RESUMO
Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them acquisition of a fibroblastic-like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis. J. Cell. Physiol. 232: 1368-1386, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Processamento Alternativo/genética , Caderinas/genética , Transição Epitelial-Mesenquimal/genética , Adulto , Processamento Alternativo/efeitos dos fármacos , Sequência de Aminoácidos , Antígenos CD , Sequência de Bases , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/química , Caderinas/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Meios de Cultivo Condicionados/farmacologia , Epididimo/efeitos dos fármacos , Epididimo/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Humanos , Masculino , Modelos Biológicos , Invasividade Neoplásica , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
The establishment, remodeling and maintenance of tissular architecture during animal development, and even across juvenile to adult life, are deeply regulated by a delicate interplay of extracellular signals, cell membrane receptors and intracellular signal messengers. It is well known that cell adhesion molecules (cell-cell and cell-extracellular matrix) play a critical role in these processes. Particularly, adherens junctions (AJs) mediated by E-cadherin and catenins determine cell-cell contact survival and epithelia function. Consequently, this review seeks to encompass the complex and prolific knowledge about E-cadherin roles during physiological and pathological states, particularly focusing on the influence exerted by the thyroid hormone (TH).
Assuntos
Caderinas/metabolismo , Hormônios Tireóideos/metabolismo , Junções Aderentes/metabolismo , Animais , Caderinas/química , Caderinas/genética , Cateninas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Evolução Molecular , Transdução de SinaisRESUMO
Nonsyndromic orofacial cleft (NSOFC) is a complex disease of still unclear genetic etiology. To investigate the contribution of rare epithelial cadherin (CDH1) gene variants to NSOFC, we target sequenced 221 probands. Candidate variants were evaluated via in vitro, in silico, or segregation analyses. Three probably pathogenic variants (c.760G>A [p.Asp254Asn], c.1023T>G [p.Tyr341*], and c.2351G>A [p.Arg784His]) segregated according to autosomal dominant inheritance in four nonsyndromic cleft lip with or without cleft palate (NSCL/P) families (Lod score: 5.8 at θ = 0; 47% penetrance). A fourth possibly pathogenic variant (c.387+5G>A) was also found, but further functional analyses are needed (overall prevalence of CDH1 candidate variants: 2%; 15.4% among familial cases). CDH1 mutational burden was higher among probands from familial cases when compared to that of controls (P = 0.002). We concluded that CDH1 contributes to NSCL/P with mainly rare, moderately penetrant variants, and CDH1 haploinsufficiency is the likely etiological mechanism.
Assuntos
Encéfalo/anormalidades , Caderinas/genética , Fenda Labial/genética , Fissura Palatina/genética , Variação Genética , Alelos , Substituição de Aminoácidos , Animais , Antígenos CD , Caderinas/química , Linhagem Celular , Fenda Labial/diagnóstico , Fissura Palatina/diagnóstico , Análise Mutacional de DNA , Genótipo , Mutação em Linhagem Germinativa , Humanos , Mutação , Fases de Leitura Aberta , PenetrânciaRESUMO
Fertilization is a calcium-dependent process that involves sequential cell-cell adhesion events of spermatozoa with oviduct epithelial cells (OECs) and with cumulus-oocyte complexes (COCs). Epithelial cadherin (E-cadherin) participates in calcium-dependent somatic cell adhesion; the adaptor protein ß-catenin binds to the E-cadherin cytoplasmic domain and links the adhesion protein to the cytoskeleton. The study was conducted to immunodetect E-cadherin and ß-catenin in bovine gametes and oviduct (tissue sections and OEC monolayers), and to assess E-cadherin participation in fertilization-related events. Epithelial cadherin was found in spermatozoa, oocytes, cumulus cells, and OEC. In acrosome-intact noncapacitated spermatozoa, E-cadherin was mainly localized in the apical ridge and acrosomal cap (E1-pattern; 84 ± 9%; mean ± standard deviation of the mean). After sperm treatment with heparin to promote capacitation, the percentage of cells with E1-pattern (56 ± 12%) significantly decreased; concomitantly, the percentage of spermatozoa depicting an E-cadherin staining pattern similar to E1-pattern but showing a signal loss in the acrosomal cap (E2-pattern: 40 ± 11%) increased. After l-α-lysophosphatidylcholine-induced acrosome reaction, E-cadherin signal was mainly localized in the inner acrosomal membrane (E3-pattern: 67 ± 22%). In IVM COC, E-cadherin was immunodetected in the plasma membrane of cumulus cells and oocytes, but was absent in the polar body. The 120 KDa mature protein form was found in protein extracts from spermatozoa, oocytes, cumulus cells, and OEC. ß-Catenin distribution followed E-cadherin's in all cells evaluated. Epithelial cadherin participation in cell-cell interaction was evaluated using specific blocking monoclonal antibody DECMA-1. Sperm incubation with DECMA-1 impaired sperm-OEC binding (the number of sperm bound to OEC: DECMA-1 = 6.7 ± 6.1 vs. control = 29.6 ± 20.1; P < 0.001), fertilization with COC (% fertilized COC: DECMA-1 = 68.8 ± 10.4 vs. control = 90.7 ± 3.1; P < 0.05) or denuded oocytes (% fertilized oocytes: DECMA-1 = 57.0 ± 15.2 vs. control = 89.2 ± 9.8; P < 0.05) and binding to the oolemma (the number of sperm bound to oolemma: DECMA-1 = 2.2 ± 1.1 vs. control = 11.1 ± 4.8; P < 0.05). This study describes, for the first time, the presence of E-cadherin in bovine spermatozoa, COC, and OEC, and shows evidence of its participation in sperm interaction with the oviduct and the oocyte during fertilization.
Assuntos
Caderinas/metabolismo , Bovinos , Tubas Uterinas/citologia , Fertilização/fisiologia , Óvulo/metabolismo , Animais , Caderinas/química , Técnicas de Cocultura/veterinária , Tubas Uterinas/fisiologia , Feminino , Masculino , Óvulo/química , Espermatozoides/fisiologia , beta CateninaRESUMO
Cry proteins from Bacillus thuringiensis are insecticidal PFTs (pore-forming toxins). In the present study, we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin receptor, but before membrane insertion. Both pre-pores actively induce pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analysed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations that are resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore-formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target membrane. We propose that not only different insect targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected for in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data assign a functional role for the protoxin fragment of Cry PFTs that was not understood previously. Most PFTs produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of the general mechanism of action of other PFTs.
Assuntos
Proteínas de Bactérias/metabolismo , Caderinas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Caderinas/química , Membrana Celular , Endotoxinas/química , Ensaio de Imunoadsorção Enzimática , Proteínas Hemolisinas/química , Manduca/metabolismo , Microvilosidades , Ligação Proteica , Receptores de Superfície Celular , Tripsina/metabolismoRESUMO
Disruption/denudation of the ependymal lining has been associated with the pathogenesis of various human CNS disorders, including hydrocephalus, spina bifida aperta, and periventricular heterotopia. It has been traditionally considered that ependymal denudation is a consequence of mechanical forces such as ventricular enlargement. New evidence indicates that ependymal disruption can precede ventricular dilation, but the cellular and molecular mechanisms involved in the onset of ependymal denudation are unknown. Here, we present a novel model to study ependymal cell pathophysiology and demonstrate that selective disruption of N-cadherin-based adherens junctions is sufficient to provoke progressive ependymal denudation. Blocking N-cadherin function using specific peptides that interfere with the histidine-alanine-valine extracellular homophilic interaction domain caused early pathologic changes characterized by disruption of zonula adherens and abnormal intracellular accumulation of N-cadherin. These changes then triggered massive apoptosis of ependymal cells and denudation of brain ventricular walls. Because no typical extrinsic mechanical factors such as elevated pressure or stretching forces are involved in this model, the critical role of N-cadherin-based adherens junctions in ependymal survival/physiology is highlighted. Furthermore, the results suggest that abnormal adherens junctions between ependymal cells should be considered as key components of the pathogenesis of CNS disorders associated with ependymal denudation.
Assuntos
Junções Aderentes/metabolismo , Antígenos CD/metabolismo , Apoptose/fisiologia , Encéfalo/citologia , Caderinas/metabolismo , Epêndima/metabolismo , Junções Aderentes/efeitos dos fármacos , Análise de Variância , Animais , Anticorpos/farmacologia , Antígenos CD/química , Antígenos CD/imunologia , Apoptose/efeitos dos fármacos , Caderinas/química , Caderinas/imunologia , Bovinos , Relação Dose-Resposta a Droga , Impedância Elétrica , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Epêndima/citologia , Epêndima/ultraestrutura , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica de Transmissão , Técnicas de Cultura de Órgãos , Peptídeo Hidrolases/imunologia , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Fatores de TempoRESUMO
In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like ß-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins.
Assuntos
Proteínas de Bactérias/química , Caderinas/química , Proteínas de Ligação ao Cálcio/metabolismo , Lectinas/química , Lectinas/metabolismo , Polissacarídeos/metabolismo , Rhizobium leguminosarum/metabolismo , Sequência de Aminoácidos , Cálcio/química , Proteínas de Ligação ao Cálcio/química , Calorimetria/métodos , Dados de Sequência Molecular , Polissacarídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Solventes/químicaRESUMO
Suboptimal intake of Zn is one of the most common nutritional problems worldwide. Previously, we have shown that Zn deficiency (ZD) produces oxidative and nitrosative stress in the lung of rats. We analyse the effect of moderate ZD on the expression of several intermediate filaments of the cytoskeleton, as well as the effect of restoring Zn during the refeeding period. Adult male rats were divided into three groups: Zn-adequate control (CO) group; ZD group; Zn-refeeding group. CerbB-2 and proliferating cell nuclear antigen (PCNA) expression was increased in the ZD group while the other parameters did not change. During the refeeding time, CerbB-2, cytokeratins, vimentin and PCNA immunostaining was higher than that in the CO group. The present findings indicate that the overexpression of some markers could lead to the fibrotic process in the lung. Perhaps ZD implications must be taken into account in health interventions because an inflammation environment is associated with ZD in the lung.
Assuntos
Matriz Extracelular/química , Matriz Extracelular/metabolismo , Pulmão/metabolismo , Zinco/deficiência , Animais , Biomarcadores/análise , Peso Corporal , Caderinas/química , Caderinas/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Queratinas/química , Queratinas/metabolismo , Masculino , Ratos , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Objetivos: Comparar a expressão imuno-histoquímica da E-caderina e da Beta-catenina de lesões escamosaspré-neoplásicas e neoplásicas de mucosa oral de amostras emblocadas em parafina. Materiais e métodos:Foram selecionadas 15 amostras de mucosa oral de pacientes apresentando hiperplasia com ou sem displasialeve (grupo 1); 5 amostras apresentando displasia moderada, acentuada ou carcinoma in situ (grupo 2); e12 amostras apresentando carcinoma de células escamosas invasor (grupo 3). Essas amostras foram submetidasà técnica de imuno-histoquímica com anticorpos primários monoclonais anti-E-caderina e anti-Betacatenina.A leitura em microscopia óptica compreendeu a expressão tecidual desses marcadores no epitélioescamoso das amostras de mucosa oral lesadas ou não. A expressão imuno-histoquímica dessas moléculasde adesão foi classificada, segundo a sua intensidade de marcação tecidual, em negativa, positiva fraca epositiva forte. Resultados: A expressão de E-caderina foi forte em 93,3% dos casos do grupo 1 (hiperplasia/displasia leve), e 100% dos casos demonstraram forte expressão para a Beta-catenina nesse mesmo grupo. Contudo, no grupo 3 (carcinoma de célula escamosa), somente 42% dos casos foram fortemente positivospara E-caderina e 25% deles para Beta-catenina. Conclusões: A E-caderina e a Beta-catenina diminuíram asua expressão segundo a progressão tumoral do carcinoma de mucosa oral, reforçando um dos mecanismosrelacionados com a sua carcinogênese.
Objectives: To compare the immunohistochemical expression of E-cadherin and Beta-catenin in squamous pre-malignant and malignant lesions of formalin fixed paraffin embedded buccal mucosa samples. Materials e methods: Selected 15 samples of buccal mucosa of patients with hyperplasia with or without mild dysplasia (group 1), 5 samples showing moderate dysplasia, severe or carcinoma in situ (group 2) and 12 samples presenting invasive squamous cell carcinoma (group 3). These samples were subjected to immunohistochemistry with anti-E-cadherin and anti-Beta-catenin monoclonal antibodies. The expression of these markers in tissue samples injured or not were analyzed in accordance of positivity that was observed in epithelium stratum. The immunohistochemical expression of these adhesion molecules was classified according to their intensity in negative, weak positive and strong positive. Results: The expression of E-cadherin was strong at 93.3% of patients in group 1, and 100% of the cases showed strong expression of Beta-catenin in the same group. However, in group 3, only 42% of cases were strongly positive for E-cadherin and 25% of them to Beta-catenin. Conclusions: The E-cadherin and Beta-catenin decreased their expression according to tumor progression, from hiperplasia/mild dysplasia lesion to buccal invasive carcinoma and this fact may be related of the carcinogenesis mechanisms.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Caderinas/química , Carcinoma de Células Escamosas/patologia , Mucosa Bucal/patologia , Neoplasias Bucais/patologia , beta Catenina/química , Fatores Etários , Distribuição de Qui-Quadrado , Estudos Prospectivos , Fatores SexuaisRESUMO
Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R(1)) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R(1) promotes cleavage of the amino-terminal end, including helix alpha-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R(1) fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.