RESUMO
CONCLUSION: This study provides evidence that histamine induced MUC5AC mRNA expression through the activation of the core region of its promoter. It may also help in approaching new therapeutic strategies in airway mucins hypersecretory diseases. OBJECTIVE: Mucin hypersecretion characterizes several respiratory diseases. Production of MUC5AC, a major gel forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators. Histamine is associated with MUC5AC up-regulation during the early phase of allergic respiratory diseases. The goal of the present study was to identify whether histamine may induce MUC5AC gene expression both at mRNA and protein levels and to elucidate its mechanism. METHODS: Guinea pigs were sensitized and challenged with dermatophagoides farinae (Der f) extract. Human lung mucoepidermoid carcinoma cell line (NCI-H292) was used. The regulatory mechanism of MUC5AC by histamine and H1R was investigated using RT-PCR, immunofluorescence, and MUC5AC promoter-driven luciferase reporter assay. RESULTS: The MUC5AC expression levels were increased by histamine treatment in either nasal tissues of Der f challenged guinea pigs or NCI-H292 cells, whereas the MUC5AC protein over-production induced by histamine administration was significantly inhibited by H1R antagonist chlorpheniramine. It was found that histamine enhanced the activation of the proximal core region of the MUC5AC promoter, which was significantly blocked by chlorpheniramine, as indicated by luciferase reporter assays.
Assuntos
Regulação da Expressão Gênica , Histamina/metabolismo , Mucina-5AC/metabolismo , Animais , Linhagem Celular Tumoral , Clorfeniramina , Cobaias , Antagonistas dos Receptores Histamínicos H1 , Humanos , Masculino , Mucosa Nasal/metabolismo , Regiões Promotoras Genéticas , Regulação para CimaRESUMO
The human E-cadherin is a single transmembrane domain protein involved in Ca(2+)-dependent cell-cell adhesion. In a previous study, we demonstrated that all of four potential N-glycosylation sites in E-cadherin are occupied by N-glycans in human breast carcinoma cells in vivo and the elimination of N-glycan at Asn-633 dramatically affected E-cadherin expression and made it degraded. In this study we investigated the molecular mechanism of E-cadherin, which lacks N-glycosylation at Asn-633 (M4), degradation and the role of the N-glycan at Asn-633 in E-cadherin folding. We treated cells stably expressed M4 E-cadherin with MG123, DMM, respectively. Either MG132 or DMM could efficiently block degradation of M4 E-cadherin. M4 E-cadherin was recognized as the substrate of ERAD and was retro-translocated from ER lumen to cytoplasm by p97. It was observed that the ration of M4 E-cadherin binding to calnexin was significantly increased compared with that of other variants, suggesting that it was a misfolded protein, though cytoplasmic domain of M4 E-cadherin could associate with beta-catenin. Furthermore, we found that N-glycans of M4 E-cadherin were modified in immature high mannose type, suggesting that it could not depart to Golgi apparatus. In conclusion, this study revealed that N-glycosylation at Asn-633 is essential for E-cadherin expression, folding and trafficking.
Assuntos
Caderinas/química , Caderinas/metabolismo , Retículo Endoplasmático/metabolismo , Asparagina/química , Sequência de Bases , Sítios de Ligação/genética , Caderinas/genética , Linhagem Celular Tumoral , Primers do DNA/genética , Feminino , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina/metabolismoRESUMO
E-cadherin mediates calcium-dependent cell-cell adhesion between epithelial cells. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N-glycosylation in E-cadherin-mediated cell-cell adhesion was investigated by site-directed mutagenesis. In MDA-MB-435 cells, all four potential N-glycosylation sites of human E-cadherin were N-glycosylated. Removal of N-glycan at Asn-633 dramatically affected E-cadherin stability. In contrast, mutant E-cadherin lacking the other three N-glycans showed similar protein stability in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn-554 and Asn-566 were found to affect E-cadherin-mediated calcium-dependent cell-cell adhesion, and removal of either of the two N-glycans caused a significant decrease in calcium-dependent cell-cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N-glycans on E-cadherin resulted in elevated tyrosine phosphorylation level of beta-catenin and reduced beta- and alpha-catenins at AJs. These findings demonstrate that N-glycosylation may affect the adhesive function of E-cadherin through modifying the composition of AJs.
Assuntos
Junções Aderentes/química , Neoplasias da Mama/patologia , Caderinas/metabolismo , Caderinas/fisiologia , Adesão Celular , Linhagem Celular Tumoral , Feminino , Glicosilação , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Polissacarídeos , alfa Catenina/metabolismo , beta Catenina/metabolismoRESUMO
MAGI-2, a multidomain scaffolding protein, contains nine potential protein-protein interaction modules, including a GuK domain, two WW domains and six PDZ domains. In this study, we examined eight human hepatocarcinoma cell lines (HHCCs) and found that MAGI-2 was expressed only in 7721 cells. After 7721, 7404 and 97H cells were transfected with myc-MAGI-2 plasmid, their migration and proliferation was significantly inhibited, which was associated with downregulation of p-FAK and p-Akt. It is known that p-FAK is a substrate of PTEN and p-Akt can be regulated by PTEN via PIP(3). We demonstrated that PTEN was upregulated after myc-MAGI-2 transfection, which was due to the enhancement of PTEN protein stability rather than mRNA levels. Furthermore, MAGI-2-induced inhibition of cell migration and proliferation was attenuated in 7721 cells with PTEN silence or in PTEN-null cell line U87MG, and PTEN transfection could restore the effect of MAGI-2 in U87MG cells. Finally, the molecular association between PTEN and MAGI-2 was confirmed. Our results suggested that PTEN played a critical role in MAGI-2-induced inhibition of cell migration and proliferation in HHCCs.
Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Movimento Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Guanilato Quinases , Humanos , Modelos Biológicos , Fosforilação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas/fisiologia , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
DNA methylation, which affects gene expression and chromatin stability, is catalyzed by DNA methyltransferases (DNMTs) of which DNMT1 possesses most abundant activity. PI3K/PKB pathway is an important pathway involved in cell proliferation, viability, and metabolism and often disrupted in cancer. Here we investigated the impact of PKB on DNMT1 and DNA methylation. Positive correlation between PKB-Ser473-phosphorylation and DNMT1 protein level in 17 human cell lines (p<0.01) and in 27 human bladder cancer tissues (p<0.05) was found. With activator, inhibitor, siRNA and constitutively active or dominant-negative plasmids of PKB, we found that PKB increased the protein level of DNMT1 without coordinate mRNA change, which was specific rather than due to cell-cycle change. PKB enhanced DNMT1 protein stability independent of de novo synthesis of any protein, which was attributed to down-regulation of N-terminal-120-amino-acids-dependent DNMT1 degradation via ubiquitin-proteasome pathway. Gsk3beta inhibitor rescued the decrease of DNMT1 by PKB inhibition, suggesting that Gsk3beta mediated the stabilization of DNMT1 by PKB. Then role of PKB regulating DNMT1 was investigated. Inhibition of PKB caused observable DNA hypomethylation and chromatin decondensation and DNMT1 overexpression partially reversed cell growth inhibition by PKB inhibition. In conclusion, our results suggested that PKB enhanced DNMT1 stability and maintained DNA methylation and chromatin structure, which might contribute to cancer cell growth.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ciclo Celular , Proliferação de Células , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Estabilidade Enzimática , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HeLa , Humanos , Proteínas Mutantes/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Ubiquitina/metabolismoRESUMO
We present here an effective technique for the large-scale separation and identification of N-linked glycoproteins from Chang liver cells, the human normal liver cells. To enrich N-linked glycoproteins from the whole cells, a procedure containing the lysis of human liver cells, the solubilization of total proteins, lectin affinity chromatography including Concanavalin A and wheat germ agglutinin was established. Furthermore, captured N-linked glycoproteins were separated by 2-DE, and identified by MS and database searching. Finally, we found 63 N-glycoproteins in Chang liver cells. In addition, using the above method, we identified 7 remarkably up-regulated glycoproteins from MHCC97-H cells, highly metastatic liver cancer cells, compared to Chang liver cells. These up-regulated glycoproteins were associated with liver cancer and might be used as biomarkers for tumor diagnosis. Results showed that we established a high-throughput proteomic analysis for separating N-linked glycoproteins from human liver cells. This strategy greatly improved the glycoprotein analysis method associated with proteome-wide glycosylation changes related to liver cancer. Our work was part of the HUPO Human Liver Proteome Project (HLPP) studies and was supported by CHINA HUPO.
Assuntos
Cromatografia de Afinidade/métodos , Concanavalina A/metabolismo , Glicoproteínas/análise , Glicoproteínas/metabolismo , Hepatócitos/metabolismo , Proteômica/métodos , Aglutininas do Germe de Trigo/metabolismo , Células Cultivadas , Eletroforese em Gel Bidimensional , Glicoproteínas/química , Hepatócitos/química , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para CimaRESUMO
The tumor suppressor PTEN gene maps to chromosome 10q23.3 and encodes a dual specificity phosphatase. Mutations of this gene had been found in a variety of human tumors. In the present study, we analyzed the structure and expression of the PTEN gene in 34 hepatocellular carcinoma tissues and two hepatoma cell lines. We found neither homozygous nor hemizygous deletions in these samples. We, however, found point mutations in 4 of the 34 tissue samples. Five of ten hepatocellular carcinoma tissues showed reduced PTEN expression at mRNA level. HepG2 and SMMC-7721 hepatoma cells showed decreased PTEN expression at both mRNA and protein levels compared with immortalized L02 hepatic cells. PTEN mRNA in SMMC-7721 hepatoma cells could be reduced by TGF-betaI treatment. We also found that the phosphorylation levels of FAK in both of the hepatoma cell lines were higher than that in L02 hepatic cells. Transient expression of the PTEN gene in SMMC-7721 and HepG2 hepatoma cells resulted in decreased FAK phosphorylation. The level of FAK tyrosine phosphorylation appeared to be inversely correlated with the level of the PTEN protein. In summary, our results indicated that the function of the PTEN gene in hepatocarcinomas may be impaired mainly through point mutations and expression deficiency and that the defect of PTEN in tumor cells could alter the phosphorylation of FAK.
Assuntos
Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Monoéster Fosfórico Hidrolases/genética , Mutação Puntual , Proteínas Tirosina Quinases/metabolismo , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , PTEN Fosfo-Hidrolase , Fosforilação , RNA Mensageiro/análise , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1RESUMO
PTEN is a major tumor suppressor gene that encodes a dual-specificity phosphatase with high sequence similarity to the cytoskeletal protein tensin. PTEN may be involved in the formation and disassembly of focal adhesion and affect cell migration. In the present study, PTEN expression plasmid was constructed and transfected into the hepatoma cell line SMMC-7721 to analyze the alterations of cell motility and FAK tyrosine phosphorylation. It was observed that the overexpression of PTEN gene significantly inhibited cell motility on extracellular matrix (Fn), and the cell migration on fibronectin was reduced by 35%. Similarly, at 30-min and 60-min, the cell spreading on Fn but not on polylysine was inhibited by 29% and 26% respectively. The data obtained from immunoprecipitation and immunoblotting analyses showed that the overexpression of PTEN did not affect FAK expression but resulted in a decrease in FAK tyrosine phosphorylation. The level of FAK phosphorylation was inversely correlated with the level of PTEN protein in three cell lines. It was also found that the overexpression of PTEN led to growth inhibition, with the number of cells in S phase reduced by 16%. These results indicate that PTEN exerts its tumor-suppressive effects on hepatocellular carcinoma cells through the inhibition of cell motility and cell cycle progression.