RESUMEN
Cellular cholesterol plays an important role in influenza A virus (IAV) endocytosis and replication. However, how IAV infection regulates cholesterol biosynthesis remains poorly understood. Here, we report that IAV infection activates SREBP2 and induces the expression of HMGCR, a rate-limiting enzyme in cholesterol synthesis pathway. SREBP2 deficiency suppresses IAV-induced HMGCR expression and virus replication. Mechanistically, IAV infection activates JAK2 and STAT3, inhibition of JAK2 and STAT3 activity by their inhibitors or by gene knockout downregulates IAV-induced SREBP2 and HMGCR expression and IAV replication, reduces the content of cellular cholesterol and virus binding to host cells. Exogenous cholesterol reverses the inhibitory effect of S3I-201 and STAT3 deficiency on virus replication. STAT3 or JAK2 overexpression increases the expression of SREBP2 and its downstream target genes, leading to increased IAV replication. These observations collectively suggest that STAT3 activation facilitates IAV replication by inducing SREBP2 expression and increasing cholesterol biosynthesis.
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Caspase-8, an aspartate-specific cysteine protease that primarily functions as an initiator caspase to induce apoptosis, can downregulate innate immunity in part by cleaving RIPK1 and IRF3. However, patients with caspase-8 mutations or deficiency develop immunodeficiency and are prone to viral infections. The molecular mechanism underlying this controversy remains unknown. Whether caspase-8 enhances or suppresses antiviral responses against influenza A virus (IAV) infection remains to be determined. Here, we report that caspase-8 is readily activated in A549 and NL20 cells infected with the H5N1, H5N6, and H1N1 subtypes of IAV. Surprisingly, caspase-8 deficiency and two caspase-8 inhibitors, Z-VAD and Z-IETD, do not enhance but rather downregulate antiviral innate immunity, as evidenced by decreased TBK1, IRF3, IκBα, and p65 phosphorylation, decreased IL-6, IFN-ß, MX1, and ISG15 gene expression; and decreased IFN-ß production but increased virus replication. Mechanistically, caspase-8 cleaves and inactivates CYLD, a tumor suppressor that functions as a deubiquitinase. Caspase-8 inhibition suppresses CYLD cleavage, RIG-I and TAK1 ubiquitination, and innate immune signaling. In contrast, CYLD deficiency enhances IAV-induced RIG-I and TAK1 ubiquitination and innate antiviral immunity. Neither caspase-3 deficiency nor treatment with its inhibitor Z-DEVD affects CYLD cleavage or antiviral innate immunity. Our study provides evidence that caspase-8 activation in two human airway epithelial cell lines does not silence but rather enhances innate immunity by inactivating CYLD.
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Caspasa 8 , Proteína 58 DEAD Box , Enzima Desubiquitinante CYLD , Inmunidad Innata , Virus de la Influenza A , Gripe Humana , Quinasas Quinasa Quinasa PAM , Ubiquitinación , Humanos , Enzima Desubiquitinante CYLD/metabolismo , Enzima Desubiquitinante CYLD/genética , Caspasa 8/metabolismo , Caspasa 8/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/inmunología , Virus de la Influenza A/inmunología , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Células A549 , Animales , Transducción de Señal/inmunología , Receptores InmunológicosRESUMEN
Many DNA viruses develop various strategies to inhibit cell death to facilitate their replication. However, whether influenza A virus (IAV), a fast-replicating RNA virus, attenuates cell death remains unknown. Here, we report that IAV infection induces TAK1 phosphorylation in a murine alveolar epithelial cell line (LET1) and a murine fibroblastoma cell line (L929). The TAK1-specific inhibitor 5Z-7-Oxzeneonal (5Z) and TAK1 knockout significantly enhance IAV-induced apoptosis, as evidenced by increased PARP, caspase-8, and caspase-3 cleavage. TAK1 inhibition also increases necroptosis as evidenced by increased RIPK1S166, RIPK3T231/S232, and MLKLS345 phosphorylation. Mechanistically, TAK1 activates IKK, which phosphorylates RIPK1S25 and inhibits its activation. TAK1 also activates p38 and its downstream kinase MK2, which phosphorylates RIPK1S321 but does not affect RIPK1 activation. Further investigation revealed that the RIPK1 inhibitor Nec-1 and RIPK1 knockout abrogate IAV-induced apoptosis and necroptosis; re-expression of wild-type but not kinase-dead (KD)-RIPK1 restores IAV-induced cell death. ZBP1 knockout abrogates IAV-induced cell death, whereas RIPK3 knockout inhibits IAV-induced necroptosis but not apoptosis. 5Z treatment enhances IAV-induced cell death and slightly reduces the inflammatory response in the lungs of H1N1 virus-infected mice and prolongs the survival of IAV-infected mice. Our study provides evidence that IAV activates TAK1 to suppress RIPK1-dependent apoptosis and necroptosis, and that RIPK3 is required for IAV-induced necroptosis but not apoptosis in epithelial cells.
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Apoptosis , Quinasas Quinasa Quinasa PAM , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Fosforilación , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/patología , Línea Celular , Virus de la Influenza A/fisiología , Ratones Endogámicos C57BL , HumanosRESUMEN
BACKGROUND: The Porcine Epidemic Diarrhea Virus (PEDV) has caused significant economic losses in the global swine industry. As a potential drug for treating diarrhea, the antiviral properties of attapulgite deserve further study. METHODS: In this study, various methods such as RT-qPCR, Western blot, viral titer assay, Cytopathic Effect, immunofluorescence analysis and transmission electron microscopy were used to detect the antiviral activity of attapulgite and to assess its inhibitory effect on PEDV. RESULTS: When exposed to the same amount of virus, there was a significant decrease in the expression of the S protein, resulting in a viral titer reduction from 10-5.613 TCID50/mL to 10-2.90 TCID50/mL, which represents a decrease of approximately 102.6 folds. Results of cytopathic effect and indirect immunofluorescence also indicate a notable decrease in viral infectivity after attapulgite treatment. Additionally, it was observed that modified materials after acidification had weaker antiviral efficacy compared to powdered samples that underwent ultrasonic disintegration, which showed the strongest antiviral effects. CONCLUSION: As a result, Attapulgite powders can trap and adsorb viruses to inhibit PEDV in vitro, leading to loss of viral infectivity. This study provides new materials for the development of novel disinfectants and antiviral additives.
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Antivirales , Virus de la Diarrea Epidémica Porcina , Compuestos de Silicona , Virus de la Diarrea Epidémica Porcina/efectos de los fármacos , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/fisiología , Animales , Antivirales/farmacología , Compuestos de Silicona/farmacología , Compuestos de Silicona/química , Chlorocebus aethiops , Compuestos de Magnesio/farmacología , Porcinos , Células Vero , Carga Viral/efectos de los fármacos , Efecto Citopatogénico Viral/efectos de los fármacos , Enfermedades de los Porcinos/virología , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Microscopía Electrónica de TransmisiónRESUMEN
Lipophagy is a subset of selective autophagy that specifically degrades lipid droplets and plays an important role in obesity. Leflunomide treatment in rheumatoid arthritis (RA) patients has been associated with weight loss and decreased blood glucose levels, which cannot be attributed to its known side effects. Our prior studies showed that A77 1726, the active metabolite of leflunomide, acts as an inhibitor of S6K1 to sensitize the insulin receptor and control hyperglycemia. Whether the anti-obesity effect of leflunomide is mediated by targeting S6K1 and its underlying mechanisms remain unclear. Here, we report that A77 1726 induced LC3 lipidation and increased the formation of autophagosomes and lipoautolysosomes in 3T3-L1 adipocytes by activating TGF-ß-activated kinase 1 (TAK1), AMP-activated kinase (AMPK), and Unc-51 like autophagy-activated kinase 1 (ULK1). A77 1726 reduced the content of lipid droplets in 3T3-L1 adipocytes, which was blocked by bafilomycin or by beclin-1 knockdown. Similar observations were made in murine adipocytes differentiated from S6K1-/- embryonic fibroblasts (MEFs). Leflunomide treatment restricted bodyweight gains in ob/ob mice and reduced the visceral fat deposit and the size of adipocytes. Leflunomide treatment induced autophagy in adipose and liver tissues and reduced hepatic lipid contents. Consistently, S6K1 knockout increased the levels of LC3 lipidation in the liver, muscle, and fat of S6K-/- mice. Leflunomide treatment and S6K1 deficiency both induced TAK1, AMPK, and ULK1 phosphorylation in these tissues. These observations collectively suggest that leflunomide controls obesity in part by activating AMPK and inducing lipophagy. Our study provides insights into the mechanisms of leflunomide-mediated anti-obesity activity.
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Proteínas Quinasas Activadas por AMP , Autofagia , Ratones , Humanos , Animales , Leflunamida/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Obesidad/tratamiento farmacológicoRESUMEN
B-Raf kinase inhibitors such as vemurafenib (PLX4032) and dabrafenib have limited therapeutic efficacy on BRAF-mutated thyroid cancer. Cancer stem cells (CSCs) play important roles in tumor recurrence, drug resistance, and metastasis. Whether CSCs play a role in dampening the antitumor activity of B-Raf kinase inhibitors remains unknown. Here, we report that vemurafenib (PLX4032) induced the expression of several stemness-related genes including Gli1, Snail, BMI1, and SOX2 in two anaplastic thyroid cancer cell lines, SW1736 and 8505C, but decreased the expression of these genes in A375 cells, a human melanoma cell line. PLX4032 promoted thyroid cancer stem cell self-renewal, as evidenced by increased numbers of aldehyde dehydrogenase-positive cells and thyrospheres. Mechanistically, PLX4032 activates the PI-3 and mitogen-activated protein kinase pathways through HER3 to cross-activate Gli1, a transcription factor of the sonic hedgehog (Shh) pathway. GANT61, a specific inhibitor of Gli1, blocked the expression of the stemness-related genes in PLX4032-treated thyroid cancer cells in vitro and in vivo in two thyroid cancer xenograft models. GANT61 treatment alone weakly inhibited SW1736 tumor growth but enhanced the antitumor activity of PLX4032 when used in combination. Our study provides mechanistic insights into how thyroid cancer poorly responds to B-Raf kinase inhibitors and suggests that targeting B-Raf and the Shh pathway in combination may overcome thyroid cancer drug resistance.
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Proteínas Hedgehog , Neoplasias de la Tiroides , Humanos , Vemurafenib/farmacología , Vemurafenib/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/uso terapéutico , Autorrenovación de las Células , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Indoles/farmacología , Indoles/uso terapéutico , Línea Celular Tumoral , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias de la Tiroides/genética , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P â+ âM227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P â+ âM227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.
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Subtipo H9N2 del Virus de la Influenza A , Gripe Aviar , Infecciones por Orthomyxoviridae , Humanos , Animales , Ratones , Cobayas , Subtipo H9N2 del Virus de la Influenza A/genética , Virulencia , Hemaglutininas , Proteínas Virales/genética , Proteínas Virales/metabolismo , Mutación , Mamíferos/metabolismoRESUMEN
Autophagy is a highly conserved cellular process that profoundly impacts the efficacy of genotoxic chemotherapeutic drugs. TGF-ß-activated kinase 1 (TAK1) is a serine/threonine kinase that activates several signaling pathways involved in inducing autophagy and suppressing cell death. Xanthine oxidoreductase (XOR) is a rate-limiting enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid and hydrogen peroxide in the purine catabolism pathway. Recent studies showed that uric acid can bind to TAK1 and prolong its activation. We hypothesized that genotoxic drugs may induce autophagy and apoptosis resistance by activating TAK1 through XOR-generated uric acid. Here, we report that gemcitabine and 5-fluorouracil (5-FU), two genotoxic drugs, induced autophagy in HeLa and HT-29 cells by activating TAK1 and its two downstream kinases, AMP-activated kinase (AMPK) and c-Jun terminal kinase (JNK). XOR knockdown and the XOR inhibitor allopurinol blocked gemcitabine-induced TAK1, JNK, AMPK, and Unc51-like kinase 1 (ULK1)S555 phosphorylation and gemcitabine-induced autophagy. Inhibition of the ATM-Chk pathway, which inhibits genotoxic drug-induced uric acid production, blocked gemcitabine-induced autophagy by inhibiting TAK1 activation. Exogenous uric acid in its salt form, monosodium urate (MSU), induced autophagy by activating TAK1 and its downstream kinases JNK and AMPK. Gene knockdown or the inhibitors of these kinases blocked gemcitabine- and MSU-induced autophagy. Inhibition of autophagy by allopurinol, chloroquine, and 5Z-7-oxozeaenol (5Z), a TAK1-specific inhibitor, enhanced gemcitabine-induced apoptosis. Our study uncovers a previously unrecognized role of XOR in regulating genotoxic drug-induced autophagy and apoptosis and has implications for designing novel therapeutic strategies for cancer treatment.
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Ácido Úrico , Xantina Deshidrogenasa , Humanos , Ácido Úrico/farmacología , Ácido Úrico/metabolismo , Xantina Deshidrogenasa/genética , Xantina Deshidrogenasa/metabolismo , Alopurinol , Proteínas Quinasas Activadas por AMP/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Autofagia , Daño del ADN , ApoptosisRESUMEN
Autophagy plays an important role in defending against invading microbes. However, numerous viruses can subvert autophagy to benefit their replication. Porcine epidemic diarrhoea virus (PEDV) is an aetiological agent that causes severe porcine epidemic diarrhoea. How PEDV infection regulates autophagy and its role in PEDV replication are inadequately understood. Herein, we report that PEDV induced complete autophagy in Vero and IPEC-DQ cells, as evidenced by increased LC3 lipidation, p62 degradation, and the formation of autolysosomes. The lysosomal protease inhibitors chloroquine (CQ) or bafilomycin A and Beclin-1 or ATG5 knockdown blocked autophagic flux and inhibited PEDV replication. PEDV infection activated AMP-activated protein kinase (AMPK) and c-Jun terminal kinase (JNK) by activating TGF-beta-activated kinase 1 (TAK1). Compound C (CC), an AMPK inhibitor, and SP600125, a JNK inhibitor, inhibited PEDV-induced autophagy and virus replication. AMPK activation led to increased ULK1S777 phosphorylation and activation. Inhibition of ULK1 activity by SBI-0206965 (SBI) and TAK1 activity by 5Z-7-Oxozeaenol (5Z) or by TAK1 siRNA led to the suppression of autophagy and virus replication. Our study provides mechanistic insights into PEDV-induced autophagy and how PEDV infection leads to JNK and AMPK activation.
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Virus de la Diarrea Epidémica Porcina , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Autofagia , Beclina-1 , Cloroquina , Quinasas Quinasa Quinasa PAM , Virus de la Diarrea Epidémica Porcina/fisiología , Inhibidores de Proteasas , ARN Interferente Pequeño , Porcinos , Replicación ViralRESUMEN
XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Factor 1 Regulador del Interferón , Infecciones por Virus ARN , Virus ARN , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/inmunología , Humanos , Inmunidad Innata , Factor 1 Regulador del Interferón/inmunología , Ratones , Ratones Noqueados , Infecciones por Virus ARN/inmunología , Replicación ViralRESUMEN
The H5N1 subtype of the avian influenza virus causes sporadic but fatal infections in humans. H5N1 virus infection leads to the disruption of the alveolar epithelial barrier, a pathologic change that often progresses into acute respiratory distress syndrome (ARDS) and pneumonia. The mechanisms underlying this remain poorly understood. Here we report that H5N1 viruses downregulate the expression of intercellular junction proteins (E-cadherin, occludin, claudin-1, and ZO-1) in several cell lines and the lungs of H5N1 virus-infected mice. H5N1 virus infection activates TGF-ß-activated kinase 1 (TAK1), which then activates p38 and ERK to induce E3 ubiquitin ligase Itch expression and to promote occludin ubiquitination and degradation. Inhibition of the TAK1-Itch pathway restores the intercellular junction structure and function in vitro and in the lungs of H5N1 virus-infected mice. Our study suggests that H5N1 virus infection impairs the alveolar epithelial barrier by downregulating the expression of intercellular junction proteins at the posttranslational level.
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Células Epiteliales Alveolares , Subtipo H5N1 del Virus de la Influenza A , Ubiquitina-Proteína Ligasas , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , Animales , Uniones Intercelulares/genética , Uniones Intercelulares/metabolismo , Uniones Intercelulares/virología , Pulmón/patología , Pulmón/virología , Ratones , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
DNA damage by genotoxic drugs such as gemcitabine and 5-fluorouracil (5-FU) activates the ataxia telangiectasia, mutated (ATM)-Chk pathway and induces the expression of NKG2D ligands such as the MHC class I-related chain A and B (MICA/B). The mechanisms underlying this remain incompletely understood. Here we report that xanthine oxidoreductase (XOR), a rate-limiting enzyme that produces uric acid in the purine catabolism pathway, promotes DNA damage-induced MICA/B expression. Inhibition of the ATM-Chk pathway blocks genotoxic drug-induced uric acid production, TGF-ß-activated kinase 1 (TAK1) activation, ERK phosphorylation, and MICA/B expression. Inhibition of uric acid production by the XOR inhibitor allopurinol blocks DNA damage-induced TAK1 activation and MICA/B expression in genotoxic drug-treated cells. Exogenous uric acid activates TAK1, NF-κB, and the MAP kinase pathway. TAK1 inhibition blocks gemcitabine- and uric acid-induced MAP kinase activation and MICA/B expression. Exogenous uric acid in its salt form, monosodium urate (MSU), induces MICA/B expression and sensitizes tumor cells to NK cell killing. MSU immunization with irradiated murine breast cancer cell line RCAS-Neu retards breast cancer growth in syngeneic breast cancer models and delays breast cancer development in a somatic breast cancer model. Our study suggests that uric acid accumulation plays an important role in activating TAK1, inducing DNA damage-induced MICA/B expression, and enhancing antitumor immunity.
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Subfamilia K de Receptores Similares a Lectina de Células NK , Ácido Úrico , Animales , ADN , Daño del ADN , Ligandos , Quinasas Quinasa Quinasa PAM , Ratones , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Ácido Úrico/farmacologíaRESUMEN
A179L, a non-structural protein of African swine fever virus (ASFV), is capable of suppressing apoptosis by binding the BH3 domain of the pro-apoptotic Bcl-2 family proteins via a conserved ligand binding groove. Our present study aims to determine if A179L affects necroptosis, the second form of programmed cell death induced by DNA and RNA viruses. Here we report that A179L enhanced TNF-α or TSZ (TNF-α, Smac, and Z-Vad)-induced receptor-interacting protein kinase (RIPK1), RIPK3, and mixed lineage kinase domain like peudokinase (MLKL) phosphorylation in L929 cells, a murine fibrosarcoma cell line. Sytox green staining revealed that A179L significantly increased the number of necroptotic cells in TSZ-treated L929 cells. Using human herpes simplex virus 1 (HSV-1) to model DNA virus-induced cell death, we found that A179L blocked the HSV-1-induced cleavage of poly (ADP-ribose) polymerase (PARP), caspase 8, and caspase 3 and decreased the number of apoptotic cells in HSV-1-infected IPEC-DQ cells, a porcine intestinal epithelial cell line. In contrast, A179L transfection of IPEC-DQ cells enhanced HSV-1-induced RIPK1, RIPK3, and MLKL phosphorylation and increased the number of necroptotic cells. Consistently, A179L also suppressed apoptosis but enhanced the necroptosis induced by two RNA viruses, Sendai virus (SeV) and influenza virus (IAV). Our study uncovers a previously unrecognized role of A179L in regulating cell death and suggests that A179L re-directs its anti-apoptotic activity to necroptosis.
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Virus de la Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/fisiopatología , Proteínas Reguladoras de la Apoptosis/metabolismo , Necroptosis , Proteínas Virales/metabolismo , Fiebre Porcina Africana/genética , Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Interacciones Huésped-Patógeno , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Porcinos , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Virales/genéticaRESUMEN
The genotype S (G57) H9N2 virus, which first emerged in 2007 with the substitution of the G1-like PB2 gene for F98-like ones, has become the predominant genotype in the past 10 years. However, whether this substitution plays a role in the fitness of genotype S H9N2 viruses remains unknown. Comparison of the PB2 genes of F98-like and G1-like viruses revealed a close homology in amino acid sequences but great variations at nucleotide levels. We then determined if the packaging region, a unique sequence in each segment utilized for the assembly of the vRNA into virions, played a role in the fitness of the S genotype. The chimeric H9N2 virus with PB2 segments of the G1-like packaging regions significantly increased viral protein levels and polymerase activity. Substituting the packaging regions in the two terminals of F98-like PB2 with the sequence of G1-like further improved its competitive advantage. Substitution of the packaging regions of F98-like PB2 with those of G1-like sequences increased the infectivity of the chimeric virus in the lungs and brains of chicken at 3 days post infection (dpi) and extended the lengths of virus shedding time. Our study suggests that the packaging regions of the G1-like PB2 gene contribute to improve the survival advantage of the genotype S H9N2 virus in China.
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The sonic hedgehog (Shh) pathway is highly activated in a variety of malignancies and plays important roles in tumorigenesis, tumor growth, drug resistance, and metastasis. Our recent study showed that the inhibitors of the Shh pathway such as cyclopamine (CP), a Smothened (SMO) inhibitor, and GANT61, a Gli1 inhibitor, have modest inhibitory effects on thyroid tumor cell proliferation and tumor growth. The objective of this study was to determine whether autophagy was induced by inhibition of the Shh pathway and could negatively regulate GANT61-induced apoptosis. Here we report that inhibition of the Shh pathway by Gli1 siRNA or by cyclopamine and GANT61 induced autophagy in SW1736 and KAT-18 cells, two anaplastic thyroid cancer cell lines; whereas Gli1 overexpression suppressed autophagy. Mechanistic investigation revealed that inhibition of the Shh pathway activated TAK1 and its two downstream kinases, the c-Jun-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). GANT61-induced autophagy was blocked by TAK1 siRNA and the inhibitors of TAK1 (5Z-7-oxozeaenol, 5Z), JNK (SP600125), and AMPK (Compound C, CC). Inhibition of autophagy by chloroquine and 5Z and by TAK1 and Beclin-1 siRNA enhanced GANT61-induced apoptosis and its antiproliferative activity. Our study has shown that inhibition of the Shh pathway induces autophagy by activating TAK1, whereas autophagy in turn suppresses GANT61-induced apoptosis. We have uncovered a previously unrecognized role of TAK1 in Shh pathway inhibition-induced autophagy and apoptosis.
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Proteínas Hedgehog/antagonistas & inhibidores , Glándula Tiroides/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Apoptosis , Autofagia , Proteínas Hedgehog/metabolismo , Humanos , Transducción de Señal , TransfecciónRESUMEN
The sonic hedgehog (Shh) pathway plays important roles in tumorigenesis, tumor growth, drug resistance, and metastasis. We and others have reported earlier that this pathway is highly activated in thyroid cancer. However, its role in thyroid cancer stem cell (CSC) self-renewal and tumor development remains incompletely understood. B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and SRY-Box Transcription Factor 2 (SOX2) are two CSC-related transcription factors that have been implicated in promoting CSC self-renewal. The objective of our current investigation was to determine the role of the Shh pathway in regulating BMI1 and SOX2 expression in thyroid cancer and promoting thyroid tumor growth and development. Here we report that inhibition of the Shh pathway by Gli1 siRNA or by cyclopamine and GANT61 reduced BMI1 and SOX2 expression in SW1736 and KAT-18 cells, two anaplastic thyroid cancer cell lines. The opposite results were obtained in cells overexpressing Gli1 or its downstream transcription factor Snail. The Shh pathway regulated SOX2 and BMI1 expression at a transcriptional and post-transcriptional level, respectively. GANT61 treatment suppressed the growth of SW1736 CSC-derived tumor xenografts but did not significantly inhibit the growth of tumors grown from bulk tumor cells. Clinicopathological analyses of thyroid tumor specimens by immunohistochemical (IHC) staining revealed that BMI1 and SOX2 were highly expressed in thyroid cancer and correlated with Gli1 expression. Our study provides evidence that activation of the Shh pathway leads to increased BMI1 and SOX2 expression in thyroid cancer and promotes thyroid CSC-driven tumor initiation. Targeting the Shh pathway may have therapeutic value for treating thyroid cancer and preventing recurrence.
RESUMEN
Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.
Asunto(s)
Antivirales/farmacología , Crotonatos/farmacología , Virus del Moquillo Canino/efectos de los fármacos , Hidroxibutiratos/farmacología , Nitrilos/farmacología , Nucleótidos de Pirimidina/biosíntesis , Toluidinas/farmacología , Animales , Compuestos de Bifenilo/farmacología , Chlorocebus aethiops , Crotonatos/antagonistas & inhibidores , Medios de Cultivo Condicionados , Dihidroorotato Deshidrogenasa , Virus del Moquillo Canino/fisiología , Hidroxibutiratos/antagonistas & inhibidores , Imidazoles/farmacología , Quinasas Janus/antagonistas & inhibidores , Leflunamida/metabolismo , Nitrilos/antagonistas & inhibidores , Proteínas de la Nucleocápside/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Fosforilación , Piperazinas/farmacología , ARN Interferente Pequeño/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Toluidinas/antagonistas & inhibidores , Uridina/farmacología , Células Vero , Proteínas de la Matriz Viral/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Autophagy plays an important role in restricting the growth of invading intracellular microbes. Salmonella (S) Typhimurium, an intracellular pathogen that causes gastroenteritis and food poisoning in humans, evades autophagic detection by multiple mechanisms. There has been growing interest in developing autophagy inducers as novel antimicrobial agents for treating intracellular bacterial infections. We recently reported that A77 1726, the active metabolite of the anti-inflammatory drug leflunomide, induces autophagy by activating AMP-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase 1 (ULK1). Our present study aims to determine if A77 1726 was able to restrict intracellular Salmonella growth by inducing autophagy. We first confirmed the ability of A77 1726 to induce autophagy by activating the AMPK-ULK1 axis in uninfected RAW264.7 (a murine macrophage cell line) and HeLa cells (a human cervical carcinoma cell line). A77 1726 enhanced autophagy in S. Typhimurium-infected cells, as evidenced by increased levels of LC3 lipidation and increased numbers of autophagosomes and autolysosomes. Confocal microscopy revealed that A77 1726 induced xenophagy in macrophages, as evidenced by an increased number of LC3-coated bacteria in the cytoplasm. A77 1726 significantly decreased the number of intracellular S. Typhimurium in macrophages. Taken together, our study has demonstrated the ability of A77 1726 to restrict intracellular S. Typhimurium growth in vitro by enhancing xenophagy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia/efectos de los fármacos , Crotonatos/farmacología , Hidroxibutiratos/farmacología , Macrófagos/microbiología , Nitrilos/farmacología , Salmonella typhimurium/crecimiento & desarrollo , Toluidinas/farmacología , Proteínas Quinasas Activadas por AMP/genética , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Células HeLa , Humanos , Macrófagos/efectos de los fármacos , Ratones , Fosforilación , Células RAW 264.7 , Salmonella typhimurium/efectos de los fármacos , Transducción de SeñalRESUMEN
Janus kinase (JAK) inhibitors have been developed as novel immunomodulatory drugs and primarily used for treating rheumatoid arthritis and other inflammatory diseases. Recent studies have suggested that this category of anti-inflammatory drugs could be potentially useful for the control of inflammation "storms" in respiratory virus infections. In addition to their role in regulating immune cell functions, JAK1 and JAK2 have been recently identified as crucial cellular factors involved in influenza A virus (IAV) replication and could be potentially targeted for antiviral therapy. Gingerenone A (Gin A) is a compound derived from ginger roots and a dual inhibitor of JAK2 and p70 S6 kinase (S6K1). Our present study aimed to determine the antiviral activity of Gin A on influenza A virus (IAV) and to understand its mechanisms of action. Here, we reported that Gin A suppressed the replication of three IAV subtypes (H1N1, H5N1, H9N2) in four cell lines. IAV replication was also inhibited by Ruxolitinib (Rux), a JAK inhibitor, but not by PF-4708671, an S6K1 inhibitor. JAK2 overexpression enhanced H5N1 virus replication and attenuated Gin A-mediated antiviral activity. In vivo experiments revealed that Gin A treatment suppressed IAV replication in the lungs of H5N1 virus-infected mice, alleviated their body weight loss, and prolonged their survival. Our study suggests that Gin A restricts IAV replication by inhibiting JAK2 activity; Gin A could be potentially useful for the control of influenza virus infections.
Asunto(s)
Antivirales/farmacología , Diarilheptanoides/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H9N2 del Virus de la Influenza A/efectos de los fármacos , Janus Quinasa 2/antagonistas & inhibidores , Células A549 , Animales , Línea Celular , Perros , Femenino , Células HEK293 , Humanos , Imidazoles/farmacología , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H9N2 del Virus de la Influenza A/crecimiento & desarrollo , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Nitrilos , Piperazinas/farmacología , Pirazoles/farmacología , Pirimidinas , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
Astaxanthin (AST) is a carotenoid with therapeutic values on hyperglycemia and diabetic complications. The mechanisms of action of AST remain incompletely understood. p70 S6 kinase 1 (S6K1) is a serine/threonine kinase that phosphorylates insulin receptor substrate 1 (IRS-1)S1101 and desensitizes the insulin receptor (IR). Our present study aims to determine if AST improves glucose metabolisms by targeting S6K1. Western blot analysis revealed that AST inhibited the phosphorylation of two S6K1 substrates, S6S235/236 and IRS-1S1101, but enhanced the phosphorylation of AKTT308, AKTS473, and S6K1T389 by feedback activation of the phosphatidylinositol-3 (PI-3) kinase in 3T3-L1 adipocytes and L6 myotubes. In vitro kinase assays revealed that AST inhibited S6K1 activity with an IC50 value of approximately 13.8 µM. AST increased insulin-induced IR tyrosine phosphorylation and IRS-1 binding to the p85 subunit of PI-3 kinase. Confocal microscopy revealed that AST increased the translocation of the glucose transporter 4 (GLUT4) to the plasma membrane in L6 cells. Glucose uptake assays using a fluorescent dye, 2-NBDG (2-N-(Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), revealed that AST increased glucose uptake in 3T3-L1 adipocytes and L6 myotubes under insulin resistance conditions. Our study identifies S6K1 as a previously unrecognized molecular target of AST and provides novel insights into the mechanisms of action of AST on IR sensitization.