RESUMO
BACKGROUND: Cyclooxygenase 2 (COX-2) and matrix metalloproteinases (MMPs) have been implicated in tissue injury and fibrogenesis in animal models but little is known regarding their role in hepatitis C virus (HCV) related liver disease in humans. AIMS: To characterise the intrahepatic expression pattern of COX-2 and MMPs in chronic HCV infection and determine whether HCV core and NS5A proteins could promote their expression in cultured hepatocyte derived cell lines. PATIENTS: Thirty two anti-HCV+ and 10 anti-HCV- patients were studied. METHODS: Western blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunohistochemistry were used to assess the expression pattern of COX-2 and MMPs in liver biopsy samples from all patients. COX-2 gene expression and MMP-9 protein levels were also determined by immunoblot, RT-PCR, and luciferase assays in core and NS5A transfected hepatocyte derived cells. RESULTS: The intrahepatic expression level of COX-2, MMP-2, and MMP-9 was significantly higher in HCV+ than in HCV- patients, increasing with the fibrotic stage of liver disease. We further demonstrated that COX-2 mRNA, protein, and activity were induced in resting and activated core and NS5A transfectants. Both viral proteins induced transcriptional activity of the COX-2 gene promoter whereas core, but not NS5A, exerted an inducer effect on MMP-9 protein levels in cultured hepatocyte derived cells. CONCLUSIONS: Intrahepatic COX-2, MMP-2, and MMP-9 overexpression is associated with progressive hepatic fibrosis in chronic HCV infection, suggesting their pathogenic role in fibrogenesis. HCV core and NS5A proteins were able to upregulate COX-2 and MMP-9 gene expression in hepatocyte derived cells, providing a potential mechanism for hepatic fibrosis during chronic HCV infection.
Assuntos
Hepatite C Crônica/enzimologia , Isoenzimas/metabolismo , Fígado/enzimologia , Metaloproteinases da Matriz/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Adulto , Linhagem Celular , Ciclo-Oxigenase 2 , Progressão da Doença , Feminino , Hepatite C Crônica/virologia , Humanos , Isoenzimas/genética , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz/genética , Proteínas de Membrana , Pessoa de Meia-Idade , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transfecção , Regulação para Cima , Proteínas do Core Viral/genética , Proteínas do Core Viral/fisiologia , Carga Viral , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/fisiologiaRESUMO
Co-infection with hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1) is relatively common. However, the impact of this co-infection on the clinical outcome of HIV infection has not been elucidated. We herein demonstrate that the HBV X protein (HBx) superinduces ongoing HIV-1 replication and HIV-1 long terminal repeat (LTR) transcription by synergizing with Tat protein and with T-cell activation signals. Although HBx cooperated with mitogenic stimuli in the induction of reporter plasmids harboring the HIV-1 kappaB enhancer, in both a NF-kappaB-dependent manner and a NF-AT-dependent manner, deletion of this element from the LTR did not affect the HBx-mediated up-regulation in the presence of Tat and/or mitogens. In contrast, mutation of the proximal LTR Sp1-binding sites abolished the HBx-mediated synergistic activation, but only when it was accompanied by deletion of the kappaB enhancer. When HBx was targeted to the nucleus, its ability to synergize with cellular activation stimuli was maintained. Furthermore, mutations of HBx affecting its interaction with the basal transcription machinery abrogated the synergistic activation by HBx, suggesting that this protein exerts its function by acting as a nuclear co-activator. These results indicate that HBx could contribute to a faster progression to AIDS in HBV-HIV co-infected individuals.
Assuntos
Repetição Terminal Longa de HIV/genética , HIV-1/fisiologia , Ativação Linfocitária , Proteínas Nucleares , Regiões Promotoras Genéticas , Linfócitos T/imunologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Replicação Viral/fisiologia , Sequência de Bases , Sítios de Ligação , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Elementos Facilitadores Genéticos , Produtos do Gene tat/fisiologia , HIV-1/genética , Humanos , Células Jurkat , NF-kappa B/genética , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Fatores de Transcrição NFATC , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Virais Reguladoras e Acessórias , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND/AIMS: The hepatitis B virus HBx protein is associated with the development of hepatocellular carcinoma (HCC). However, its possible contribution to tumor spreading has not been explored. The migration of tumor cells through the extracellular matrix (ECM) represents a crucial step in tumor metastasis. Our aim was to study the effect of HBx on the integrin-mediated cell-ECM interaction, and its possible consequences for cell migration. METHODS: Cell-ECM interaction was evaluated by static adhesion experiments, using blocking and stimulating anti-beta1 integrin mAbs. ECM receptor expression was analyzed by flow cytometry. The cellular distribution of the activated beta1 integrin subunit was determined by immunofluorescence analysis, and cell motility was determined by wound-healing assays. RESULTS: HBx-bearing cells showed decreased adhesion to fibronectin, which correlated with a decreased expression of the alpha5 integrin subunit. The activated beta1 subunit was redistributed to the tips of pseudopodial protrusions of HBx-bearing cells, whereas it was evenly localized in the control cells. HBx-induced cell migration was abrogated by irreversible stimulation of beta1 integrins. CONCLUSIONS: These results suggest that HBx might play a role in tumor spreading by modulating the adhesion-deadhesion balance of the cells in the primary tumor site and favoring integrin-mediated cell migration.
Assuntos
Matriz Extracelular/fisiologia , Integrinas/fisiologia , Transativadores/farmacologia , Antígenos CD/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Fibronectinas/fisiologia , Integrina alfa1 , Integrina alfa5 , Integrina beta1/fisiologia , Transfecção , Proteínas Virais Reguladoras e AcessóriasRESUMO
Human hepatocytes infected by hepatitis B virus (HBV) produce the proinflammatory cytokine, tumor necrosis factor (TNF-). In this study, we explored the mechanism of induction of TNF- synthesis by HBV. We found that the stable HBV-transfected hepatoma cell line, 2. 2.15, expressed high-molecular-weight (HMW) TNF- mRNAs, which were absent in the parent HepG2 cells. Treatment of 2.2.15 cells with interferon alfa (IFN-) and/or interleukin-1beta (IL-1beta) reduced both viral gene transcription and TNF- mRNA expression. Transient or stable transfection of hepatocyte-derived cell lines with HBV X protein (HBx) expression vectors induced the production of biologically active TNF-. In these cells, the HBx-induced TNF- was detected both as cell-associated and soluble forms. Luciferase gene-expression assays showed that the TNF- gene promoter contained target sequences for HBx trans-activation within the proximal region of the promoter. These results indicate that the hepatocyte TNF- synthesis induced by HBV is transcriptionally up-regulated by HBx. Thus, HBx may have a role in the induction of the intrahepatic inflammatory processes that take place during acute and chronic hepatitis B.
