RESUMEN
Tat transactivating activity regulated by NAD(+) -dependent histone deacetylase sirtuin1 (SIRT1) connects HIV transcription with the metabolic state of the cell. Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD(+) biosynthesis. Nampt, SIRT1, and AMPK were involved in inhibiting HIV-1 transactivation through redox-regulated pathway. Tanshinone II A is a main lipid-soluble monomer derivative from the root of Salvia miltiorrhiza (Danshen) and tanshinone II A possess a variety of biological activities through redox signaling pathway. Here we investigated the effect of tanshinone II A on Tat-induced HIV-1 transactivation and the redox signaling pathway involved in it. As the results were shown, tanshinone II A reversed Tat-induced reactive oxygen species (ROS) production and down-regulation of glutathione (GSH) levels in TZM-bl cells through up-regulation of Nrf2 expression. Tanshinone II A reversed Tat-induced inhibition of SIRT1 activity but not SIRT1 protein expression. Tanshinone II A reversed Tat-induced inhibition of Nampt protein expression and depletion of NAD(+) levels in TZM-bl cells in a dose-dependent manner. Tanshinone II A-evoked Nampt expression was mediated by AMPK signaling pathway. Tanshinone II A inhibited Tat-induced HIV-1 LTR transactivation dependent on AMPK-Nampt pathway. Collectively, our data provide new insights into understanding of the molecular mechanisms of tanshinone II A inhibited Tat-regulated transcription, suggesting that targeting AMPK/Nampt/SIRT1 pathway could serve as new anti-HIV-1 agents.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Abietanos/farmacología , Fármacos Anti-VIH/farmacología , Citocinas/metabolismo , VIH-1/efectos de los fármacos , Nicotinamida Fosforribosiltransferasa/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Viral/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Citocinas/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática , Regulación Viral de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Duplicado del Terminal Largo de VIH/efectos de los fármacos , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , VIH-1/metabolismo , VIH-1/patogenicidad , Células HeLa , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Nicotinamida Fosforribosiltransferasa/genética , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genética , Sirtuina 1/metabolismo , Transfección , Activación Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and may contribute to the development and progression of many infective diseases including human immunodeficiency virus 1 (HIV-1) infection. The Tat protein is fundamental to viral gene expression. In this study, our goal was to investigate the regulation of a specific miRNA (known as miR-217) in multinuclear activation of galactosidase indicator (MAGI) cells and explore the mechanisms by which miR-217 influenced Tat-induced HIV-1 transactivation through down-regulation of SIRT1 expression. We showed that miR-217 was up-regulated when Tat was expressed in multinuclear activation of galactosidase indicator cells. Forced expression of "miR-217 mimics" increased Tat-induced LTR transactivation. In addition, miR-217 significantly inhibited SIRT1 protein expression by acting on the 3'-UTR of the SIRT1 mRNA. In turn, the decrease in SIRT1 protein abundance provoked by miR-217 affected two important types of downstream signaling molecules that were regulated by Tat. Lower expression of SIRT1 caused by miR-217 enhanced Tat-induced phosphorylation of IKK and p65-NFkB and also exacerbated the loss of AMPK phosphorylation triggered by Tat. Our results uncover previously unknown links between Tat and a specific host cell miRNA that targets SIRT1. We also demonstrate that this regulatory mechanism impinges on p65-NFkB and AMPK signaling: two important host cell pathways that influence HIV-1 pathogenesis. Our results also suggest that strategies to augment SIRT1 protein expression by down-regulation of miR-217 may have therapeutic benefits to prevent HIV-1 replication.
Asunto(s)
Regulación hacia Abajo/genética , Duplicado del Terminal Largo de VIH/genética , MicroARNs/metabolismo , Sirtuina 1/genética , Activación Transcripcional/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Adenilato Quinasa/metabolismo , Activación Enzimática , Células HeLa , Humanos , MicroARNs/genética , FN-kappa B/metabolismo , Sirtuina 1/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
EZH2 plays a major role in HIV-1 latency, however, the molecular linkage between Tat-induced HIV-1 transactivation and EZH2 activity is not fully understood. It was shown Tat induced HIV-1 transactivation through inhibiting EZH2 activity. Tat decreased the levels of H3K27me3 and EZH2 occupy at the long terminal repeat (LTR) of HIV-1. We further showed for the first time that transfected with Tat construct resulted in an increase in phosphorylated EZH2 (p-EZH2), mediated by active Akt. ROS/Akt-dependent p-EZH2 was correlated with Tat-induced transactivation. Our study reveals that novel mechanisms allow Tat-induced HIV-1 transactivation by ROS/Akt-dependent downregulating the EZH2 epigenetic silencing machinery.
Asunto(s)
VIH-1/fisiología , Complejo Represivo Polycomb 2/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/agonistas , Especies Reactivas de Oxígeno/agonistas , Transducción de Señal , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/agonistas , Adenosina/análogos & derivados , Adenosina/farmacología , Sustitución de Aminoácidos , Inmunoprecipitación de Cromatina , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/farmacología , Genes Reporteros/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-1/enzimología , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Complejo Represivo Polycomb 2/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Viral/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Tat's transactivating activity is controlled by sirtuin 1 (SIRT1) that connects HIV transcription with the metabolic state of the cell. Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme in the salvaging pathway for the synthesis of nicotinamide adenine dinucleotide (NAD(+)) that is involved in energy metabolism. Host encoded microRNAs (miRNAs) may influence viral replication. In this study, our goal was aimed to investigate the regulation of miR-182 in TZM-bl cells and explore the mechanisms by which miR-182 influenced Tat-induced HIV-1 transactivation through targeting at down-regulation of NAMPT expression. We showed that miR-182 was up-regulated when Tat was expressed in TZM-bl cells. MiR-182 significantly inhibited NAMPT protein expression by acting on the 3'-UTR of the NAMPT mRNA. MiR-182 was involved in Tat-induced NAD(+) depletion, down-regulation of SIRT1 protein expression and activity, increased acetylation of p65. Forced expression of "miR-182 mimics" increased Tat-induced LTR transactivation. Our results uncover previously unknown links between Tat and a specific host cell miRNA that targets NAMPT. Our results suggest that strategies to augment NAMPT protein expression by down-regulation of miR-182 may have therapeutic benefits to prevent HIV-1 replication.
Asunto(s)
Citocinas/genética , Duplicado del Terminal Largo de VIH/genética , MicroARNs/fisiología , Nicotinamida Fosforribosiltransferasa/genética , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/fisiología , Regiones no Traducidas 3' , Acetilación , Citocinas/metabolismo , Regulación hacia Abajo , Expresión Génica , Regulación Viral de la Expresión Génica , VIH-1/genética , Células HeLa , Humanos , Nicotinamida Fosforribosiltransferasa/metabolismo , Procesamiento Proteico-Postraduccional , Interferencia de ARN , Sirtuina 1/genética , Sirtuina 1/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
MicroRNAs (miRNAs) regulate gene expression and may contribute to HIV-1 infection. In this study, our goal was to investigate the mechanisms by which miR-34a influenced Tat-induced HIV-1 transactivation through the SIRT1/NFκB pathway. We showed that Tat induced up-regulation of miR-34a expression in TZM-bl cells. MiR-34a significantly inhibited SIRT1 expression. Overexpression of miR-34a increased Tat-induced LTR transactivation. Forced expression of miR-34a decreased SIRT1 protein expression and consequently diminished Tat-induced acetylation of p65, while treatment with a miR-34a inhibitor had the opposite effect. These results suggest that regulating SIRT1 by down-regulation of miR-34a levels may be a therapeutic strategy against HIV-1 replication.
Asunto(s)
Duplicado del Terminal Largo de VIH/genética , MicroARNs/metabolismo , FN-kappa B/metabolismo , Sirtuina 1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Western Blotting , Línea Celular Tumoral , Humanos , MicroARNs/genética , FN-kappa B/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sirtuina 1/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
AIMS: Transcription is a crucial step for human immunodeficiency virus 1 (HIV-1) gene expression in infected host cells. The HIV-1 Tat activates the nuclear factor-kappa B (NF-κB) signaling transduction pathway, which is necessary for viral replication. Epigallocatechin-3-gallate (EGCG) has antioxidant, anti-inflammatory, and anti-viral properties. In this study, we investigated the effects of EGCG on Tat-induced HIV-1 transactivation and potential mechanisms by which EGCG inhibited activation of NF-κB pathway. MAIN METHODS: HeLa-CD4-long terminal repeat (LTR)-ß-gal (MAGI) cells were transfected with Tat plasmid. Tat-induced HIV-1 LTR transactivation was determined by MAGI cell assay. The reactive oxygen species (ROS) levels and glutathione (GSH) levels were measured. In addition, the protein expressions were assayed by western blotting. KEY FINDINGS: Tat caused a significant decrease in the intracellular glutathione (GSH) levels, a mild increase in the expression of nuclear levels of NF-E2-related factor-2 (Nrf2), a significant increase in the levels of NF-κB (phosphorylation of p65 and IKK) and a significant increase in ROS production. EGCG supplementation significantly improved the changes associated with Tat-induced oxidative stress by increasing nuclear levels of Nrf2, decreasing levels of NF-κB and ROS production. EGCG reversed Tat-mediated AKT activation and AMPK inhibition in MAGI cells. EGCG inhibited Tat-induced LTR transactivation in a dose-dependent manner. SIGNIFICANCE: The results suggest that Nrf2 signaling pathway may be the primary target for prevention of Tat-induced HIV-1 transactivation by EGCG, and EGCG also reduce NF-κB activation by inhibiting AKT signaling pathway and activating AMPK signaling pathway.