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Antiretroviral therapy has effectively suppressed HIV infection and replication and prolonged the lifespan of HIV-infected individuals. In the meantime, various complications including type 2 diabetes associated with the long-term antiviral therapy have shown steady increases. Metformin has been the front-line anti-hyperglycemic drug of choice and the most widely prescribed medication for the treatment of type 2 diabetes. However, little is known about the effects of Metformin on HIV infection and replication. In this study, we showed that Metformin treatment enhanced HIV gene expression and transcription in HIV-transfected 293T and HIV-infected Jurkat and human PBMC. Moreover, we demonstrated that Metformin treatment resulted in increased CREB expression and phosphorylation, and TBP expression. Furthermore, we showed that Metformin treatment increased the recruitment of phosphorylated CREB and TBP to the HIV LTR promoter. Lastly, we showed that inhibition of CREB phosphorylation/activation significantly abrogated Metformin-enhanced HIV gene expression. Taken together, these results demonstrated that Metformin treatment increased HIV transcription, gene expression, and production through increased CREB phosphorylation and recruitment to the HIV LTR promoter. These findings may help design the clinical management plan and HIV cure strategy of using Metformin to treat type 2 diabetes, a comorbidity with an increasing prevalence, in people living with HIV.
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Diabetes Mellitus Tipo 2 , Infecciones por VIH , Metformina , Humanos , Infecciones por VIH/tratamiento farmacológico , Duplicado del Terminal Largo de VIH/genética , Leucocitos Mononucleares , Fosforilación , Metformina/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Expresión GénicaRESUMEN
The balance of microbial communities in the gut is extremely important for normal physiological function. Disruption of the balance is often associated with various disorders and diseases. Both HIV infection and cocaine use are known to change the gut microbiota and the epithelial barrier integrity, which contribute to inflammation and immune activation. Our recent study shows that Tat expression and cocaine exposure result in changes of genome-wide DNA methylation and gene expression and lead to worsen the learning and memory impairments. In the current study, we extended the study to determine effects of Tat and cocaine on the gut microbiota composition. We found that both Tat expression and cocaine exposure increased Alteromonadaceae in 6-month-old female/male mice. In addition, we found that Tat, cocaine, or both increased Alteromonadaceae, Bacteroidaceae, Cyanobiaceae, Erysipelotrichaceae, and Muribaculaceae but decreased Clostridiales_vadinBB60_group, Desulfovibrionaceae, Helicobacteraceae, Lachnospiraceae, and Ruminococcaceae in 12-month-old female mice. Lastly, we analyzed changes of metabolic pathways and found that Tat decreased energy metabolism and nucleotide metabolism, and increased lipid metabolism and metabolism of other amino acids while cocaine increased lipid metabolism in 12-month-old female mice. These results demonstrated that Tat expression and cocaine exposure resulted in significant changes of the gut microbiota in an age- and sex-dependent manner and provide additional evidence to support the bidirectional gut-brain axis hypothesis.
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HIV infection of astrocytes leads to restricted gene expression and replication but abundant expression of HIV early genes Tat, Nef and Rev. A great deal of neuroHIV research has so far been focused on Tat protein, its effects on astrocytes, and its roles in neuroHIV. In the current study, we aimed to determine effects of Nef expression on astrocytes and their function. Using transfection or infection of VSVG-pseudotyped HIV viruses, we showed that Nef expression down-modulated glial fibrillary acidic protein (GFAP) expression. We then showed that Nef expression also led to decreased GFAP mRNA expression. The transcriptional regulation was further confirmed using a GFAP promoter-driven reporter gene assay. We performed transcription factor profiling array to compare the expression of transcription factors between Nef-intact and Nef-deficient HIV-infected cells and identified eight transcription factors with expression changes of 1.5-fold or higher: three up-regulated by Nef (Stat1, Stat5, and TFIID), and five down-regulated by Nef (AR, GAS/ISRE, HIF, Sp1, and p53). We then demonstrated that removal of the Sp1 binding sites from the GFAP promoter resulted in a much lower level of the promoter activity and reversal of Nef effects on the GFAP promoter, confirming important roles of Sp1 in the GFAP promoter activity and for Nef-induced GFAP expression. Lastly, we showed that Nef expression led to increased glutamate uptake and decreased glutamate release by astrocytes and increased astrocyte proliferation. Taken together, these results indicate that Nef leads to down-modulation of GFAP expression and alteration of glutamate metabolism in astrocytes, and astrocyte proliferation and could be an important contributor to neuroHIV.
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HIV infects astrocytes in a restricted manner but leads to abundant expression of Nef, a major viral factor for HIV replication and disease progression. However, the roles of Nef in HIV gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells remain largely unclear. In this study, we attempted to address these issues by transfecting human primary astrocytes with HIV molecular clones with intact Nef and without Nef (a nonsense Nef mutant) and comparing gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells MT4. First, we found that lack of Nef expression led to increased extracellular virus production from astrocytes and intracellular viral protein and RNA expression in astrocytes. Using a HIV LTR-driven luciferase reporter gene assay, we showed that ectopic Nef expression alone inhibited the HIV LTR promoter activity in astrocytes. Consistent with the previously established function of Nef, we showed that the infectivity of HIV derived from astrocytes with Nef expression was significantly higher than that with no Nef expression. Next, we performed the co-culture assay to determine HIV transfer from astrocytes transfected to MT4. We showed that lack of Nef expression led to significant increase in HIV transfer from astrocytes to MT4 using two HIV clones. We also used Nef-null HIV complemented with Nef in trans in the co-culture assay and demonstrated that Nef expression led to significantly decreased HIV transfer from astrocytes to MT4. Taken together, these findings support a negative role of Nef in HIV replication and pathogenesis in astrocytes.
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Infecciones por VIH , VIH-1 , Humanos , Linfocitos T , Astrocitos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Replicación Viral/genética , VIH-1/genética , Linfocitos T CD4-Positivos , Infecciones por VIH/genética , Expresión GénicaRESUMEN
To date, there are a small number of nuclear-restricted proteins that have been reported to play a role in NF-κB signaling. However, the exact molecular mechanisms are not fully understood. Tip110 is a nuclear protein that has been implicated in multiple biological processes. In a previous study, we have shown that Tip110 interacts with oncogenic ubiquitin specific peptidase 15 (USP15) and that ectopic expression of Tip110 leads to re-distribution of USP15 from the cytoplasm to the nucleus. USP15 is known to regulate NF-κB activity through several mechanisms including modulation of IκBα ubiquitination. These findings prompted us to investigate the role of Tip110 in the NF-κB signaling pathway. We showed that Tip110 regulates NF-κB activity. The expression of Tip110 potentiated TNF-α-induced NF-κB activity and deletion of the nuclear localization domain in Tip110 abrogated this potentiation activity. We then demonstrated that Tip110 altered IκBα phosphorylation and stability in the presence of TNF-α. Moreover, we found that Tip110 and USP15 opposingly regulated NF-κB activity by targeting IκBα protein stability. We further showed that Tip110 altered the expression of NF-κB-dependent proinflammatory cytokines. Lastly, by using whole-transcriptome analysis of Tip110 knockout mouse embryonic stem cells, we found several NF-κB and NF-κB-related pathways were dysregulated. Taken together, these findings add to the nuclear regulation of NF-κB activity by Tip110 through IκBα stabilization and provide new evidence to support the role of Tip110 in controlling cellular processes such as cancers that involve proinflammatory responses.
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Cocaine use is a major comorbidity of HIV-associated neurocognitive disorder (HAND). In this study, we show that cocaine exposure worsens the learning and memory of doxycycline-inducible and brain-specific HIV Tat transgenic mice (iTat) and results in 14,838 hypermethylated CpG-related differentially methylated regions (DMRs) and 15,800 hypomethylated CpG-related DMRs, which are linked to 52 down- and 127 upregulated genes, respectively, in the hippocampus of iTat mice. These genes are mostly enriched at the neuronal function-, cell morphology-, and synapse formation-related extracellular matrix (ECM) receptor-ligand interaction pathway and mostly impacted in microglia. The accompanying neuropathological changes include swollen dendritic spines, increased synaptophysin expression, and diminished glial activation. We also find that sex (female) and age additively worsen the behavioral and pathological changes. These findings together indicate that chronic cocaine and long-term Tat expression interactively contribute to HAND, likely involving changes of DNA methylation and ECM receptor-ligand interactions.
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Cocaína , Animales , Cocaína/farmacología , ADN/metabolismo , Metilación de ADN/genética , Femenino , Expresión Génica , Ligandos , Trastornos de la Memoria/genética , Ratones , Ratones Transgénicos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.
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Replicón , SARS-CoV-2 , Antivirales/farmacología , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
"Bugs as drugs" in medicine encompasses the use of microbes to enhance the efficacy of vaccination, such as the delivery of vaccines by Leishmania-the protozoan etiological agent of leishmaniasis. This novel approach is appraised in light of the successful development of vaccines for Covid-19. All relevant aspects of this pandemic are summarized to provide the necessary framework in contrast to leishmaniasis. The presentation is in a side-by-side matching format with particular emphasis on vaccines. The comparative approach makes it possible to highlight the timeframe of the vaccine workflows condensed by the caveats of pandemic urgency and, at the same time, provides the background of Leishmania behind its use as a vaccine carrier. Previous studies in support of the latter are summarized as follows. Leishmaniasis confers life-long immunity on patients after cure, suggesting the effective vaccination is achievable with whole-cell Leishmania. A new strategy was developed to inactivate these cells in vitro, rendering them non-viable, hence non-disease causing, albeit retaining their immunogenicity and adjuvanticity. This was achieved by installing a dual suicidal mechanism in Leishmania for singlet oxygen (1O2)-initiated inactivation. In vitro cultured Leishmania were genetically engineered for cytosolic accumulation of UV-sensitive uroporphyrin I and further loaded endosomally with a red light-sensitive cationic phthalocyanine. Exposing these doubly dye-loaded Leishmania to light triggers intracellular production of highly reactive but extremely short-lived 1O2, resulting in their rapid and complete inactivation. Immunization of susceptible animals with such inactivated Leishmania elicited immunity to protect them against experimental leishmaniasis. Significantly, the inactivated Leishmania was shown to effectively deliver transgenically add-on ovalbumin (OVA) to antigen-presenting cells (APC), wherein OVA epitopes were processed appropriately for presentation with MHC molecules to activate epitope-specific CD8+ T cells. Application of this approach to deliver cancer vaccine candidates, e.g., enolase-1, was shown to suppress tumor development in mouse models. A similar approach is predicted to elicit lasting immunity against infectious diseases, including complementation of the spike protein-based vaccines in use for COVID-19. This pandemic is devastating, but brings to light the necessity of considering many facets of the disease in developing vaccination programs. Closer collaboration is essential among those in diverse disciplinary areas to provide the roadmap toward greater success in the future. Highlighted herein are several specific issues of vaccinology and new approaches worthy of consideration due to the pandemic.
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Background: Astrocytes become activated with certain infections, and this might alter the brain to trigger or worsen depressed mood. Indeed, astrocytes are chronically activated in people with HIV infection (PWH), who are much more frequently depressed than people without HIV (PWoH). A particularly disabling component of depression in PWH is apathy, a loss of interest, motivation, emotion, and goal-directed behavior. We tested the hypothesis that depression and apathy in PWH would be associated with higher levels of a biomarker of astrocyte activation, glial fibrillary acidic protein (GFAP), in cerebrospinal fluid (CSF). Methods: We evaluated PWH in a prospective observational study using the Beck Depression Inventory-II (BDI-II) and additional standardized assessments, including lumbar puncture. We measured GFAP in CSF with a customized direct sandwich ELISA method. Data were analyzed using ANOVA and multivariable regression. Results: Participants were 212 PWH, mean (SD) age 40.9±9.14 years, median (IQR) nadir and current CD4 199 (57, 326) and 411 (259, 579), 65.1% on ART, 67.3% virally suppressed. Higher CSF GFAP correlated with worse total BDI-II total scores (Pearson correlation r=0.158, p-value=0.0211), and with worse apathy scores (r=0.205, p=0.0027). The correlation between apathy/depression and GFAP was not in fluenced by other factors such as age or HIV suppression status. Conclusions: Astrocyte activation, reflected in higher levels of CSF GFAP, was associated with worse depression and apathy in PWH. Interventions to reduce astrocyte activation -- for example, using a peptide-1 receptor (GLP-1R) agonist -- might be studied to evaluate their impact on disabling depression in PWH.
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HIV-1 Tat-interacting protein of 110 kDa (Tip110; p110nrb/SART3) has been identified to be important for HIV gene transcription and several host gene expression. In this study, we showed that Tip110 was present in the 7SK snRNP through direct binding to MEPCE, a component of the 7SK snRNP complex. In addition, we found a positive association between Tip110 expression, change of HEXIM1 from dimer/oligomer to monomer, and release of HEXIM1 and P-TEFb from the 7SK snRNP complex. A similar association was also noted specifically in nuclear matrix as well as in chromatin where the free HEXIM1 and 7SK snRNP-bound HEXIM1 are located. Moreover, we demonstrated that Tip110 expression was linked to the glutathione metabolic pathway and the intracellular redox level, which in turn regulated HEXIM1 dimerization/oligomerization. Lastly, we performed the FRET microscopic analysis and confirmed the direct relationship between Tip110 expression and HEXIM1 dimerization/oligomerization in vivo. Taken together, these results identified a new mechanism governing HEXIM1 dimerization/oligomerization and the release of HEXIM1 and P-TEFb from the 7SK snRNP complex. These results also yield new insights to the roles of Tip110 in HIV gene transcription and replication.
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HIV-associated neurocognitive disorders (HAND) in the era of combination antiretroviral therapy are primarily manifested as impaired behaviours, glial activation/neuroinflammation and compromised neuronal integrity, for which there are no effective treatments currently available. In the current study, we used doxycycline-inducible astrocyte-specific HIV Tat transgenic mice (iTat), a surrogate HAND model, and determined effects of PNU-125096, a positive allosteric modulator of α7 nicotinic acetylcholine receptor (α7 nAChR) on Tat-induced behavioural impairments and neuropathologies. We showed that PNU-125096 treatment significantly improved locomotor, learning and memory deficits of iTat mice while inhibited glial activation and increased PSD-95 expression in the cortex and hippocampus of iTat mice. Using α7 nAChR knockout mice, we showed that α7 nAChR knockout eliminated the protective effects of PNU-125096 on iTat mice. In addition, we showed that inhibition of p38 phosphorylation by SB239063, a p38 MAPK-specific inhibitor exacerbated Tat neurotoxicity in iTat mice. Last, we used primary mouse cortical individual cultures and neuron-astrocytes co-cultures and in vivo staining of iTat mouse brain tissues and showed that glial activation was directly involved in the interplay among Tat neurotoxicity, α7 nAChR activation and the p38 MAPK signalling pathway. Taken together, these findings demonstrated for the first time that α7 nAChR activation led to protection against HAND and suggested that α7 nAChR modulator PNU-125096 holds significant promise for development of therapeutics for HAND.
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Complejo SIDA Demencia/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/toxicidadRESUMEN
This special issue contains 10 invited review papers that highlighted and extended the presentations at the NIDA-sponsored workshop "Unraveling NeuroAIDS in the Presence of Substance Use Disorders" at the 25th Society on NeuroImmune Pharmacology conference in 2019. The topics covered by these papers focused on the interactive, additive or synergistic effects of substance use disorders (SUD) with HIV infection on the immune system and on neuropathogenesis. These papers reviewed four categories of substances of abuse (opioids, tobacco, stimulants, and cannabis) and how comorbid HIV infection (including models with HIV proteins, HIV transgenic rodent models and SIV) might further impact the dysregulated dopaminergic and immune systems, and the subsequent neuropathogenesis and behavioral disorders known as HIV-associated neurological disorders (HAND). These reviews provided detailed background knowledge regarding how each of these addictive substances and HIV individually or collectively affected the immune system at the cellular, molecular and system levels, and the subsequent clinical and behavioral outcomes. The authors also identified gaps, confounds or constraints in the current disease models and approaches, and proposed future research directions.
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Complejo SIDA Demencia/epidemiología , Congresos como Asunto , Infecciones por VIH/epidemiología , Trastornos Relacionados con Sustancias/epidemiología , Complejo SIDA Demencia/diagnóstico , Complejo SIDA Demencia/terapia , Infecciones por VIH/diagnóstico , Infecciones por VIH/terapia , Humanos , Trastornos Relacionados con Sustancias/diagnóstico , Trastornos Relacionados con Sustancias/terapia , Revisiones Sistemáticas como Asunto/métodosRESUMEN
Tobacco smoking is highly prevalent among HIV-infected individuals. Chronic smokers with HIV showed greater cognitive deficits and impulsivity, and had more psychopathological symptoms and greater neuroinflammation than HIV non-smokers or smokers without HIV infection. However, preclinical studies that evaluated the combined effects of HIV-infection and tobacco smoking are scare. The preclinical models typically used cell cultures or animal models that involved specific HIV viral proteins or the administration of nicotine to rodents. These preclinical models consistently demonstrated that nicotine had neuroprotective and anti-inflammatory effects, leading to cognitive enhancement. Although the major addictive ingredient in tobacco smoking is nicotine, chronic smoking does not lead to improved cognitive function in humans. Therefore, preclinical studies designed to unravel the interactive effects of chronic tobacco smoking and HIV infection are needed. In this review, we summarized the preclinical studies that demonstrated the neuroprotective effects of nicotine, the neurotoxic effects of the HIV viral proteins, and the scant literature on nicotine or tobacco smoke in HIV transgenic rat models. We also reviewed the clinical studies that evaluated the neurotoxic effects of tobacco smoking, HIV infection and their combined effects on the brain, including studies that evaluated the cognitive and behavioral assessments, as well as neuroimaging measures. Lastly, we compared the different approaches between preclinical and clinical studies, identified some gaps and proposed some future directions. Graphical abstract Independent and combined effects of HIV and tobacco/nicotine. Left top and bottom panels: Both clinical studies of HIV infected persons and preclinical studies using viral proteins in vitro or in vivo in animal models showed that HIV infection could lead to neurotoxicity and neuroinflammation. Right top and bottom panels: While clinical studies of tobacco smoking consistently showed deleterious effects of smoking, clinical and preclinical studies that used nicotine show mild cognitive enhancement, neuroprotective and possibly anti-inflammatory effects. In the developing brain, however, nicotine is neurotoxic. Middle overlapping panels: Clinical studies of persons with HIV who were smokers typically showed additive deleterious effects of HIV and tobacco smoking. However, in the preclinical studies, when nicotine was administered to the HIV-1 Tg rats, the neurotoxic effects of HIV were attenuated, but tobacco smoke worsened the inflammatory cascade.
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Encéfalo/efectos de los fármacos , Infecciones por VIH/epidemiología , Nicotina/administración & dosificación , Fumar Tabaco/epidemiología , Animales , Encéfalo/diagnóstico por imagen , Ensayos Clínicos como Asunto/métodos , Cognición/efectos de los fármacos , Cognición/fisiología , Evaluación Preclínica de Medicamentos/métodos , Infecciones por VIH/diagnóstico por imagen , Humanos , Fumar Tabaco/efectos adversosRESUMEN
HIV infects the central nervous system and causes HIV/neuroAIDS, which is predominantly manifested in the form of mild cognitive and motor disorder in the era of combination antiretroviral therapy. HIV Tat protein is known to be a major pathogenic factor for HIV/neuroAIDS through a myriad of direct and indirect mechanisms. However, most, if not all of studies involve short-time exposure of recombinant Tat protein in vitro or short-term Tat expression in vivo. In this study, we took advantage of the doxycycline-inducible brain-specific HIV-1 Tat transgenic mouse model, fed the animals for 12 months, and assessed behavioral, pathological, and epigenetic changes in these mice. Long-term Tat expression led to poorer short-and long-term memory, lower locomotor activity and impaired coordination and balance ability, increased astrocyte activation and compromised neuronal integrity, and decreased global genomic DNA methylation. There were sex- and brain region-dependent differences in behaviors, pathologies, and epigenetic changes resulting from long-term Tat expression. All these changes are reminiscent of accelerated aging, raising the possibility that HIV Tat contributes, at least in part, to HIV infection-associated accelerated aging in HIV-infected individuals. These findings also suggest another utility of this model for HIV infection-associated accelerated aging studies.
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In the original article the name of author Chris Sanborn was misspelled. It is correct here. Also, in the Acknowledgments section there was an error in the NIH/NINDS grant numbers.
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Astrocytes are susceptible to HIV infection and potential latent HIV reservoirs. Tat is one of three abundantly expressed HIV early genes in HIV-infected astrocytes and has been shown to be a major pathogenic factor for HIV/neuroAIDS. In this study, we sought to determine if and how Tat expression would affect HIV infection and latency in astrocytes. Using the glycoprotein from vesicular stomatitis virus-pseudotyped red-green HIV (RGH) reporter viruses, we showed that HIV infection was capable of establishing HIV latency in astrocytes. We also found that Tat expression decreased the generation of latent HIV-infected cells. Activation of latent HIV-infected astrocytes showed that treatment of GSK126, a selective inhibitor of methyltransferase enhancer of zeste homolog 2 (Ezh2) that is specifically responsible for tri-methylation of histone 3 lysine 27 (H3K27me3), led to activation of significantly more latent HIV-infected Tat-expressing astrocytes. Molecular analysis showed that H3K27me3, Ezh2, MeCP2, and Tat all exhibited a similar bimodal expression kinetics in the course of HIV infection and latency in astrocytes, although H3K27me3, Ezh2, and MeCP2 were expressed higher in Tat-expressing astrocytes and their expression were peaked immediately preceding Tat expression. Subsequent studies showed that Tat expression alone was sufficient to induce H3K27me3 expression, likely through its regulation of Ezh2 and MeCP2 expression. Taken together, these results showed for the first time that Tat expression induced H3K27me3 expression and contributed to HIV latency in astrocytes and suggest a new role and novel mechanism for Tat in HIV latency.
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Astrocitos/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Histonas/genética , Interacciones Huésped-Patógeno/genética , Proteína 2 de Unión a Metil-CpG/genética , Latencia del Virus/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Astrocitos/efectos de los fármacos , Astrocitos/virología , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Histonas/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Indoles/farmacología , Células Jurkat , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Metilación , Piridonas/farmacología , Transducción de Señal , Vesiculovirus/genética , Vesiculovirus/metabolismo , Latencia del Virus/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Proteína Fluorescente RojaRESUMEN
Tip110, an important regulator of several oncogenic proteins, was significantly downregulated in human metastatic melanoma cells exposed to a hypoxic condition. Therefore, in this study, we set to determine whether differential expression of Tip110 could be an important indicator for melanoma tumorigenesis and metastasis. We found that in melanoma, but not in other cancer types, Tip110 knockdown enhanced significant expression and secretion of IL-8 and melanoma cells invasions. This induction was further potentiated under hypoxia and by inflammatory cytokine and found independent of TNF-α autocrine signaling. We further showed that Tip110 knockdown-mediated IL-8 induction involved IL-8 mRNA stability. Furthermore, the transcriptomic profiling data and survival from 455 melanoma patients demonstrated that the correlation between Tip110 expression and the clinical outcomes in melanoma was stage-dependent. These findings uncover important roles of Tip110 in melanoma tumorigenesis and metastasis through regulation of IL-8 and hope to provide new clues for future therapeutic strategies.
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Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Perfilación de la Expresión Génica/métodos , Interleucina-8/genética , Melanoma/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-8/química , Interleucina-8/metabolismo , Masculino , Melanoma/genética , Melanoma/patología , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Estabilidad del ARNRESUMEN
Tip110 plays important roles for stem cell pluripotency and hematopoiesis. However, little is known about the regulatory mechanisms of Tip110 expression in this process. In this study, we first showed that constitutive Tip110 expression was cell proliferation and differentiation dependent and self-regulated in both human cord blood CD34+ cells. Using a series of molecular techniques, we found that ectopic Tip110 expression led to increased constitutive Tip110 expression through its 3'-untranslated region (3'UTR), specifically through preferential usage of proximal polyadenylation sites within its 3'UTR in cells, including human cord blood CD34+ cells, which indeed led to an increased number of CD34+ cells during differentiation of those cells. Lastly, we showed that Tip110 protein interacted with cleavage stimulation factor 64 (CstF64) protein and that more CstF64 was recruited to the promixal polyadenylation site than the distal polyadenylation site within its 3'UTR. These finding together demonstrates that constitutive Tip110 expression is regulated, at least in part, through its interaction with CstF64, recruitment of CstF64 to, and selective usage of those two polyadenylation sites within its 3'UTR.