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1.
J Virol ; 92(13)2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29669827

RESUMO

Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrated the important relationship among osteopontin (OPN), the ER, and LDs. OPN is a secreted phosphoprotein, and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage, and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors integrin αVß3 and CD44. However, the role of HCV-induced OPN in the HCV life cycle has not been elucidated. In this study, we showed a significant reduction in HCV replication, assembly, and infectivity in HCV-infected cells transfected with small interfering RNA (siRNA) against OPN, αVß3, and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B, and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating a possible role for OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity, and assembly through binding to αVß3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly.IMPORTANCE Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in the HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated that endogenous OPN associates with HCV NS3, NS5A, NS5B, and core proteins, which are in close proximity to the ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors αVß3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal roles in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.


Assuntos
Hepatite C/virologia , Receptores de Hialuronatos/metabolismo , Integrina alfaVbeta3/metabolismo , Gotículas Lipídicas/metabolismo , Osteopontina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus , Replicação Viral , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Replicação do DNA , Hepacivirus/fisiologia , Hepatite C/metabolismo , Humanos , Receptores de Hialuronatos/genética , Integrina alfaVbeta3/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Osteopontina/genética , RNA Viral , Células Tumorais Cultivadas , Proteínas não Estruturais Virais/genética
2.
J Biol Chem ; 291(7): 3254-67, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26698881

RESUMO

Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with chronic HCV.


Assuntos
Proteínas de Transporte/metabolismo , Hepacivirus/fisiologia , Hepatócitos/virologia , Inflamassomos/metabolismo , Lipogênese , Proteína de Ligação a Elemento Regulador de Esterol 1/agonistas , Proteína de Ligação a Elemento Regulador de Esterol 2/agonistas , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Caspase 1/química , Caspase 1/genética , Caspase 1/metabolismo , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Indução Enzimática/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Complexo de Golgi/virologia , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/metabolismo , Hepatite C Crônica/patologia , Hepatite C Crônica/fisiopatologia , Hepatite C Crônica/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Inflamassomos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Hepatopatia Gordurosa não Alcoólica/etiologia , Pró-Proteína Convertases/química , Pró-Proteína Convertases/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Interferência de RNA , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
4.
J Biol Chem ; 288(52): 36994-7009, 2013 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-24240095

RESUMO

Osteopontin (OPN) is a secreted phosphoprotein, originally characterized in malignant-transformed epithelial cells. OPN is associated with tumor metastasis of several tumors and is overexpressed in hepatocellular carcinoma (HCC) tissue involving HCC invasion and metastasis. Importantly, OPN is significantly up-regulated in liver injury, inflammation, and hepatitis C virus (HCV)-associated HCC. However, the underlying mechanisms of OPN activation and its role in HCV-mediated liver disease pathogenesis are not known. In this study, we investigated the mechanism of OPN activation in HCV-infected cells. We demonstrate that HCV-mediated Ca(2+) signaling, elevation of reactive oxygen species, and activation of cellular kinases such as p38 MAPK, JNK, PI3K, and MEK1/2 are involved in OPN activation. Incubation of HCV-infected cells with the inhibitors of AP-1 and Sp1 and site-directed mutagenesis of AP-1- and Sp1-binding sites on the OPN promoter suggest the critical role of AP-1 and Sp1 in OPN promoter activation. In addition, we show the in vivo interactions of AP-1 and Sp1 with the OPN promoter using chromatin immunoprecipitation assay. We also show the calpain-mediated processing of precursor OPN (∼75 kDa) into ∼55-, ∼42-, and ∼36-kDa forms of OPN in HCV-infected cells. Furthermore, we demonstrate the critical role of HCV-induced OPN in increased phosphorylation of Akt and GSK-3ß followed by the activation of ß-catenin, which can lead to EMT of hepatocytes. Taken together, these studies provide an insight into the mechanisms of OPN activation that is relevant to the metastasis of HCV-associated HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transição Epitelial-Mesenquimal , Hepatite C/metabolismo , Hepatócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Osteopontina/metabolismo , Sinalização do Cálcio/genética , Calpaína/genética , Calpaína/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hepacivirus , Hepatite C/genética , Hepatite C/patologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Metástase Neoplásica , Osteopontina/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição AP-1 , beta Catenina/genética , beta Catenina/metabolismo
6.
Arch Virol ; 159(5): 1017-25, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24240483

RESUMO

Chronic hepatitis C is a lethal blood-borne infection often associated with a number of pathologies such as insulin resistance and other metabolic abnormalities. Insulin is a key hormone that regulates the expression of metabolic pathways and favors homeostasis. In this study, we demonstrated the molecular mechanism of hepatitis C virus (HCV) nonstructural protein 5A (NS5A)-induced metabolic dysregulation. We showed that transient expression of HCV NS5A in human hepatoma cells increased lipid droplet formation through enhanced lipogenesis. We also showed increased transcriptional expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and diacylglycerol acyltransferase-1 (DGAT-1) in NS5A-expressing cells. On the other hand, there was significantly reduced transcriptional expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor γ (PPARγ) in cells expressing HCV NS5A. Furthermore, increased gluconeogenic gene expression was observed in HCV-NS5A-expressing cells. In addition, it was also shown that HCV-NS5A-expressing hepatoma cells show serine phosphorylation of IRS-1, thereby hampering metabolic activity and contributing to insulin resistance. Therefore, this study reveals that HCV NS5A is involved in enhanced gluconeogenic and lipogenic gene expression, which triggers metabolic abnormality and impairs insulin signaling pathway.


Assuntos
Gluconeogênese/fisiologia , Hepacivirus/metabolismo , Lipogênese/fisiologia , Proteínas não Estruturais Virais/fisiologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Neoplasias Hepáticas/metabolismo , Transdução de Sinais
7.
J Gen Virol ; 93(Pt 2): 235-246, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21994322

RESUMO

Interleukin-1ß (IL-1ß) is a potent pro-inflammatory cytokine involved in the pathogenesis of HCV, but the sensors and underlying mechanisms that facilitate HCV-induced IL-1ß proteolytic activation and secretion remains unclear. In this study, we have identified a signalling pathway leading to IL-1ß activation and secretion in response to HCV infection. Previous studies have shown the induction and secretion of IL-1ß through the inflammasome complex in macrophages/monocytes. Here, we report for the first time the induction and assembly of the NALP3-inflammasome complex in human hepatoma cells infected with HCV (JFH-1). We demonstrate that activation of IL-1ß in HCV-infected cells involves the proteolytic processing of pro-caspase-1 into mature caspase-1 in a multiprotein inflammasome complex. Next, we demonstrate that HCV is sensed by NALP3 protein, which recruits the adaptor protein ASC for the assembly of the inflammasome complex. Using a small interfering RNA approach, we further show that components of the inflammasome complex are involved in the activation of IL-1ß in HCV-infected cells. Our study also demonstrates the role of reactive oxygen species in HCV-induced IL-1ß secretion. Collectively, these observations provide an insight into the mechanism of IL-1ß processing and secretion, which is likely to provide novel strategies for targeting the viral or cellular determinants to arrest the progression of liver disease associated with chronic HCV infection.


Assuntos
Caspase 1/metabolismo , Hepacivirus/imunologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Multimerização Proteica , Transdução de Sinais
8.
J Biol Chem ; 285(8): 5171-7, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20028980

RESUMO

Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (K(i) = 380 +/- 160 microM) and 2-phosphoascorbate (K(i) = 3.2 +/- 0.85 microM) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia.


Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Francisella tularensis/enzimologia , Macrófagos/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/fisiologia , Francisella tularensis/genética , Deleção de Genes , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo
9.
PLoS One ; 9(1): e87464, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24498111

RESUMO

Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVß3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Hepacivirus/metabolismo , Hepatite C Crônica/metabolismo , Hepatócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/biossíntese , Osteopontina/biossíntese , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Células Hep G2 , Hepatite C Crônica/patologia , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Receptores de Hialuronatos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Transdução de Sinais
10.
PLoS One ; 8(2): e56367, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23437118

RESUMO

Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-ß1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-ß1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-ß1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-ß1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-ß1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-ß1 is critical for the induction of alpha smooth muscle actin (α-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-ß1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-ß1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-ß1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection.


Assuntos
Hepacivirus/fisiologia , Células Estreladas do Fígado/patologia , Células Estreladas do Fígado/virologia , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta1/genética , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Análise Mutacional de DNA , Furina/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células Estreladas do Fígado/metabolismo , Humanos , Luciferases/metabolismo , Vison , Dados de Sequência Molecular , Transdução de Sinais/genética , Ativação Transcricional/genética , Fator de Crescimento Transformador beta1/metabolismo , Replicação Viral
11.
Diabetes ; 60(4): 1258-69, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21346177

RESUMO

OBJECTIVE: Hyperuricemia is strongly associated with obesity and metabolic syndrome and can predict visceral obesity and insulin resistance. Previously, we showed that soluble uric acid directly stimulated the redox-dependent proinflammatory signaling in adipocytes. In this study we demonstrate the role of hyperuricemia in the production of key adipokines. RESEARCH DESIGN AND METHODS: We used mouse 3T3-L1 adipocytes, human primary adipocytes, and a mouse model of metabolic syndrome and hyperuricemia. RESULTS: Uric acid induced in vitro an increase in the production (mRNA and secreted protein) of monocyte chemotactic protein-1 (MCP-1), an adipokine playing an essential role in inducing the proinflammatory state in adipocytes in obesity. In addition, uric acid caused a decrease in the production of adiponectin, an adipocyte-specific insulin sensitizer and anti-inflammatory agent. Uric acid-induced increase in MCP-1 production was blocked by scavenging superoxide or by inhibiting NADPH oxidase and by stimulating peroxisome-proliferator-activated receptor-γ with rosiglitazone. Downregulation of the adiponectin production was prevented by rosiglitazone but not by antioxidants. In obese mice with metabolic syndrome, we observed hyperuricemia. Lowering uric acid in these mice by inhibiting xanthine oxidoreductase with allopurinol could improve the proinflammatory endocrine imbalance in the adipose tissue by reducing production of MCP-1 and increasing production of adiponectin. In addition, lowering uric acid in obese mice decreased macrophage infiltration in the adipose tissue and reduced insulin resistance. CONCLUSIONS: Hyperuricemia might be partially responsible for the proinflammatory endocrine imbalance in the adipose tissue, which is an underlying mechanism of the low-grade inflammation and insulin resistance in subjects with the metabolic syndrome.


Assuntos
Tecido Adiposo/metabolismo , Hiperuricemia/metabolismo , Síndrome Metabólica/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Adiponectina/sangue , Tecido Adiposo/fisiopatologia , Animais , Células Cultivadas , Quimiocina CCL2/sangue , Humanos , Hiperuricemia/sangue , Imuno-Histoquímica , Síndrome Metabólica/sangue , Camundongos , Camundongos Obesos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiobarbitúricos/metabolismo , Ácido Úrico/metabolismo
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