Plakoglobin Gene Research Articles

CD8+ T Cell-Dependent Antitumor Activity InVivo of a Mass Spectrometry-Identified Neoepitope despite Undetectable CD8+ Immunogenicity InVitro.

Identification of neoepitopes that can control tumor growth invivo remains a challenge even 10 y after the first genomics-defined cancer neoepitopes were identified. In this study, we identify a neoepitope, resulting from a mutation in the junction plakoglobin (Jup) gene (chromosome 11), from the mouse colon cancer line MC38-FABF (C57BL/6). This neoepitope, Jup mutant (JupMUT), was detected during mass spectrometry of MHC class I-eluted peptides from the tumor. JupMUT has a predicted binding affinity of 564 nM for the Kb molecule and a higher predicted affinity of 82 nM for Db. However, whereas structural modeling of JupMUT and its unmutated counterpart Jup wild-type indicates that there are little conformational differences between the two epitopes bound to Db, large structural divergences are predicted between the two epitopes bound to Kb. Together with invitro binding data with RMA-S cells, these data suggest that Kb rather than Db is the relevant MHC class I molecule of JupMUT. Immunization of naive C57BL/6 mice with JupMUT elicits CD8-dependent tumor control of a MC38-FABF challenge. Despite the CD8 dependence of JupMUT-mediated tumor control invivo, CD8+ T cells from JupMUT-immunized mice do not produce higher levels of IFN-γ than do naive mice. The structural and immunological characteristics of JupMUT are substantially different from those of many other neoepitopes that have been shown to mediate tumor control.

Premature Termination Codon in 5' Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation.

Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5′-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5′-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5′-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5′ region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5′-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5′-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5′-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.

Lack of plakoglobin impairs integrity and wound healing in corneal epithelium in mice

We generated cornea-specific plakoglobin (Jup; junctional plakoglobin) knockout mice in order to investigate the function of plakoglobin on the maintenance of the homeostasis of corneal epithelium in mice. Cornea epithelium-specific conditional knockouts (JupCEΔ/CEΔ) (cKO) were obtained by breeding keratin12-Cre (Krt12-Cre) mice to Jup-floxed (Jupf/f) mice. Light and transmission electron microscopic and immunohistochemical analyses were carried out to determine consequence of the loss of plakoglobin on maintaining corneal epithelium integrity under mechanical stress, e.g., brushing and wound healing. Immunohistochemistry analysis demonstrated that, although Jup ablation did not affect BrdU incorporation, basal cell-like cells labeled for keratin 14 were ectopically present in the supra-basal layer in mutant corneal epithelium, suggestive of altered cell differentiation. Plakoglobin-deficient epithelium exhibits increased fragility against mechanical intervention when compared to wild-type controls under identical treatment. Closure of an epithelial defect was significantly delayed in JupCEΔ/CEΔ epithelium. Our findings indicate that the lack of plakoglobin significantly affects corneal epithelium differentiation, as well as its structural integrity. Plakoglobin is essential to the maintenance of the structure of the corneal epithelium and its wound healing.Homeostasis of the corneal epithelium is critical in exerting a barrier function to noxious external stimuli. This study shows that that plakoglobin, a member of the catenin protein family, is essential to the maintenance of homeostasis of the corneal epithelium in mice. Corneal epithelium-specific deletion of the plakoglobin gene impairs tissue integrity against mechanical intervention and also suppresses wound healing without affecting cell proliferation.

Clinical utility gene card for: arrhythmogenic right ventricular cardiomyopathy (ARVC).

Name of the disease (synonyms) Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable disease characterized by structural and functional abnormalities of the right ventricle (RV), with or without concomitant left ventricular (LV) disease. The diagnosis ARVC is made when a patient fulfils the recently revised criteria. Criteria encompass global and/or regional dysfunction and structural changes; repolarization abnormalities; depolarization and conduction abnormalities; arrhythmias; family history/the results of genetic testing; and tissue characterization by endomyocardial biopsy. Either localized or diffuse atrophy, with subsequent replacement by fibrous and fatty tissue mainly of the RV outflow tract, RV inflow tract and RV apex (‘triangle of dysplasia’) represent the histopathological characteristics of ARVC. Synonyms: arrhythmogenic right ventricular dysplasia (ARVD); arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D); arrhythmogenic cardiomyopathy (ACM); arrhythmogenic left-dominant cardiomyopathy; and arrhythmogenic LV cardiomyopathy (ALVC). OMIM# of the disease 107970; 600996; 602086; 602087; 604400; 604401; 607450; 609040; 610193; 610476; 611528. Name of the analyzed genes or DNA/chromosome segments (1) Cytoskeletal protein genes: (a) Desmin gene (DES), locus 2q35#; (b) Titin gene (TTN), locus 2q31.2#. (2) Nuclear envelope protein genes: (a) Lamin A/C gene (LMNA), locus 1q22#. (3) Desmosomal protein genes: (a) Desmocollin 2 gene (DSC2), locus 18q12.1; (b) Desmoglein 2 gene (DSG2), locus 18q12.1; (c) Desmoplakin gene (DSP), locus 6p24.3; (d) Junction plakoglobin gene (JUP), locus 17q21.2; (e) Plakophilin 2 gene (PKP2), locus 12p11.21. (4) Calcium/sodium-handling genes: (a) Phospholamban gene (PLN), locus 6q22.31#; (b) Ryanodine receptor 2 gene (RYR2), locus 1q43*. (5) Other genes: (a) Alpha-T-catenin (CTNNA3), locus 10q21.3#; (b) Transforming growth factor-β3 (TGFβ3), locus 14q24.3*; (c) Transmembrane protein 43 (TMEM43), locus 3p25.1. #Indicates gene not yet annotated as ARVC related in the OMIM database; *indicates the involvement of these genes is based on single publications and therefore controversial. OMIM# of the gene(s) (1) Cytoskeletal protein genes: (a) 125660 and (b) 188840#. (2) Nuclear envelope protein genes: (a) 150330#. (3) Desmosomal protein genes: (a) 125645; (b) 125671; (c) 125647; (d) 173325; and (e) 602861. (4) Calcium/sodium-handling genes: (a) 172405# and (b) 180902*. (5) Other genes: (a) 607667#; (b) 190230*; and (c) 612048. #Indicates gene not yet annotated as ARVC related in the OMIM database; *denotes the involvement of these genes is based on single publications and therefore controversial.

Aryl Hydrocarbon Receptor Negatively Regulates Expression of the Plakoglobin Gene (Jup)

Plakoglobin is an important component of intercellular junctions, including both desmosomes and adherens junctions, which is known as a tumor suppressor. Although mutations in the plakoglobin gene (Jup) and/or changes in its protein levels have been observed in various disease states, including cancer progression or cardiovascular defects, the information about endogenous or exogenous stimuli orchestrating Jup expression is limited. Here we show that the aryl hydrocarbon receptor (AhR) may regulate Jup expression in a cell-specific manner. We observed a significant suppressive effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a model toxic exogenous activator of the AhR signaling, on Jup expression in a variety of experimental models derived from rodent tissues, including contact-inhibited rat liver progenitor cells (where TCDD induces cell proliferation), rat and mouse hepatoma cell models (where TCDD inhibits cell cycle progression), cardiac cells derived from the mouse embryonic stem cells, or cardiomyocytes isolated from neonatal rat hearts. The small interfering RNA (siRNA)-mediated knockdown of AhR confirmed its role in both basal and TCDD-deregulated Jup expression. The analysis of genomic DNA located ~2.5kb upstream of rat Jup gene revealed a presence of evolutionarily conserved AhR binding motifs, which were confirmed upon their cloning into luciferase reporter construct. The siRNA-mediated knockdown of Jup expression affected both proliferation and attachment of liver progenitor cells. The present data indicate that the AhR may contribute to negative regulation of Jup gene expression in rodent cellular models, which may affect cell adherence and proliferation.

High Motility of Triple-negative Breast Cancer Cells Is Due to Repression of Plakoglobin Gene by Metastasis Modulator Protein SLUG

One of highly pathogenic breast cancer cell types are the triple negative (negative in the expression of estrogen, progesterone, and ERBB2 receptors) breast cancer cells. These cells are highly motile and metastatic and are characterized by high levels of the metastasis regulator protein SLUG. Using isogenic breast cancer cell systems we have shown here that high motility of these cells is directly correlated with the levels of the SLUG in these cells. Because epithelial/mesenchymal cell motility is known to be negatively regulated by the catenin protein plakoglobin, we postulated that the transcriptional repressor protein SLUG increases the motility of the aggressive breast cancer cells through the knockdown of the transcription of the plakoglobin gene. We found that SLUG inhibits the expression of plakoglobin gene directly in these cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high cancer cells elevated the levels of plakoglobin. Blocking of SLUG function with a double-stranded DNA decoy that competes with the E2-box binding of SLUG also increased the levels of plakoglobin mRNA, protein, and promoter activity in the SLUG-high triple negative breast cancer cells. Overexpression of SLUG in the SLUG-deficient cells elevated the motility of these cells. Knockdown of plakoglobin in these low motility non-invasive breast cancer cells rearranged the actin filaments and increased the motility of these cells. Forced expression of plakoglobin in SLUG-high cells had the reverse effects on cellular motility. This study thus implicates SLUG-induced repression of plakoglobin as a motility determinant in highly disseminating breast cancer.

Loss of plakoglobin promotes decreased cell-cell contact, increased invasion, and breast cancer cell dissemination in vivo

IntroductionThe majority of deaths from breast cancer are a result of metastases; however, little is understood about the genetic alterations underlying their onset. Genetic profiling has identified the adhesion molecule plakoglobin as being three-fold reduced in expression in primary breast tumors that have metastasized compared with nonmetastatic tumors. In this study, we demonstrate a functional role for plakoglobin in the shedding of tumor cells from the primary site into the circulation.MethodsWe investigated the effects of plakoglobin knockdown on breast cancer cell proliferation, migration, adhesion, and invasion in vitro and on tumor growth and intravasation in vivo. MCF7 and T47D cells were stably transfected with miRNA sequences targeting the plakoglobin gene, or scramble vector. Gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Cell proliferation, adhesion, migration, and invasion were measured by cell counting, flow cytometry, and scratch and Boyden Chamber assays. For in vivo experiments, plakoglobin knockdown and control cells were inoculated into mammary fat pads of mice, and tumor growth, shedding of tumor cells into the bloodstream, and evidence of metastatic bone lesions were monitored with caliper measurement, flow cytometry, and microcomputed tomography (μCT), respectively.ResultsPlakoglobin and γ-catenin expression were reduced by more than 80% in all knockdown cell lines used but were unaltered after transfection with the scrambled sequence. Reduced plakoglobin resulted in significantly increased in MCF7 and T47D cell proliferation in vitro and in vivo, compared with control, with significantly more tumor cells being shed into the bloodstream of mice bearing plakoglobin knockdown tumors. In addition, plakoglobin knockdown cells showed a >250% increase in invasion through basement membrane and exhibited reduced cell-to-cell adhesion compared with control cells.ConclusionDecreased plakoglobin expression increases the invasive behavior of breast cancer cells. This is the first demonstration of a functional role for plakoglobin/γ-catenin in the metastatic process, indicating that this molecule may represent a target for antimetastatic therapies.

Analysis of a Jup hypomorphic allele reveals a critical threshold for postnatal viability

Mutations in the human Jup gene cause arrhythmogenic right ventricular cardiomyopathy (ARVC), a heart muscle disease that often leads to sudden cardiac death. Inactivation of the murine Jup gene (also known as plakoglobin) results in embryonic lethality due to cardiac rupture. In an effort to generate a conditional knockout allele, a neomycin cassette was introduced into the murine plakoglobin (PG) gene. This allele (PG F(N)) functions as a hypomorph when combined with a null allele (PG Δ). About half of the PG F(N)/Δ animals were smaller than their littermates and died before weaning age, whereas the remaining PG F(N)/Δ animals survived. Despite the reduced levels of PG in the heart, there were no signs of cardiomyopathy or cardiac dysfunction as determined by echocardiography. Importantly, the PG homolog, β-catenin (CTNNB1), was increased in the PG F(N)/Δ hearts. In addition to its structural role as part of the N-cadherin/catenin adhesion complex, β-catenin is a downstream effector of Wnt signaling. However, no change in β-catenin/TCF reporter activity was observed in PG F(N)/Δ embryos suggesting that excess β-catenin was not likely causing increased transcription of Wnt/β-catenin target genes. These data suggest novel function(s) for PG beyond the heart and define a critical threshold of PG expression that is necessary for postnatal survival.

Abstract 1287: High mobility of triple-negative breast cancer cells is due to repression of plakoglobin gene by SLUG

Abstract One of highly pathogenic breast cancer cell types are the SLUG-high claudin-low triple negative (negative in the expression of estrogen, progesterone and ERBB2 receptors) breast cancer (TNBC) cells. These cells are highly metastatic and have low levels of the motility regulatory catenin plakoglobin. In order to link high levels of the transcriptional repressor SLUG in the TNBC cells with low levels of plakoglobin we found that SLUG inhibits the expression of plakoglobin gene directly in these cells and thus, among other downstream effects, help disseminating these tumor cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high cancer cells elevated the levels of plakoglobin. Overexpression of SLUG in the SLUG-deficient cells elevated the invasiveness and motility of these cells. On the other hand, knockdown of plakoglobin in these low motility non-invasive breast cancer cells did not affect the ability of the cancer cells to penetrate Matrigel matrix but increase the growth and migration rates of these cells. This study thus implicates high levels of SLUG and low levels of plakoglobin as determinants in the progression of highly disseminating breast cancer. Supported in parts by the DOD-CDMRP grants BC050641, BC086542 and BC103645 to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1287. doi:1538-7445.AM2012-1287

Evidence for genetic heterogeneity in Carvajal syndrome

Carvajal syndrome is a rare syndrome with woolly hair, palmoplantar keratosis and dilated cardiomyopathy. The inheritance of the mutation is autosomal recessive. As a causal gene, the desmoplakin gene (DSP) has so far been identified; it encodes an essential component of desmosomes, a cell-cell structure aimed at keeping cells attached to each other in tissues in which cells are often exposed to strong shear forces. Recently, familial cases of an autosomal dominant Carvajal syndrome were documented with a new feature: hypo/oligodontia. A mutation in the DSP gene was also evidenced in these latter cases. A patient was seen for cardiogenetic consultation at the University Hospital of Lyon with cardiac failure involving first degree atrioventricular block, complete left bundle branch block, non-compaction of the apex of the left ventricle and a dilated cardiomyopathy. A coronarography disclosed a complete thrombosis of the right coronary artery. At examination, he had also woolly hair, mild palmoplantar keratosis and missing teeth (essentially molars and premolars). His family history was uninformative. His DNA was screened for mutations in the DSP and plakoglobin genes but no mutation could be found. This case suggests that Carvajal syndrome with hypo/oligodontia is a heterogeneous condition in which genes other than DSP might be involved, although we cannot rule out a mutation in this gene consisting in a deletion of a single exon or a gene rearrangement.

P2-01-20: Reduced Plakoglobin Expression Increases the Metastatic Potential of Breast Cancer Cells.

Abstract The majority of deaths from breast cancer are a result of metastases, however, little is understood about the genetic alterations underlying their onset. A recent study of human breast cancer biopsies showed alterations of expression of 9 genes in primary tumours that had metastasized compared to benign tumours. Of these, plakoglobin was the most altered, with metastatic tumours showing a 3x decrease in expression compared to benign. Furthermore, expression of plakoglobin is reduced upon autocrine production of human growth hormone, which is associated with increased breast tumour cell invasion in vivo. Plakoglobin codes for the adhesion protein gamma catenin, an integral part of the cadherin-catenin complex involved in cell-to-cell adhesion. The aim of the current study is to investigate the effects of plakoglobin knockdown on cell proliferation, migration, adhesion and invasion in vitro and in vivo. The non-metastatic human breast cancer cell lines MCF7 and T47D that express high levels of plakoglobin and gamma-catenin were stably transfected with miRNA to two different regions of the plakoglobin gene, or scramble vector, to produce plakoglobin knockdown and controls for each cell line. Plakoglobin gene expression was monitored by qPCR and gamma-catenin expression by Western blot. Cell proliferation was assessed 24, 48, 72 and 96 hours post seeding; cell migration and invasion were measured over 24 and 48 hours using scratch and modified Boyden Chamber assays. Cell-cell adhesion was assessed by spheroid formation in agar matrix. For in vivo experiments, MCF7 plakoglobin knockdown and control cells were inoculated into the 5th and 10th mammary fat pads of 12-week old female balb/c mice (n=10 per group). Tumour growth was monitored by caliper measurements and local invasion was investigated 6 weeks after tumour inoculation on histological sections. Shedding of tumour cells into the blood stream and evidence of metastatic bone lesions was monitored by flow cytometry and μCT, respectively. Plakoglobin and gamma catenin expression were reduced by more than 80% in all knockdown cell lines used but were unaltered following transfection with the scrambled vector. Furthermore, knockdown of plakoglobin did not effect expression of its binding partner, e-cadherin. Reduced expression of plakoglobin resulted in a more than 3-fold increase in MCF7 and T47D cell proliferation in vitro (p<0.005) and a 2-fold increase in MCF-7 proliferation in vivo (P<0.05), compared with control. In addition plakoglobin knockdown cells showed a 6 - 15 fold increase in invasion through basement membrane (p<0.005), a 2-fold increase in migration (p<0.005) and exhibited reduced cell-to-cell adhesion compared with control cells. Data show that decreased expression of plakoglobin increases the early pro-metastatic behaviour of cells including increased cell proliferation, migration and invasion. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-20.

Abstract B35: SLUG-induced plakoglobin gene repression in triplenegative breast cancer cells

Abstract Breast cancer with the triple-negative phenotype (TNBC) are ER-negative, PR-negative and ERBB2 (HER2)-negative and represent one of the most aggressive and difficult to treat subtypes of human breast cancer. TNBC is highly over-represented in African American breast cancer patients and determining the genetic and molecular basis for this incidence and the biological basis for the aggressiveness of TNBCs are largely unknown and of high priority. The transcription factor SLUG, which controls epithelial to mesenchymal transition, stem cell phenotypes and therapeutic responsiveness, is highly expressed in the basal-type TNBC cells, making it a candidate master regulator of the TNBC phenotype. Plakoglobin, also known as γ-catenin or JUP, is a member of the armadillo motifcontaining proteins. This protein is a critical component of the desmosomal structure conferring structural integrity and resistance from mechanical stress to epithelial cells in tissues. We report here that SLUG inhibits the expression of several desmosomal proteins including the plakoglobin gene directly and thus, among other downstream effects, help disseminating the TNBC cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high TNBC cells elevated the levels of plakoglobin. Inhibition of SLUG activity with a molecular decoy in the TNBC cells abrogated the inhibitory effect of SLUG on plakoglobin gene expression. Although overexpression of SLUG in the SLUG-deficient cells elevated the invasiveness and motility of these cells, knockdown of plakoglobin only affected the growth and migration rates of these cells. This study thus implicates high levels of SLUG and low levels of plakoglobin as determinants in the progression of highly disseminating breast cancer of the TNBC type. Supported by the DOD-CDMRP BCRP Grants W81XWH-06-1-0466, W81XWH-08-1-0446, BC086542, and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B35.

Restrictive loss of plakoglobin in cardiomyocytes leads to arrhythmogenic cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable myocardial disorder associated with fibrofatty replacement of myocardium and ventricular arrhythmia. A subset of ARVC is categorized as Naxos disease, which is characterized by ARVC and a cutaneous disorder. A homozygous loss-of-function mutation of the Plakoglobin (Jup) gene, which encodes a major component of the desmosome and the adherens junction, had been identified in Naxos patients, although the underlying mechanism remained elusive. We generated Jup mutant mice by ablating Jup in cardiomyocytes. Jup mutant mice largely recapitulated the clinical manifestation of human ARVC: ventricular dilation and aneurysm, cardiac fibrosis, cardiac dysfunction and spontaneous ventricular arrhythmias. Ultra-structural analyses revealed that desmosomes were absent in Jup mutant myocardia, whereas adherens junctions and gap junctions were preserved. We found that ventricular arrhythmias were associated with progressive cardiomyopathy and fibrosis in Jup mutant hearts. Massive cell death contributed to the cardiomyocyte dropout in Jup mutant hearts. Despite the increase of β-catenin at adherens junctions in Jup mutant cardiomyoicytes, the Wnt/β-catenin-mediated signaling was not altered. Transforming growth factor-beta-mediated signaling was found significantly elevated in Jup mutant cardiomyocytes at the early stage of cardiomyopathy, suggesting an important pathogenic pathway for Jup-related ARVC. These findings have provided further insights for the pathogenesis of ARVC and potential therapeutic interventions.

Defining the Roles of .ALPHA.-Catenin in Cell Adhesion and Cytoskeleton Organization: Isolation of F9 Cells Completely Lacking Cadherin-catenin Complex

To define the roles of α-catenin in cell-cell adhesion, the E-cadherin, α-catenin, β-catenin, and/or plakoglobin genes were inactivated in F9 teratocarcinoma cells. An E-cadherin-α-catenin fusion protein (Eα) restored full cell-adhesion function and organized the actin-based cytoskeleton and ZO-1, an actin filament binding protein, in F9 cells lacking all endogenous cadherin-catenin complex components. There were two types of cadherin-based cell-adhesion junctions in parental F9 cells, those with ZO-1 and those without ZO-1, and only junctions with ZO-1 were associated with thick actin bundles. Additionally, ZO-1 localized to most Eα-based cell-adhesion junctions. These data demonstrated that Eα supported cadherin-based cell adhesion and recruited actin bundles and ZO-1 to cell-cell contact sites in the absence of cytoplasmic α-catenin. Moreover, the C-terminal half of α-catenin was involved in the formation of cell-adhesion junctions with ZO-1.

A New Hypo/Oligodontia Syndrome

Dental agenesis is either syndromic or non-syndromic. Here, we describe a familial case with Carvajal/Naxos syndrome associating woolly hair, palmoplantar keratoderma, and biventricular dilated cardiomyopathy. In addition to these signs, all three affected family members had hypo/oligodontia ranging from absence of the lower left second molar to 15 missing teeth, the typical pattern of oligodontia being absent 2nd premolars and absent 2nd and 3rd molars. Mutation screening in the desmoplakin gene (DSP) revealed a de novo missense mutation (c.1790 C>T, p.Ser597Leu) changing a serine residue conserved in all vertebrates. In addition, this variation was absent from 100 control DNA samples. There were no mutations in the plakoglobin gene. This familial case report and two other previous reports demonstrate that autosomal-dominant mutations in the DSP gene are associated with hypo/oligodontia in the setting of Carvajal/Naxos syndrome. This study suggests that dentists discovering oligo/hypodontia should screen for woolly hair and palmoplantar keratoderma because of the probable cardiac involvement with an inherent high risk of severe cardiomyopathy. In addition, this study reveals the role of desmosomes in the development of teeth and suggests that other genes encoding proteins of the desmosome could be involved in oligo/hypodontia.

Diagnostic value of integrin α3, β4, and β5 gene expression levels for the clinical outcome of tongue squamous cell carcinoma

The objective of the current study was to identify biomarkers that reflect the clinical course of squamous cell carcinoma of the tongue (TSCC). TSCC tissue samples from 66 patients were subjected to gene expression analysis by real-time polymerase chain reaction. Eleven integrin family genes and 14 genes used for normalization, including housekeeping genes and genes that encode desmosomal, cytoskeletal, and extracellular matrix molecules, were considered. Multivariate statistical analysis was performed on 154 expression ratios of integrin genes with clinical parameters. In principal-component analysis, the first principal component was related to the outcome of death, and the second principal component mainly reflected the tendency for cervical lymph node (LN) metastasis. The former axis consisted of the variance of the integrin beta4 gene (ITGB4) and ITGB5 expression levels, and the latter axis agreed with the expression level of the integrin alpha3 gene (ITGA3). Multivariate logistic regression analysis with cervical LN metastasis as the response variable concordantly identified ITGA3/junction plakoglobin gene (JUP) expression (P=.02) and ITGB5/paxillin gene (PXN) expression (P=.04) as significant factors. Only ITGB4/JUP expression was identified as a significant factor in terms of the outcome of death (P<.00049) by a Cox proportional hazards model. The group with high ITGB4/JUP levels exhibited a significantly high death rate on a Kaplan-Meier curve (P<.0001; Wilcoxon and log-rank tests). The expression levels of ITGA3, ITGB4, and ITGB5 with functional normalization by desmosomal or cytoskeletal molecule genes were selected as candidate biomarkers for cervical LN metastasis or for the outcome of death in TSCC.

Desmosomal gene evaluation in Boxers with arrhythmogenic right ventricular cardiomyopathy

To sequence the exonic and splice site regions of the 4 desmosomal genes associated with the human form of familial arrhythmogenic right ventricular cardiomyopathy (ARVC) in Boxers with ARVC and identify a causative mutation. 10 unrelated Boxers with ARVC and 2 unaffected Labrador Retrievers (control dogs). Exonic and splice site regions of the 4 genes encoding the desmosomal proteins plakophilin-2, plakoglobin, desmoplakin, and desmoglein-2 were sequenced. Sequences were compared for nucleotide sequence changes between affected dogs and the published sequences for clinically normal dogs and between affected dogs and the control dogs. Base-pair changes were considered to be causative for ARVC if they were detected in an affected dog but not in unaffected dogs, and if they involved a conserved amino acid and changed that amino acid to one of a different polarity, acid-base status, or structure. A causative mutation for ARVC in Boxers was not identified, although single nucleotide polymorphisms were detected in some affected dogs within exon 3 of the plakophilin-2 gene; exon 3 of the plakoglobin gene; exons 3 and 7 of the desmoglein-2 gene; and exons 6, 14, 15, and 24 of the desmoplakin gene. None of these changed the amino acid of the respective protein. Mutations within the desmosomal genes associated with the development of ARVC in humans do not appear to be causative for ARVC in Boxers. Genomewide scanning for genetic loci of interest in dogs should be pursued.

Magnetic Resonance Imaging Characteristics in Carvajal Syndrome (Variant of Naxos Disease)

Naxos disease is an autosomal recessively inherited familial syndrome that is characterized by woolly hair, palmoplantar keratoderma, and a cell adhesion cardiomyopathy, specifically an arrhythmogenic right ventricular dysplasia.1 This cardiocutaneous syndrome was first reported in the Hellenic island of Naxos. Cases also have been reported in other Hellenic islands, as well as in Turkey, Israel, and Saudi Arabia.2 Apart from a small minority that show woolly hair and a few ECG or echocardiographic abnormalities not fulfilling the criteria for arrhythmogenic right ventricular dysplasia/cardiomyopathy, heterozygotes display normal phenotype.3 A variant of Naxos disease, reported as Carvajal syndrome, has been described in families from India and Ecuador. Clinically, it presents with the same cutaneous phenotype and predominantly left ventricular involvement. A 2-bp deletion in the plakoglobin gene has been identified as the cause of Naxos disease. In Carvajal syndrome, 2 different mutations of the desmoplakin gene have been found as causative genes.3 These mutations in genes encoding desmosomal proteins cause defects in the linking sites of these proteins and consequently can interrupt the contiguous chain of cell adhesion, particularly under conditions of increased mechanical stress or stretch, leading to cell death, progressive loss of myocardium, and fibrofatty replacement. The affected members of both syndromes have woolly hair at birth, whereas palmoplantar keratoderma appears during the first year of life, when infants start using their extremities.3 In Naxos disease, the cardiomyopathy clinically manifests in adolescence. Patients may develop progressive heart disease involving the right or both ventricles. Symptoms of right heart failure are found in the final …

DNMT3A and DNMT3B mediate autocrine hGH repression of plakoglobin gene transcription and consequent phenotypic conversion of mammary carcinoma cells

Directed by microarray analyses, we report that autocrine human growth hormone (hGH) increased the mRNA and protein expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B in mammary carcinoma cells. Autocrine hGH stimulation of DNMT3A and DNMT3B expression was mediated by JAK2 and Src kinases, and treatment of mammary carcinoma cells with the DNMT inhibitor, 5'-aza-2'-deoxycytidine (AZA), abrogated autocrine hGH-stimulated cellular proliferation, apoptosis and anchorage-independent growth. AZA reversed the epitheliomesenchymal transition of mammary carcinoma cells induced by autocrine hGH, to an epithelioid morphology and abrogated cell migration stimulated by autocrine hGH. Autocrine hGH-stimulated hypermethylation of the first exon of the PLAKOGLOBIN gene and AZA abrogated the ability of autocrine hGH to repress plakoglobin gene transcription. Small interfering RNA (siRNA)-mediated depletion of the individual DNMT molecules did not release autocrine hGH repression of PLAKOGLOBIN promoter activity nor did individual DNMT depletion affect autocrine hGH-stimulated migration. However, concomitant siRNA-mediated depletion of both DNMT3A and DNMT3B abrogated hypermethylation of the PLAKOGLOBIN gene stimulated by autocrine hGH and subsequent repression of plakoglobin gene transcription and increased cell migration. Thus, the autocrine hGH-stimulated increases in DNMT3A and DNMT3B expression mediate repression of plakoglobin gene transcription by direct hypermethylation of its promoter and consequent phenotypic conversion of mammary carcinoma cells. Autocrine hGH, therefore, utilizes DNA methylation as a mechanism to exert its oncogenic effects in mammary carcinoma cells.

Case report of a Spanish patient with arrhythmogenic right ventricular cardiomyopathy and palmoplantar keratoderma without plakoglobin and desmoplakin gene modifications

We report a case of a 43 year old man from Spain, who has been diagnosed with Naxos disease. It is a hereditary disorder characterized by palmoplantar keratoderma, woolly hair and cardiomyopathy, which has been associated with a mutation in plakoglobin encoding gene in chromosome 17q21. In the patient, the direct sequencing of the plakoglobin gene discarded TG deletion at 2157 characteristic of Naxos disease. Analysis of the reported desmoplakin mutations associated with Carvajal Syndrome, another ARVC disease, that it is also accompanied with a skin and hair disorder, also failed to reveal mutations in desmoplakin gene. These results suggest the existence of other causative genes and/or other putative sites in desmoplakin/plakoglobin encoding genes than those recently published.