interacted with desmosomal proteins desmocollin 3a and desmoplakin, whereas the armadillo repeat domain binds to non-desmosomal cadherins. Head and armadillo repeat domains both interacted with plakoglobin by binding to different sites. Our data suggest that, in addition to plakoglobin, p0071 is the second armadillo protein present in both types of adhesive junctions and may play a role in regulating crosstalk between adherens junctions and desmosomes.

    Funded by: NIAMS NIH HHS: AR43380; NIDCR NIH HHS: P01 DE12328

    Journal of cell science 2003;116;Pt 7;1219-33

  • Rab8B GTPase and junction dynamics in the testis.

    Lau AS and Mruk DD

    Population Council, Center for Biomedical Research, New York, New York 10021, USA.

    Throughout spermatogenesis, germ cells migrate from the basal to the adluminal compartment while remaining attached to Sertoli cells via actin-based adherens and intermediate filament-based anchoring junctions. However, the events that trigger deadhesion and adhesion remain largely unknown. As part of our continued effort in elucidating the mechanism of germ cell movement, we have examined the role of Rab8B, a GTPase probably participating in intracellular trafficking events at the site of the adherens junction. By RT-PCR Rab8B mRNA was found in the brain, testis, heart, kidney, and spleen. Immunohistochemical studies revealed that Rab8B was concentrated predominantly in the basal compartment, localizing to a similar site at which immunoreactive E-cadherin was found. Additional experiments demonstrated that Rab8B associated with the actin, intermediate filament, and microtubule cytoskeletal networks. When Sertoli cells were cultured at high density or germ cells were cocultured with Sertoli cells, Rab8B increased significantly during junction assembly. Moreover, inclusion of germ cell-conditioned medium in Sertoli cell cultures resulted in stimulation of Rab8B expression. Conversely, treatment of adult rats with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide reduced Rab8B mRNA and protein levels, coinciding with the time of germ cell loss from the epithelium. Taken collectively, these studies suggest that Rab8B participates in adherens junction dynamics in the testis.

    Endocrinology 2003;144;4;1549-63

  • RICS, a novel GTPase-activating protein for Cdc42 and Rac1, is involved in the beta-catenin-N-cadherin and N-methyl-D-aspartate receptor signaling.

    Okabe T, Nakamura T, Nishimura YN, Kohu K, Ohwada S, Morishita Y and Akiyama T

    Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

    Cadherin adhesion molecules are believed to be important for synaptic plasticity. beta-Catenin, which links cadherins and the actin cytoskeleton, is a modulator of cadherin adhesion and regulates synaptic structure and function. Here we show that beta-catenin interacts with a novel GTPase-activating protein, named RICS, that acts on Cdc42 and Rac1. The RICS-beta-catenin complex was found to be associated with N-cadherin, N-methyl-d-aspartate receptors, and postsynaptic density-95, and localized to the postsynaptic density. Furthermore, the GTPase-activating protein activity of RICS was inhibited by phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II. These results suggest that RICS is involved in the synaptic adhesion- and N-methyl-d-aspartate-mediated organization of cytoskeletal networks and signal transduction. Thus, RICS may regulate dendritic spine morphology and strength by modulating Rho GTPases.

    The Journal of biological chemistry 2003;278;11;9920-7

  • PTP1B modulates the association of beta-catenin with N-cadherin through binding to an adjacent and partially overlapping target site.

    Xu G, Arregui C, Lilien J and Balsamo J

    Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA.

    The nonreceptor tyrosine phosphatase PTP1B associates with the cytoplasmic domain of N-cadherin and may regulate cadherin function through dephosphorylation of beta-catenin. We have now identified the domain on N-cadherin to which PTP1B binds and characterized the effect of perturbing this domain on cadherin function. Deletion constructs lacking amino acids 872-891 fail to bind PTP1B. This domain partially overlaps with the beta-catenin binding domain. To further define the relationship of these two sites, we used peptides to compete in vitro binding. A peptide representing the most NH(2)-terminal 8 amino acids of the PTP1B binding site, the region of overlap with the beta-catenin target, effectively competes for binding of beta-catenin but is much less effective in competing PTP1B, whereas two peptides representing the remaining 12 amino acids have no effect on beta-catenin binding but effectively compete for PTP1B binding. Introduction into embryonic chick retina cells of a cell-permeable peptide mimicking the 8 most COOH-terminal amino acids in the PTP1B target domain, the region most distant from the beta-catenin target site, prevents binding of PTP1B, increases the pool of free, tyrosine-phosphorylated beta-catenin, and results in loss of N-cadherin function. N-cadherin lacking this same region of the PTP1B target site does not associate with PTP1B or beta-catenin and is not efficiently expressed at the cell surface of transfected L cells. Thus, interaction of PTP1B with N-cadherin is essential for its association with beta-catenin, stable expression at the cell surface, and consequently, cadherin function.

    Funded by: NEI NIH HHS: EY 12132

    The Journal of biological chemistry 2002;277;51;49989-97

  • Beta-catenin interacts with low-molecular-weight protein tyrosine phosphatase leading to cadherin-mediated cell-cell adhesion increase.

    Taddei ML, Chiarugi P, Cirri P, Buricchi F, Fiaschi T, Giannoni E, Talini D, Cozzi G, Formigli L, Raugei G and Ramponi G

    Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, 50134 Florence, Italy.

    Beta-catenin plays a dual role as a major constituent of cadherin-based adherens junctions and also as a transcriptional coactivator. In normal ephitelial cells, at adherens junction level, beta-catenin links cadherins to the actin cytoskeleton. The structure of adherens junctions is dynamically regulated by tyrosine phosphorylation. In particular, cell-cell adhesion can be negatively regulated through the tyrosine phosphorylation of beta-catenin. Furthermore, the loss of beta-catenin-cadherin association has been correlated with the transition from a benign tumor to an invasive, metastatic cancer. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitous PTP implicated in the regulation of mitosis and cytoskeleton rearrangement. Here we demonstrate that the amount of free cytoplasmic beta-catenin is decreased in NIH3T3, which overexpresses active LMW-PTP, and this results in a stronger association between cadherin complexes and the actin-based cytoskeleton with respect to control cells. Confocal microscopy analysis shows that beta-catenin colocalizes with LMW-PTP at the plasma membrane. Furthermore, we provide evidence that beta-catenin is able to associate with LMW-PTP both in vitro and in vivo. Moreover, overexpression of active LMW-PTP strongly potentiates cadherin-mediated cell-cell adhesion, whereas a dominant-negative form of LMW-PTP induces the opposite phenotype, both in NIH3T3 and in MCF-7 carcinoma cells. On the basis of these results, we propose that the stability of cell-cell contacts at the adherens junction level is positively influenced by LMW-PTP expression, mainly because of the beta-catenin and LMW-PTP interaction at the plasma membrane level with consequent dephosphorylation.

    Cancer research 2002;62;22;6489-99

  • A signaling pathway leading to metastasis is controlled by N-cadherin and the FGF receptor.

    Suyama K, Shapiro I, Guttman M and Hazan RB

    Derald H Ruttenberg Cancer Center, The Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA.

    The intracellular signaling events causing tumor cells to become metastatic are not well understood. N-cadherin and FGF-2 synergistically increase migration, invasion, and secretion of extracellular proteases in breast tumor cells. Here, we define a metastatic signaling cascade activated by N-cadherin and FGF-2. In the presence of N-cadherin, FGF-2 caused sustained activation of the MAPK-ERK pathway, leading to MMP-9 gene transcription and cellular invasion. N-cadherin prevented the FGF receptor (FGFR) from undergoing ligand-induced internalization, resulting in increased FGFR-1 stability. Association of FGFR-1 with N-cadherin was mediated by the first two Ig-like domains of FGFR-1. These results suggest that protection of the FGFR-1 from ligand-induced downregulation by N-cadherin enhances receptor signaling and provides a mechanism by which tumor cells can acquire metastatic properties.

    Funded by: NCI NIH HHS: R01CA90872

    Cancer cell 2002;2;4;301-14

  • Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization.

    Tran NL, Adams DG, Vaillancourt RR and Heimark RL

    Cancer Biology Graduate Program, University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA.

    Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin.catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin.catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.

    Funded by: NCI NIH HHS: CA5666, T32CA09213; NIA NIH HHS: AG19710

    The Journal of biological chemistry 2002;277;36;32905-14

  • Expression of adhesion and extracellular matrix molecules in the developing human brain.

    Anlar B, Atilla P, Cakar AN, Kose MF, Beksaç MS, Dagdeviren A and Akçören Z

    Department of Pediatric Neurology, Hacettepe University, Ankara, Turkey. banlar@hacettepe.edu.tr

    Cell adhesion molecules and extracellular matrix molecules have important roles in cell migration and connection. Their developmental expression has not been fully described in humans. In this report, these molecules were examined by immunohistochemistry in frontal tissue samples from 14- to 28-week-old fetuses aborted for obstetric reasons (n = 20) and four fetuses with nervous system abnormalities. Neural cell adhesion molecule (NCAM), tenascin, and laminin were expressed after 17 weeks. Neural cell adhesion molecule was observed in the neuropil, whereas tenascin and laminin also had cellular and vascular expression. Thrombospondin and fibronectin, apparent after 14 weeks, showed a redistribution from periventricular to outer cortical layers after midgestation. N-cadherin and integrin were observed in mid- and late gestation. Maternal or environmental conditions seemed to influence the pattern of expression. Fetuses with nervous system abnormalities had altered expression of several molecules. The descriptive data obtained in this study might constitute a basis for further studies investigating the role of these molecules in developmental abnormalities of the brain.

    Journal of child neurology 2002;17;9;707-13

  • Recruitment of the kainate receptor subunit glutamate receptor 6 by cadherin/catenin complexes.

    Coussen F, Normand E, Marchal C, Costet P, Choquet D, Lambert M, Mège RM and Mulle C

    Centre National de la Recherche Scientifique Unité Mixte de Recherche 5091, Institut François Magendie, Paris 75005, France.

    Kainate receptors modulate synaptic transmission by acting either at presynaptic or at postsynaptic sites. The precise localization of kainate receptors as well as the mechanisms of targeting and stabilization of these receptors in neurons are largely unknown. We have generated transgenic mice expressing the kainate receptor subunit glutamate receptor 6 (GluR6) bearing an extracellular myc epitope (myc-GluR6), in forebrain neurons, in which it assembles with endogenous kainate receptor subunits. In transgenic mice crossed with GluR6-deficient mice, myc-GluR6 efficiently rescues the missing subunit. Immunoprecipitation of transgenic brain extracts with anti-myc antibodies demonstrates an interaction with cadherins, beta-catenin, and p120 catenin, as well as with the associated proteins calcium calmodulin-dependent serine kinase and Velis, but not with alpha-catenin. In glutathione S-transferase-pulldown experiments, beta-catenin interacts, although indirectly, with the last 14 aa of GluR6. Transfected myc-GluR6 colocalizes with beta-catenin at cell-cell junctions in non-neuronal cells. Finally, activation of N-cadherins by ligand-covered latex beads recruits GluR6 to cadherin/catenin complexes. These results suggest an important role for cadherin/catenin complexes in the stabilization of kainate receptors at the synaptic membrane during synapse formation and remodeling.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;15;6426-36

  • E- and N-cadherin distribution in developing and functional human teeth under normal and pathological conditions.

    Heymann R, About I, Lendahl U, Franquin JC, Obrink B and Mitsiadis TA

    Faculté d'Odontologie de Marseille, Université de la Méditerranée, Marseille, France.

    Cadherins are calcium-dependent cell adhesion molecules involved in the regulation of various biological processes such as cell recognition, intercellular communication, cell fate, cell polarity, boundary formation, and morphogenesis. Although previous studies have shown E-cadherin expression during rodent or human odontogenesis, there is no equivalent study available on N-cadherin expression in dental tissues. Here we examined and compared the expression patterns of E- and N-cadherins in both embryonic and adult (healthy, injured, carious) human teeth. Both proteins were expressed in the developing teeth during the cap and bell stages. E-cadherin expression in dental epithelium followed an apical-coronal gradient that was opposite to that observed for N-cadherin. E-cadherin was distributed in proliferating cells of the inner and outer enamel epithelia but not in differentiated cells such as ameloblasts, whereas N-cadherin expression was up-regulated in differentiated epithelial cells. By contrast to E-cadherin, N-cadherin was also expressed in mesenchymal cells that differentiate into odontoblasts and produce the hard tissue matrix of dentin. Although N-cadherin was not detected in permanent intact teeth, it was re-expressed during dentin repair processes in odontoblasts surrounding carious or traumatic sites. Similarly, N-cadherin re-expression was seen in vitro, in cultured primary pulp cells that differentiate into odontoblast-like cells. Taken together these results suggest that E- and N-cadherins may play a role during human tooth development and, moreover, indicate that N-cadherin is important for odontoblast function in normal development and under pathological conditions.

    The American journal of pathology 2002;160;6;2123-33

  • The Erbin PDZ domain binds with high affinity and specificity to the carboxyl termini of delta-catenin and ARVCF.

    Laura RP, Witt AS, Held HA, Gerstner R, Deshayes K, Koehler MF, Kosik KS, Sidhu SS and Lasky LA

    Department of Molecular Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

    Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: delta-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or delta-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and delta-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or delta-catenin complexed with beta-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherin-catenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and delta-catenin. Finally, we showed that endogenous delta-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that delta-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.

    The Journal of biological chemistry 2002;277;15;12906-14

  • Densin-180 interacts with delta-catenin/neural plakophilin-related armadillo repeat protein at synapses.

    Izawa I, Nishizawa M, Ohtakara K and Inagaki M

    Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464-8681, Japan.

    Densin-180, a protein purified from the postsynaptic density fraction of the rat forebrain, is the founding member of a newly described family of proteins termed the LAP (leucine-rich repeats and PSD-95/Dlg-A/ZO-1 (PDZ) domains) family that plays essential roles in establishment of cell polarity. To identify Densin-180-binding proteins, we screened a yeast two-hybrid library using the carboxyl-terminal fragment of Densin-180 containing PDZ domain as bait, and we isolated delta-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) as a Densin-180-interacting protein. delta-catenin/NPRAP, a member of the armadillo repeat family, is a nervous system-specific adherens junction protein originally discovered as an interactor with presenilin-1, a protein involved in Alzheimer's disease. Densin-180 PDZ domain binds the COOH terminus of delta-catenin/NPRAP containing the PDZ domain-binding sequence. Endogenous Densin-180 was co-immunoprecipitated with delta-catenin/NPRAP and N-cadherin. Although Densin-180 was reported to be a transmembrane protein, Densin-180 was not accessible to surface biotinylation in dissociated hippocampal neurons; hence Densin-180 may be a cytosolic protein. Densin-180 co-localized with delta-catenin/NPRAP at synapses in delta-catenin/NPRAP and may be involved in organization of the synaptic cell-cell junction through interaction with the delta-catenin/NPRAP-N-cadherin complex.

    The Journal of biological chemistry 2002;277;7;5345-50

  • Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

    Niessen CM and Gumbiner BM

    Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

    Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.

    Funded by: NCPDCID CDC HHS: NCI-P30-CA-08784; NIGMS NIH HHS: GM52717, R01 GM052717

    The Journal of cell biology 2002;156;2;389-399

  • Carbohydrate moieties of N-cadherin from human melanoma cell lines.

    Ciołczyk-Wierzbicka D, Gil D, Hoja-Łukowicz D, Lityńska A and Laidler P

    Institute of Medical Biochemistry, Medical College, Jagiellonian University, Kraków, Poland. mbciolcz@cyf-kr.edu.pl

    Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with alpha-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with alpha2-3 sialic acid (positive reaction with Maackia amurensis agglutinin--MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.

    Acta biochimica Polonica 2002;49;4;991-8

  • Creation of genome-wide protein expression libraries using random activation of gene expression.

    Harrington JJ, Sherf B, Rundlett S, Jackson PD, Perry R, Cain S, Leventhal C, Thornton M, Ramachandran R, Whittington J, Lerner L, Costanzo D, McElligott K, Boozer S, Mays R, Smith E, Veloso N, Klika A, Hess J, Cothren K, Lo K, Offenbacher J, Danzig J and Ducar M

    Athersys, Inc., 3201 Carnegie Ave., Cleveland, OH 44115, USA. jharrington@athersys.com

    Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.

    Nature biotechnology 2001;19;5;440-5

  • Essential tyrosine residues for interaction of the non-receptor protein-tyrosine phosphatase PTP1B with N-cadherin.

    Rhee J, Lilien J and Balsamo J

    Department of Biological Sciences, The University of Iowa, Iowa City, Iowa 52242-1342, USA.

    Expression of a dominant-negative, catalytically inactive form of the nonreceptor protein-tyrosine phosphatase PTP1B in L-cells constitutively expressing N-cadherin results in loss of N-cadherin-mediated cell-cell adhesion. PTP1B interacts directly with the cytoplasmic domain of N-cadherin, and this association is regulated by phosphorylation of tyrosine residues in PTP1B. The following three tyrosine residues in PTP1B are potential substrates for tyrosine kinases: Tyr-66, Tyr-152, and Tyr-153. To determine the tyrosine residue(s) that are crucial for the cadherin-PTP1B interaction we used site-directed mutagenesis to create catalytically inactive PTP1B constructs bearing additional single, double, or triple mutations in which tyrosine was substituted by phenylalanine. Mutation Y152F eliminates binding to N-cadherin in vitro, whereas mutations Y66F and Y153F do not. Overexpression of the catalytically inactive PTP1B with the Y152F mutation in L-cells constitutively expressing N-cadherin has no effect on N-cadherin-mediated adhesion, and immunoprecipitation reveals that the mutant Y152F PTP1B does not associate with N-cadherin in situ. Furthermore, among cells overexpressing the Y152F mutant endogenous PTP1B associates with N-cadherin and is tyrosine-phosphorylated.

    Funded by: NEI NIH HHS: EY12132

    The Journal of biological chemistry 2001;276;9;6640-4

  • Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release.

    Meigs TE, Fields TA, McKee DD and Casey PJ

    Departments of Pharmacology and Cancer Biology, and Pathology, Duke University Medical Center, Durham, NC 27710, USA.

    The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis. In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12. Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins. Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins. Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation. These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation.

    Funded by: NIGMS NIH HHS: GM 55717, R01 GM055717

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;2;519-24

  • The armadillo repeat region targets ARVCF to cadherin-based cellular junctions.

    Kaufmann U, Zuppinger C, Waibler Z, Rudiger M, Urbich C, Martin B, Jockusch BM, Eppenberger H and Starzinski-Powitz A

    Institut der Anthropologie und Humangenetik fuer Biologen, Johann-Wolfgang-Goethe-Universitaet Frankfurt, Siesmayerstrasse 70, D-60054 Frankfurt/Main, Germany.

    The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

    Journal of cell science 2000;113 ( Pt 22);4121-35

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • Functional cis-heterodimers of N- and R-cadherins.

    Shan WS, Tanaka H, Phillips GR, Arndt K, Yoshida M, Colman DR and Shapiro L

    Department of Biochemistry and Molecular Biology, Program in Cell Adhesion, The Mount Sinai School of Medicine, New York University, New York 10029, USA.

    Classical cadherins form parallel cis-dimers that emanate from a single cell surface. It is thought that the cis-dimeric form is active in cell-cell adhesion, whereas cadherin monomers are likely to be inactive. Currently, cis-dimers have been shown to exist only between cadherins of the same type. Here, we show the specific formation of cis-heterodimers between N- and R-cadherins. E-cadherin cannot participate in these complexes. Cells coexpressing N- and R-cadherins show homophilic adhesion in which these proteins coassociate at cell-cell interfaces. We performed site- directed mutagenesis studies, the results of which support the strand dimer model for cis-dimerization. Furthermore, we show that when N- and R-cadherins are coexpressed in neurons in vitro, the two cadherins colocalize at certain neural synapses, implying biological relevance for these complexes. The present study provides a novel paradigm for cadherin interaction whereby selective cis-heterodimer formation may generate new functional units to mediate cell-cell adhesion.

    Funded by: NINDS NIH HHS: NS 20147, R01 NS020147

    The Journal of cell biology 2000;148;3;579-90

  • Expression of beta1-integrins and N-cadherin in bladder cancer and melanoma cell lines.

    Laidler P, Gil D, Pituch-Noworolska A, Ciołczyk D, Ksiazek D, Przybyło M and Lityńska A

    Institute of Medical Biochemistry, Collegium Medicum, Jagiellonian University, Kraków, Poland. mblaidler@cyf-kr.edu.pl

    Changes in the expression of integrins and cadherins might contribute to the progression, invasion and metastasis of transitional cell cancer of the bladder and of melanomas. The expression of alpha5 (P < 0.001), alpha2 and beta1 (P < 0.05 - P < 0.001) integrin subunits in melanoma cells from noncutaneous metastatic sites (WM9, A375) were significantly increased as compared to cutaneous primary tumor (WM35) and metastatic (WM239) cell lines. These differences might be ascribed to the invasive character of melanoma cells and their metastasis to the noncutaneous locations. The significantly heterogeneous expression of beta1 integrin subunit in two malignant bladder cancer cell lines (T24 and Hu456) and nonsignificant differences in the expression of alpha2, alpha3, and alpha5 subunits between malignant and non-malignant human bladder cell lines do not allow an unanimous conclusion on the role of these intergrin subunits in the progression of transitional cancer of bladder. The adhesion molecule, expressed in all studied melanoma and bladder cell lines, that reacted with anti-Pan cadherin monoclonal antibodies was identified as N-cadherin except in the HCV29 non-malignant ureter cell line. However, neither this nor any other bladder or melanoma cell line expressed E-cadherin. The obtained results imply that the replacement of E-cadherin by N-cadherin accompanied by a simultaneous increase in expression of alpha2, alpha3 and alpha5 integrin subunits clearly indicates an increase of invasiveness of melanoma and, to a lesser extent, of transitional cell cancer of bladder. High expression of N-cadherin and alpha5 integrin subunit seems to be associated with the most invasive melanoma phenotype.

    Acta biochimica Polonica 2000;47;4;1159-70

  • Follicular atresia and luteolysis. Evidence of a role for N-cadherin.

    Makrigiannakis A, Coukos G, Blaschuk O and Coutifaris C

    Division of Human Reproduction, University of Pennsylvania Medical Center, Philadelphia 19104, USA.

    Studies suggest that cell-cell interactions may regulate apoptosis; and, in particular, the calcium-dependent cell adhesion molecule N-cadherin has been shown to be capable of modulating this process. Here, we review the evidence that N-cadherin is expressed by human granulosa cells (GCs) and mediates cell-cell adhesion between GCs. There is strong correlation between N-cadherin expression by granulosa or luteal cells and follicular survival in isolated follicles and archival tissue sections. There exists a strong expression of the molecule by GCs in follicles of the resting pool, of growing antral follicles, and of healthy corpora lutea. In contrast, the molecule is lost in degenerating GCs of atretic follicles and in luteal cells of the late luteal phase. Further, the experimental evidence demonstrates that cell-cell adhesion is critical to the survival of GCs and that N-cadherin-mediated cell-cell adhesion is a critical mediator of survival signals and inhibits apoptosis in these cells. Possible mechanisms by which apoptosis may be triggerred in GCs include the downregulation of N-cadherin, which is mediated, at least in part, through the enzymatic cleavage of the extracellular domain of the molecule. Collectively, these observations suggest that downregulation of N-cadherin or the absence of a functional extracellular domain of the molecule prevent GC aggregation and is associated with GC apoptosis. We propose that N-cadherin-mediated GC signaling plays a central role in follicular and luteal cell survival.

    Funded by: NICHD NIH HHS: HD31903

    Annals of the New York Academy of Sciences 2000;900;46-55

  • delta-catenin, an adhesive junction-associated protein which promotes cell scattering.

    Lu Q, Paredes M, Medina M, Zhou J, Cavallo R, Peifer M, Orecchio L and Kosik KS

    Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The classical adherens junction that holds epithelial cells together consists of a protein complex in which members of the cadherin family linked to various catenins are the principal components. delta-catenin is a mammalian brain protein in the Armadillo repeat superfamily with sequence similarity to the adherens junction protein p120(ctn). We found that delta-catenin can be immunoprecipitated as a complex with other components of the adherens junction, including cadherin and beta-catenin, from transfected cells and brain. The interaction with cadherin involves direct contact within the highly conserved juxtamembrane region of the COOH terminus, where p120(ctn) also binds. In developing mouse brain, staining with delta-catenin antibodies is prominent towards the apical boundary of the neuroepithelial cells in the ventricular zone. When transfected into Madin-Darby canine kidney (MDCK) epithelial cells delta-catenin colocalized with cadherin, p120(ctn), and beta-catenin. The Arm domain alone was sufficient for achieving localization and coimmunoprecipitation with cadherin. The ectopic expression of delta-catenin in MDCK cells altered their morphology, induced the elaboration of lamellipodia, interfered with monolayer formation, and increased scattering in response to hepatocyte growth factor treatment. We propose that delta-catenin can regulate adhesion molecules to implement the organization of large cellular arrays necessary for tissue morphogenesis.

    Funded by: NIA NIH HHS: AG06601; NIGMS NIH HHS: GM47857, R01 GM047857

    The Journal of cell biology 1999;144;3;519-32

  • Localization of human cadherin genes to chromosome regions exhibiting cancer-related loss of heterozygosity.

    Kremmidiotis G, Baker E, Crawford J, Eyre HJ, Nahmias J and Callen DF

    Department of Cytogenetics and Molecular Genetics, Women's and Children's Hospital, North Adelaide, South Australia, Australia.

    This report presents the chromosomal localization of cadherin genes. Cadherins are cellular adhesion molecules. Since disturbance of intracellular adhesion is important for invasion and metastasis of tumor cells, cadherins are considered prime candidates for tumor suppressor genes. A variety of solid tumors show loss of heterozygosity of the long arm of chromosome 16, which is indicative of the potential location of tumor suppressor genes. Refined and new localizations of six cadherin genes (CDH3, 5, 8, 11, 13, and 15) to the long arm of chromosome 16 are shown. CDH15 was localized to 16q24.3, in a region that exhibits loss of heterozygosity in a number of sporadic breast cancer tumors. Previous localization of CDH13 (H-cadherin) to 16q24 suggested this gene as a tumor suppressor candidate in the 16q24.3 loss of heterozygosity region; however, refined mapping presented in this report localizes CDH13 proximal to this region. A human EST homologous to the chicken cadherin-7 was partially sequenced and found to represent a new human cadherin. This cadherin mapped to chromosome 18q22-q23, a region that exhibits loss of heterozygosity in head and neck squamous cell carcinomas. CDH16 was localized to 8q22.1, a region exhibiting loss of heterozygosity in adult acute myeloid leukemia.

    Genomics 1998;49;3;467-71

  • Differential localization of VE- and N-cadherins in human endothelial cells: VE-cadherin competes with N-cadherin for junctional localization.

    Navarro P, Ruco L and Dejana E

    Laboratory of Vascular Biology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy. pilar@irfmn.mnegri.it

    The two major cadherins of endothelial cells are neural (N)-cadherin and vascular endothelial (VE)- cadherin. Despite similar level of protein expression only VE-cadherin is located at cell-cell contacts, whereas N-cadherin is distributed over the whole cell membrane. Cotransfection of VE-cadherin and N-cadherin in CHO cells resulted in the same distribution as that observed in endothelial cells indicating that the behavior of the two cadherins was not cell specific but related to their structural characteristics. Similar amounts of alpha- and beta-catenins and plakoglobin were associated to VE- and N-cadherins, whereas p120 was higher in the VE-cadherin complex. The presence of VE-cadherin did not affect N-cadherin homotypic adhesive properties or its capacity to localize at junctions when cotransfectants were cocultured with cells transfected with N-cadherin only. To define the molecular domain responsible for the VE-cadherin-dominant activity we prepared a chimeric construct formed by VE-cadherin extracellular region linked to N-cadherin intracellular domain. The chimera lost the capacity to exclude N-cadherin from junctions indicating that the extracellular domain of VE-cadherin alone is not sufficient for the preferential localization of the molecule at the junctions. A truncated mutant of VE-cadherin retaining the full extracellular domain and a short cytoplasmic tail (Arg621-Pro702) lacking the catenin-binding region was able to exclude N-cadherin from junctions. This indicates that the Arg621-Pro702 sequence in the VE-cadherin cytoplasmic tail is required for N-cadherin exclusion from junctions. Competition between cadherins for their clustering at intercellular junctions in the same cell has never been described before. We speculate that, in the endothelium, VE- and N-cadherin play different roles; whereas VE-cadherin mostly promotes the homotypic interaction between endothelial cells, N-cadherin may be responsible for the anchorage of the endothelium to other surrounding cell types expressing N-cadherin such as vascular smooth muscle cells or pericytes.

    The Journal of cell biology 1998;140;6;1475-84

  • Distribution of N-cadherin and NCAM in neurons and endocrine cells of the human embryonic and fetal gastroenteropancreatic system.

    Gaidar YA, Lepekhin EA, Sheichetova GA and Witt M

    Ukrainian Research Institute for Gastroenterology, Dniepropetrovsk.

    For the first time, the distribution of N-cadherin and neural cell adhesion molecule (NCAM) as well as some neuropeptides in nerve cells and endocrine cells of the human embryonic and fetal gastroenteropancreatic system has been detected in early stages (from the 6th postovulatory week onwards). Epithelial cells of the stomach and small intestine contained gastrin and somatostatin and the epithelium of the small intestine also bombesin-positive cells. Myenteric ganglionic cells showed both bombesin and VIP and were NCAM- and N-cadherin-positive at all ages studied. Some basally granulated epithelial cells of stomach, duodenum and the upper part of jejunum contained N-cadherin. The number of these cells increased from 6th to 10th postovulatory weeks. Nerve cells and the cytoplasm of individual epithelial cells of pancreatic ducts were immunoreactive for NCAM and N-cadherin. NCAM- and N-cadherin-positive cells also appeared in Langerhans islets (> 10 weeks), mainly in their peripheral part. NCAM- and N-cadherin-positive endocrine cells were less numerous than endocrine cells producing somatostatin, bombesin, and VIP, probably reflecting the features of embryonic/fetal histogenesis of Langerhans islets from epithelial endocrine cells of pancreatic ducts. NCAM and N-cadherin were localized on the surface of endocrine islets cells as well as in the cytoplasm of single islet cells. This suggests the involvement of both membrane and soluble forms of adhesion proteins in embryonic/fetal histogenesis of human pancreatic islets. The early occurrence of N-cadherin (6th postovulatory week) in enteroendocrine cells supports the existence of a common precursor. The expression of NCAM and N-cadherin in nerve cells and endocrine cells of the human fetal gastroenteropancreatic system may indicate the involvement of neuronal adhesion mechanisms in the development of neuro-endocrine complexes of fetal stomach, small intestine and pancreas.

    Acta histochemica 1998;100;1;83-97

  • Multiple cadherins are expressed in human fibroblasts.

    Matsuyoshi N and Imamura S

    Department of Dermatology, Graduate School of Medicine, Kyoto University, Japan.

    Although the cell-cell adhesiveness of fibroblasts is thought to be related to wound healing, the molecular basis of this adhesiveness is still unknown. We isolated five kinds of cadherin fragments from the cDNA of human fibroblasts by polymerase chain reaction (PCR). Two of the five were known cadherins: PC43, a protocadherin containing six cadherin repeats in the extracelluar domain, and human Fat, which is the human homologue of the Drosophila tumor suppressor Fat. The other three were novel cadherin fragments, and we named them cadherins FIB1, FIB2, and FIB3. The expressions of cadherins including E-, P-, and N-cadherin, PC43, human Fat, and cadherins FIB1, FIB2, and FIB3 were compared in human fibroblasts, human melanocytes, and human epidermal keratinocytes. The latter six cadherins were expressed in human fibroblasts, and cadherins FIB1 and FIB2 were fibroblast-specific. These results suggest that diverse cadherin molecules may contribute to the cell-cell adhesion in human fibroblasts.

    Biochemical and biophysical research communications 1997;235;2;355-8

  • Vascular endothelial cadherin (VE-cadherin): cloning and role in endothelial cell-cell adhesion.

    Ali J, Liao F, Martens E and Muller WA

    Department of Cell Physiology and Immunology, Rockefeller University, New York, USA.

    Objective: To identify proteins responsible for intercellular junction integrity in human umbilical vein endothelial cells (HUVEC), we produced a monoclonal antibody that recognized an endothelial cell-specific, junctionally restricted protein. We characterized and cloned the antigen to study its functional properties.

    Methods: The size and cellular distribution of the antigen were determined by immunofluorescence and immunoprecipitation. The molecule was cloned and transfected into cell lines, and its role in cell-cell adhesion and growth rate was determined.

    Results: Monoclonal antibody hec1 recognizes VE-cadherin, an endothelial cell-restricted cell adhesion molecule. VE-cadherin is localized to the borders between apposing endothelial cells but is diffusely distributed on subconfluent or migrating cells. Transfection of fibroblasts with VE-cadherin imparts to them the ability to adhere to each other in a calcium-dependent homophilic manner. Expression of VE-cadherin over a several-log range does not change the growth rate of these cells.

    Conclusions: Despite the fact that VE-cadherin is a "nonclassical" cadherin by structure, it functions as a classic cadherin by imparting to cells the ability to adhere in a calcium-dependent, homophilic manner. On HUVEC it appears to play a role in maintaining monolayer integrity.

    Funded by: NHLBI NIH HHS: HL09393, HL46849

    Microcirculation (New York, N.Y. : 1994) 1997;4;2;267-77

  • Shared cell adhesion molecule (CAM) homology domains point to CAMs signalling via FGF receptors.

    Doherty P, Smith P and Walsh FS

    Department of Experimental Pathology, UMDS, Guy's Hospital, London, UK.

    A number of cell adhesion molecules (CAMs) promote neurite outgrowth following transfection and expression in a variety of monolayer cells. We have shown that N-cadherin, L1 and some isoforms of NCAM can stimulate neurite outgrowth from PC12 cells and primary neurons following transfection and expression at physiologically relevant levels in NIH-3T3 cells. A number of observations suggest that these CAMs stimulate neurite outgrowth by activating a convergent second messenger pathway in neurons rather than by modulating adhesion per se, and that an early or initial step in the pathway involves activation of a tyrosine kinase. The observation that the fibroblast growth factor receptor (FGFR) contains an evolutionarily conserved sequence with homology to the above CAMs (the CAM homology domain-CHD) points to the possibility that CAMs might interact with, and signal via, FGFR tyrosine kinases. This hypothesis has been substantiated by a number of independent experimental tests. We present a speculative model in which the evolutionary conservation of a pair of complementary binding motifs can account for a direct binding interaction between FGFR and the above three CAMs.

    Perspectives on developmental neurobiology 1996;4;2-3;157-68

  • Identification of plakoglobin domains required for association with N-cadherin and alpha-catenin.

    Sacco PA, McGranahan TM, Wheelock MJ and Johnson KR

    Department of Biology, University of Toledo, Ohio 43606, USA.

    Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins, alpha-catenin, beta-catenin, and gamma-catenin (also known as plakoglobin), have been identified. The domain of the cadherin molecule important for its interaction with the catenins has been mapped to the COOH-terminal 70 amino acids, but less is known about regions of the catenins that allow them to associate with one another or with the cadherin molecule. In this study we have transfected carboxyl-terminal deletions of plakoglobin into the human fibrosarcoma HT-1080 and used immunofluorescence localization and co-immunoprecipitation to map the regions of plakoglobin that allow it to associate with N-cadherin and with alpha-catenin. Plakoglobin is an armadillo family member containing 13 weakly similar internal repeats. These data show that the alpha-catenin-binding region maps within the first repeat and the N-cadherin-binding region maps within repeats 7 and 8.

    Funded by: NIGMS NIH HHS: GM51188

    The Journal of biological chemistry 1995;270;34;20201-6

  • Expressed cadherin pseudogenes are localized to the critical region of the spinal muscular atrophy gene.

    Selig S, Bruno S, Scharf JM, Wang CH, Vitale E, Gilliam TC and Kunkel LM

    Howard Hughes Medical Institute, Children's Hospital, Boston, MA 02115, USA.

    Low-copy repeats have been associated with genomic rearrangements and have been implicated in the generation of mutations in several diseases. Here we characterize a subset of low-copy repeats in the spinal muscular atrophy (SMA) region in human chromosome 5q13. We show that this repeated sequence, named c41-cad, is a highly expressed pseudogene derived from an intact neuronal cadherin gene, Br-cadherin, situated on 5p13-14. Br-cadherin is expressed specifically in the brain, whereas the c41-cad transcripts are 10-15 times more abundant and are present in all tissues examined. We speculate that the c41-cad repeats, separately or in concert with other repeats in the SMA region, are involved in the pathogenesis of SMA by promoting rearrangements and deletions.

    Funded by: NINDS NIH HHS: NS23740, NS28877

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;9;3702-6

  • E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton.

    Hülsken J, Birchmeier W and Behrens J

    Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

    beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E-cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.

    The Journal of cell biology 1994;127;6 Pt 2;2061-9

  • Association of plakoglobin with APC, a tumor suppressor gene product, and its regulation by tyrosine phosphorylation.

    Shibata T, Gotoh M, Ochiai A and Hirohashi S

    Pathology Division, National Cancer Center Research Institute, Tokyo, Japan.

    Plakoglobin is a cytoplasmic protein localized in both adherens junctions and desmosomes. Little is known about its function, but it may play a role in maintaining cell junction integrity. A partly homologous protein, beta catenin, is localized mainly in adherens junctions and plays a key role in cell adhesion by associating with cadherins, a family of Ca2+ dependent cell-to-cell adhesion molecules. Recently the product of APC, a tumor suppressor gene, was found to associate with beta catenin. In this study we demonstrated that plakoglobin also associates with APC and that tyrosine phosphorylated plakoglobin associates with cadherins but not with APC. These results suggest that plakoglobin could play a role in mediating the signals of APC by mutual interaction and that this may be regulated by tyrosine phosphorylation.

    Biochemical and biophysical research communications 1994;203;1;519-22

  • Expression and localization of N- and E-cadherin in the human testis and epididymis.

    Andersson AM, Edvardsen K and Skakkebaek NE

    Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark.

    Cellular interactions in the testis and epididymis are an important prerequisite for spermatogenesis and sperm maturation, and involve a well-developed complex of intercellular junctions. Cadherins are cell surface proteins which mediate intercellular Ca(2+)-dependent adhesion and are believed to be fundamentally important for maintaining multicellular structures. In the present study we report the expression of a 135 kDa N-cadherin polypeptide in the human seminiferous epithelium by immunoblotting. The presence of N-cadherin was demonstrated by immunohistochemistry on the surface of spermatogonia and primary spermatocytes, and possibly also around some early spermatids, whereas late spermatids were always negative. Endothelial cells also stained for N-cadherin, whereas peritubular cells and Leydig cells did not. No expression of E-cadherin could be demonstrated in the human testis. In the human epididymis E-cadherin, but not N-cadherin, was expressed and localized to the surface of the principal epithelial cells as shown by immunohistochemistry. These observations indicate that cadherins play an important role in the organization of the seminiferous and epididymal epithelium.

    International journal of andrology 1994;17;4;174-80

  • Structure of the human N-cadherin gene: YAC analysis and fine chromosomal mapping to 18q11.2.

    Wallis J, Fox MF and Walsh FS

    Department of Experimental Pathology, UMDS, Guy's Hospital, London Bridge, United Kingdom.

    The cadherins are a large family of cell adhesion molecules involved in calcium-dependent recognition and adhesion events. We have used YAC analysis to determine the structure of the human N-cadherin gene. An 850-kb YAC was isolated and the entire N-cadherin gene mapped to a 250-kb region spanning three putative CpG islands. A PCR and cosmid subcloning strategy was used to define the boundaries for the 16 exons that compose the gene. These were shown to be not only highly conserved between mouse and human N-cadherin genes, but also similar to other cadherins. The first and second introns of the gene are large, a property conserved between the mouse and human genes. In situ hybridization with YAC DNA refined the map position of N-cadherin to human chromosome 18q11.2.

    Genomics 1994;22;1;172-9

  • Plakoglobin, or an 83-kD homologue distinct from beta-catenin, interacts with E-cadherin and N-cadherin.

    Knudsen KA and Wheelock MJ

    Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096.

    E- and N-cadherin are members of a family of calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, the transmembrane cadherins self-associate, while, intracellularly, they interact with the actin-based cytoskeleton. Several intracellular proteins, collectively termed catenins, have been noted to co-immunoprecipitate with E- and N-cadherin and are thought to be involved in linking the cadherins to the cytoskeleton. Two catenins have been identified recently: a 102-kD vinculin-like protein (alpha-catenin) and a 92-kD Drosophila armadillo/plakoglobin-like protein (beta-catenin). Here, we show that plakoglobin, or an 83-kD plakoglobin-like protein, co-immunoprecipitates and colocalizes with both E- and N-cadherin. The 83-kD protein is immunologically distinct from the 92-kD beta-catenin and, because of its molecular mass, likely represents the cadherin-associated protein called gamma-catenin. Thus, two different members of a plakoglobin family associate with N- and E-cadherin and, together with the 102-kD alpha-catenin, appear to participate in linking the cadherins to the actin-based cytoskeleton.

    Funded by: NCI NIH HHS: CA 44464; NIAMS NIH HHS: AR 37945

    The Journal of cell biology 1992;118;3;671-9

  • Extrajunctional distribution of N-cadherin in cultured human endothelial cells.

    Salomon D, Ayalon O, Patel-King R, Hynes RO and Geiger B

    Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.

    Human endothelial cells contain prominent Ca(2+)-dependent intercellular adherens-type junctions (AJ), which are associated, at their cytoplasmic surfaces, with actin, vinculin and plakoglobin. The transmembrane adhesion molecules present in these sites are members of the cadherin family, which are recognized by a pancadherin serum, directed against the conserved C terminus of these molecules. Immunoblotting analysis of cultured human endothelial cells using these antibodies revealed three immunoreactive bands with apparent molecular masses of 135, 130 and 120 kDa. Cloning and sequencing of the 135 kDa cadherin from an endothelial cDNA expression library indicated that this molecule is a typical cadherin, essentially identical to N-cadherin. Transfection of cDNA encoding this molecule into CHO cells resulted in the induction of AJ formation and an apparent epithelialization of the cells. Immunofluorescent labeling with antibodies to chicken N-cadherin indicated that the molecule is associated with intercellular junctions in the transfectants. In contrast, cultured human umbilical cord endothelial cells exhibited a largely diffuse N-cadherin labeling over the entire cell surface with only occasional enrichment in cell-cell junctions. Comparison of this pattern with the discrete junctional labeling obtained with the pan-cadherin antibody suggests that different cadherins, co-expressed in the same endothelial cells, may undergo differential surface distribution.

    Journal of cell science 1992;102 ( Pt 1);7-17

  • Human N-cadherin: nucleotide and deduced amino acid sequence.

    Reid RA and Hemperly JJ

    Becton Dickinson and Company Research Center, Research Triangle Park, NC 27709.

    Nucleic acids research 1990;18;19;5896

  • N-cadherin gene maps to human chromosome 18 and is not linked to the E-cadherin gene.

    Walsh FS, Barton CH, Putt W, Moore SE, Kelsell D, Spurr N and Goodfellow PN

    Department of Experimental Pathology, UMDS, Guy's Hospital, London, England.

    cDNA clones encoding the human N-cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe lambda N2. Comparison of the predicted protein sequences revealed greater than 91% homology between chick brain, mouse brain, and human muscle N-cadherin cDNAs over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated in the human muscle sequence. Immediately 3' of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp of 3' untranslated sequence followed. Northern analysis identified a number of major N-cadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb in C6 glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of 5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N-cadherin gene to be mapped to chromosome 18. This is distinct from the E-cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations.

    Funded by: Wellcome Trust

    Journal of neurochemistry 1990;55;3;805-12

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000033 G2C Homo sapiens Pocklington H2 Human orthologues of cluster 2 (mouse) from Pocklington et al (2006) 13
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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