RESUMEN
The mechanism by which cells undergo death determines whether dying cells trigger inflammatory responses or remain immunologically silent. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. Here, we identify a mechanism by which mitochondria and downstream proapoptotic caspases regulate the activation of antiviral immunity. In the absence of active caspases, mitochondrial outer membrane permeabilization by Bax and Bak results in the expression of type I interferons (IFNs). This induction is mediated by mitochondrial DNA-dependent activation of the cGAS/STING pathway and results in the establishment of a potent state of viral resistance. Our results show that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response and to inactivate this response in a caspase-dependent manner. This mechanism provides a dual control, which determines whether mitochondria initiate an immunologically silent or a proinflammatory type of cell death.
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Apoptosis , Caspasas/metabolismo , Interferón Tipo I/metabolismo , Transducción de Señal , Animales , ADN Mitocondrial/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Interferón Tipo I/inmunología , Ratones , Ratones Noqueados , Virosis/inmunologíaRESUMEN
The cGAS-STING pathway is essential for immune defense against microbial pathogens and malignant cells; as such, STING is an attractive target for cancer immunotherapy. However, systemic administration of STING agonists poses safety issues while intratumoral injection is limited by tumor accessibility. Here, we generated antibody-drug conjugates (ADCs) by conjugating a STING agonist through a cleavable linker to antibodies targeting tumor cells. Systemic administration of these ADCs was well tolerated and exhibited potent antitumor efficacy in syngeneic mouse tumor models. The STING ADC further synergized with an anti-PD-L1 antibody to achieve superior antitumor efficacy. The STING ADC promoted multiple aspects of innate and adaptive antitumor immune responses, including activation of dendritic cells, T cells, natural killer cells and natural killer T cells, as well as promotion of M2 to M1 polarization of tumor-associated macrophages. These results provided the proof of concept for clinical development of the STING ADCs.
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Inmunoconjugados , Neoplasias , Animales , Ratones , Inmunoterapia , Factores Inmunológicos , Neoplasias/terapia , Macrófagos Asociados a TumoresRESUMEN
Antigen receptor-mediated nuclear factor κB (NF-κB) activation relies on the formation of a large multi-protein complex that contains CARMA1, BCL10, and MALT1 (CBM complex). This signalosome is pirated in the activated B-cell-like subgroup of diffuse large B-cell lymphoma (ABC DLBCL) to drive aberrant NF-κB activation, thereby promoting cell survival and propagation. Using an unbiased proteomic approach, we screened for additional components of the CBM in lymphocytes. We found that the linear ubiquitin chain assembly complex (LUBAC), which was previously linked to cytokine-mediated NF-κB activation, dynamically integrates the CBM and marshals NF-κB optimal activation following antigen receptor ligation independently of its catalytic activity. The LUBAC also participates in preassembled CBM complex in cells derived from ABC DLBCL. Silencing the LUBAC reduced NF-κB activation and was toxic in ABC DLBCL cell lines. Thus, our findings reveal a role for the LUBAC during lymphocyte activation and in B-cell malignancy.
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Linfoma/metabolismo , FN-kappa B/química , FN-kappa B/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Ubiquitina/metabolismo , Catálisis , Línea Celular Tumoral , Humanos , Células Jurkat , Activación de Linfocitos/fisiología , Linfoma/patología , Unión Proteica , Ubiquitinación/fisiologíaRESUMEN
Thermally-induced in-situ gelation of polymers and nanogels is of significant importance for injectable non-invasive tissue engineering and delivery systems of drug delivery system. In this study, we for the first time demonstrated that the interpenetrating (IPN) nanogel with two networks of poly (N-isopropylacrylamide) (PNIPAM) and poly (N-Acryloyl-l-phenylalanine) (PAphe) underwent a reversible temperature-triggered sol-gel transition and formed a structural color gel above the phase transition temperature (Tp). Dynamic light scattering (DLS) studies confirmed that the Tp of IPN nanogels are the same as that of PNIPAM, independent of Aphe content of the IPN nanogels at pH of 6.5 â¼ 7.4. The rheological and optical properties of IPN nanogels during sol-gel transition were studied by rheometer and optical fiber spectroscopy. The results showed that the gelation time of the hydrogel photonic crystals assembled by IPN nanogel was affected by temperature, PAphe composition, concentration, and sequence of interpenetration. As the temperature rose above the Tp, the Bragg reflection peak of IPN nanogels exhibited blue shift due to the shrinkage of IPN nanogels. In addition, these colored IPN nanogels demonstrated good injectability and had no obvious cytotoxicity. These IPN nanogels will open an avenue to the preparation and thermally-induced in-situ gelation of novel NIPAM-based nanogel system.
RESUMEN
Photonic ionogels with dual electrical and optical output have been intensively studied. However, tunable temperature-responsive photonic ionogel assembled by thermosensitive nanogels has not been studied yet. Herein, an innovative approach to fabricate photonic ionogels has been developed for smart wearable devices with tunable temperature sensitivity and structural color. Firstly, poly(isopropylacrylamide-r-phenylmaleanilic acid) P(NIPAm-r-NPMA) nanogels self-assemble into photonic crystals in 2-hydroxyethyl acrylate (HEA), water, and the ionic liquid of 1-ethyl-3-methylimidazolium trifluoromethanesulfonate. And then robust photonic ionogels are developed through a polymerization of 2-hydroxyethyl acrylate crosslinked by poly(ethylene glycol) diacrylate (PEGDA). The incorporation of the ionic liquid, 1-ethyl-3-methylimidazolium trifluoromethanesulfonate, enhances the mechanical strength of photonic ionogels and tunes the temperature-sensitivity of the ionogels, making them adaptable to various environmental conditions. The findings demonstrate that these ionogels can serve dual functions in smart wearable devices, combining electrical and optical signal outputs due to the conductivity of the ionic liquid and structural color from the nanogel assembly. The resultant photonic ionogels exhibit exceptional substrate adhesion, mechanical stability, and fast resilience. More significantly, the nanogels within these ionogels serve as the building blocks of photonic crystals (PCs) endow with angle-independent coloration and enhance stretchability beyond 200 %, while the stretchability of the ionogles without the nanogels is only about 100 %. Our photonic ionogels with tunable temperature-sensitivity and dual outputs will open an avenue to the development of the innovative smart wearable devices.
RESUMEN
Thermally induced physical hydrogels formed through the sol-gel transition of nanogels usually lose structural color above phase transition temperature (Tp). Herein, temperature/pH/redox-responsive nanogels that undergo sol-gel transition still keep structural colors above the Tp have been synthesized and studied. N-isopropylacrylamide (NIPAm) was copolymerized with N-tert-butylacrylamide (TBA) and N-acrylamido-l-phenylalanine (Aphe) to form P(NIPAm/TBA/Aphe) nanogel crosslinked with N,N'-bis(acryloyl)cystine (BISS) (referred to as PNTA-BISS). PNTA-BISS nanogel with a broad range of biodegradable crosslinker BISS content can achieve a reversible sol-gel transition above the Tp, surprisingly, while PNTA nanogels with a comparable content of biodegradable N,N'-Bis(acryloyl)cystam (BAC) crosslinker (referred to as PNTA-BAC) didn't form sol-gel transition. Although BISS and BAC possess same disulfide bonds with redox properties, BISS, unlike BAC, is water-soluble and features two carboxyl groups. The mechanism by which PNTA-BISS nanogels form hydrogel photonic crystals has been deeply explored with temperature-variable NMR. The results showed the introduction of Aphe with both steric hindrance and carboxyl groups greatly slowed down the shrinkage of PNTA-BISS nanogels. Therefore, PNTA-BISS nanogels can form sol-gel transition and further structural color of hydrogel photonic crystals due to carboxyl groups above the Tp. Furthermore, the properties of biodegradable hydrogel photonic crystals above the Tp were investigated for the first time, attributed to the presence of the strong reducing agent 1,4-dithiothreitol (DTT). When loaded with doxorubicin (DOX), PNTA-BISS exhibited favorable degradation properties under the influence of DTT. In summary, the PNTA-BISS nanogel, in addition to its in-situ gelation capabilities, demonstrated degradability, potentially providing a novel nanoplatform for applications in drug delivery, biotechnology, and related fields.
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Hidrogeles , Polietilenglicoles , Nanogeles , Hidrogeles/química , PolietileneiminaRESUMEN
The protein kinase Akt (also known as protein kinase B) is a critical signaling hub downstream of various cellular stimuli such as growth factors that control cell survival, growth, and proliferation. The activity of Akt is tightly regulated, and the aberrant activation of Akt is associated with diverse human diseases including cancer. Although it is well documented that the mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of the Akt hydrophobic motif (Ser-473 in Akt1) is essential for full Akt activation, it remains unclear whether this phosphorylation has additional roles in regulating Akt activity. In this study, we found that abolishing Akt Ser-473 phosphorylation stabilizes Akt following agonist stimulation. The Akt Ser-473 phosphorylation promotes a Lys-48-linked polyubiquitination of Akt, resulting in its rapid proteasomal degradation. Moreover, blockade of this proteasomal degradation pathway prolongs agonist-induced Akt activation. These data reveal that mTORC2 plays a central role in regulating the Akt protein life cycle by first stabilizing Akt protein folding through the turn motif phosphorylation and then by promoting Akt protein degradation through the hydrophobic motif phosphorylation. Taken together, this study reveals that the Akt Ser-473 phosphorylation-dependent ubiquitination and degradation is an important negative feedback regulation that specifically terminates Akt activation.
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Regulación Enzimológica de la Expresión Génica , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transactivadores/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Modelos Biológicos , Fosforilación , Unión Proteica , Serina/química , Transducción de SeñalRESUMEN
A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.
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Adenina/análogos & derivados , Autofagia , Embrión de Mamíferos/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adenina/farmacología , Androstadienos/farmacología , Animales , Proteína 7 Relacionada con la Autofagia , Western Blotting , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Fibroblastos/citología , Fibroblastos/enzimología , Inmunoprecipitación , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/clasificación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , WortmaninaRESUMEN
Full-thickness skin wounds are common accidents. Although healing can be achieved by treatments like autologous skin grafts, donor site morbidity is hardly evitable. In this article, we provide compelling evidence demonstrating that artificial dermal template (ADT)-treated wound healing is achieved by regrowth of skin epidermis as well as adnexa without skin grafts by use of rodent models. First, by fixating a chamber to the wound edge, we confirmed that wound healing was achieved by regeneration instead of contracture. We found highly proliferative cells in adnexa in the newly formed skin. In the distal edge of newly formed skin, we identified immature hair follicles at early developing stages, suggesting they were newly regenerated. Second, we observed that the Lgr5-positive hair follicle stem cells contributed to formation of new hair follicles through a lineage tracing model. Also, Lgr6-positive cells were enriched in distal edge of newly developed skin. Finally, WNT signaling pathway mediators were highly expressed in the new skin epidermis and adnexa, implying a potential role of WNT signaling during ADT treatment-stimulated skin regrowth. Taken together, our findings demonstrated that full skin regrowth can be induced by ADT treatment alone, thus arguing for its wide clinical application in skin wound treatment.
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Trasplante de Piel/métodos , Piel Artificial , Piel/lesiones , Cicatrización de Heridas/fisiología , Heridas y Lesiones/cirugía , Animales , Biopsia con Aguja , Modelos Animales de Enfermedad , Folículo Piloso/fisiología , Inmunohistoquímica , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Regeneración/fisiología , Sensibilidad y Especificidad , Heridas y Lesiones/diagnósticoRESUMEN
OBJECTIVE: To investigate the effects of deltamethrin on the filial brain nitric oxide synthase (NOS) activity and neurobehavioral development of the exposed lactational rats. METHODS: Pregnant rats were randomizedly divided into the treated group and the control group. The treated group was administered orally with 3.35, 6.70 mg/kg deltamethrin every other day from postnatal day (PND) 1 to PND 19 while the control group was administered with the corn oil of same amount in the same period. The activity of NOS of filial brain and neurobehavioral functions of the filial rats were observed. RESULTS: The lactational survival rate (81.80%:78.60%) in both treated groups was decreased significantly (P < 0.01) compared with that in the control group. The body weight of filial rats on PND 10, 21 in 6.70 mg/kg DM treated group [(16.62 +/- 2.2 8), (31.34 +/- 6.94) g] was less than those in the control group (P < 0.05). The delayed time in the filial rats in 6.70 mg/kg group was (3.05 +/- 1.20) s and the positive rates of passive escaping response in 3.35 and 6.70 mg/kg DM treated group were 22.5% and 21.5% respectively. There was the trend of the developmental increase of the activity of filial brain NOS between PND 5 and PND 21 and the NOS activity of rat brain on PND 5 in 6.70 mg/kg group [(0.60 +/- 0.07) U.mg pro(-1).h(-1)] was lower than that in the control group (P < 0.05). CONCLUSION: Exposure to high dose of deltamethrin in lactational female rats will decrease the activity of NOS of brain and retard the neurobehavioral development of their filial rats.
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Encéfalo/efectos de los fármacos , Aprendizaje/efectos de los fármacos , Memoria/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Nitrilos/toxicidad , Efectos Tardíos de la Exposición Prenatal , Piretrinas/toxicidad , Animales , Animales Recién Nacidos , Encéfalo/enzimología , Femenino , Masculino , Embarazo , Ratas , Ratas WistarRESUMEN
During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cGAS, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras del Transporte Vesicular/química , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Quinasa I-kappa B/metabolismo , Factor 3 Regulador del Interferón/química , Interferón-alfa/biosíntesis , Interferón beta/biosíntesis , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Virus Sendai/fisiología , Serina/metabolismo , Transducción de Señal , Ubiquitinación , Vesiculovirus/fisiologíaRESUMEN
OBJECTIVE: To elucidate the mechanism of damage on central nervous system (CNS) caused by deltamethrin (DM). METHODS: The mRNA and protein expressions of brain-derived neurotrophic factor (BDNF) in the cerebral cortex and hippocampus of the rats exposed to DM were measured by retro-transcription-polymerase chain reaction (RT-PCR), dot blot, flow cytometry analysis and immunohistochemistry. RESULTS: After exposure to DM at high-dose (DM1, 25.0 mg x kg(-1) x d(-1), i.p.) once and low-dose (DM2, 12.5 mg x kg(-1) x d(-1), i.p.) for 5 days, the level of BDNF mRNA and protein expression in the cerebral cortex and hippocampus of the rats increased significantly. The levels of BDNF mRNA and protein expression in the cerebral cortex and hippocampus measured by of RT-PCR in the rats with DM1 and DM2 were higher than those in the controls by 48% and 56%, and 59% and 54%, respectively. And, those measured by dot blot in the rats with MD1 and MD2 were 186% and 161%, and 148% and 158% of those in the controls, respectively, basically similar to those measured by RT-PCR. Flow cytometric analysis showed that the levels of BDNF mRNA and protein expression in the cerebral cortex and hippocampus in the rats with DM1 and DM2 were higher than those in the controls by 53% and 89%, and 45% and 46%, respectively. Immunohistochemical analysis showed that protein expression in the cerebral cortex of the rats with DM1 and DM2 were 129% and 147% of those in the controls, same as the flow cytometric analysis, but those were significantly higher in the hippocampus mainly in the CA1 and DG areas of the rats with MD1 and the CA3 and DG areas of the rats with DM2. CONCLUSIONS: DM could induce BDNF mRNA and protein expression in the cerebral cortex and hippocampus of the rats, which could play an important role in repairing of nerve damage.
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Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Corteza Cerebral/efectos de los fármacos , Hipocampo/efectos de los fármacos , Insecticidas/toxicidad , Piretrinas/toxicidad , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Masculino , Nitrilos , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To study the effects of deltamethrin on intracellular free Ca2+ concentration and apoptosis in rat neural cells. METHODS: Wister rats were randomly divided into 4 groups(3 treated groups and 1 control). Intracellular free Ca2+ concentration in rat neural cells was measured by using the fluorescent Ca2+ indicator Fura-2/AM. Apopotic rate of neural cells was measured by using FACS420 Flow Cytometer. RESULTS: Intracellular free Ca2+ concentration at 5 h after deltamethrin exposure [hippocampus: (389.94 +/- 43.64) nmol/L, cerebral cortex: (449.33 +/- 23.23) nmol/L], at 24 h[hippocampus: (340.47 +/- 32.36) nmol/L, cerebral cortex: (311.62 +/- 25.48) nmol/L] and at 48 h[hippocampus: (287.13 +/- 24.29) nmol/L, cerebral cortex: (346.55 +/- 36.87) nmol/L] were all higher than those of the control group[hippocampus: (203.24 +/- 18.53) nmol/L, cerebral cortex: (226.85 +/- 14.81) nmol/L, P < 0.01]; Apoptotic rate in neural cells 24 h and 48 h later [hippocampus: (8.45 +/- 1.02)%, (9.44 +/- 1.14)%, cerebral cortex: (7.90 +/- 0.49)%, (8.01 +/- 0.87)%] were also higher than those of the control group[hippocampus: (2.97 +/- 0.36)%, cerebral cortex: (3.50 +/- 0.48)%, P < 0.01)] and increased with time prolonged. CONCLUSION: Exposure to high dose of deltamethrin would interfere with intracellular free Ca2+ concentration and apoptotic rate in rat neural cells, suggesting that there may be certain relation between them.
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Apoptosis/efectos de los fármacos , Calcio/metabolismo , Insecticidas/toxicidad , Neuronas/efectos de los fármacos , Nitrilos/toxicidad , Piretrinas/toxicidad , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Citometría de Flujo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To study the effects of deltamethrin (DM) on cell survival rate and intracellular Ca(2+) ([Ca(2+)]i) concentration in primary cultured astrocytes of rat. METHODS: The cell survival rate was measured by Typan Blue assay; the intracellular [Ca(2+)]i concentration was determined by the fluorescent Ca(2+) indicator Fura-2/AM. RESULTS: The survival rate of astrocytes was decreased to 91.9% after astrocytes were incubated with 1 x 10(-5) mol/L DM for 72 h (P < 0.05). The cell survival rates were 89.0%, 84.8%, 81.2% and 79.2% respectively when astrocytes were administered with 1 x 10(-4) mol/L DM for 4, 12, 24 and 72 h, which were remarkably lower than control groups (P < 0.01). Comparing with controls and before DM treatment, sharp increases in [Ca(2+)]i concentration [(451.4 +/- 42.3), (536.9 +/- 47.5) and (870.9 +/- 100.5) nmol/L respectively] were observed when astrocytes were incubated with 1 x 10(-7), 1 x 10(-6) and 1 x 10(-5) mol/L DM for 5 minutes (P < 0.01). After astrocytes were treated with 1 x 10(-8), 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol/L DM for 15 minutes, the [Ca(2+)]i concentrations were decreased to (124.3 +/- 6.0), (131.3 +/- 19.1), (118.9 +/- 1.4), (136.6 +/- 3.8) nmol/L respectively, which were significantly different from those of controls and before treatment. And this situation was almost keeping stable to 30 min. CONCLUSION: The cell survival rate was decreased and the [Ca(2+)]i concentration was temporarily increased when astrocytes were treated with DM.
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Astrocitos/efectos de los fármacos , Calcio/metabolismo , Insecticidas/toxicidad , Piretrinas/toxicidad , Animales , Astrocitos/citología , Astrocitos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Nitrilos , RatasRESUMEN
In this study we aim to elucidate the signaling pathway and biological function of autophagy induced by MNNG, a commonly used DNA alkylating agent. We first observed that MNNG is able to induce necrotic cell death and autophagy in Bax-/- Bak-/- double knockout MEFs. We analyzed the critical role of PARP-1 activation and ATP depletion in MNNG-mediated cell death and autophagy via AMPK activation and mTOR suppression. We provide evidence that suppression of AMPK blocks MNNG-induced autophagy and enhances cell death, suggesting the pro-survival function of autophagy in MNNG-treated cells. Taken together, data from this study reveal a novel mechanism in controlling MNNG-mediated autophagy via AMPK activation downstream of PARP-1 activation and ATP depletion.
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Proteínas Quinasas Activadas por AMP/metabolismo , Alquilantes/farmacología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Técnicas de Inactivación de Genes , Metilnitronitrosoguanidina/farmacología , Ratones , Modelos Biológicos , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , ARN Interferente Pequeño/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genéticaRESUMEN
Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-ß induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.
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Infecciones por VIH/inmunología , VIH/inmunología , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Células HEK293 , VIH/efectos de los fármacos , VIH/enzimología , Infecciones por VIH/enzimología , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Humanos , Interferón beta/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Nucleotidiltransferasas/genética , Retroviridae/inmunología , Infecciones por Retroviridae/enzimología , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/virología , Inhibidores de la Transcriptasa Inversa/farmacologíaRESUMEN
RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses. DOI:http://dx.doi.org/10.7554/eLife.00785.001.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Virus Sendai/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Quinasa I-kappa B/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Datos de Secuencia Molecular , Polimerizacion , Unión Proteica , Homología de Secuencia de Aminoácido , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/fisiologíaRESUMEN
It has been well documented that cells deficient in either TSC1 or TSC2 are highly sensitive to various cell death stimuli. In this study, we utilized the TSC2 (-/-) mouse embryonic fibroblasts (MEFs) to study the involvement of autophagy in the enhanced susceptibility of TSC2-null cells to cell death. We first confirmed that both TSC1-null and TSC2-null MEFs are more sensitive to apoptosis in response to amino acid starvation (EBSS) and hypoxia. Second, we found that both the basal and inducible autophagy in TSC2 (-/-) MEFs is impaired, mainly due to constitutive activation of mTORC1. Third, suppression of autophagy by chloroquine and Atg7 knockdown sensitizes TSC2 (+/+) cells, but not TSC2 (-/-) cells, to EBSS-induced cell death. Conversely, the inhibition of mTORC1 by raptor knockdown and rapamycin activates autophagy and subsequently rescues TSC2 (-/-) cells. Finally, in starved cells, nutrient supplementations (insulin-like growth factor-1 (IGF-1) and leucine) enhanced cell death in TSC2 (-/-) cells, but reduced cell death in TSC2 (+/+) cells. Taken together, these data indicate that constitutive activation of mTORC1 in TSC2 (-/-) cells leads to suppression of autophagy and enhanced susceptibility to stress-mediated cell death. Our findings thus provide new insights into the complex relationships among mTOR, autophagy and cell death, and support the possible autophagy-targeted intervention strategies for the treatment of TSC-related pathologies.
Asunto(s)
Autofagia , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proteína 7 Relacionada con la Autofagia , Muerte Celular , Línea Celular , Supervivencia Celular , Cloroquina/farmacología , Fibroblastos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos , Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Tiempo , TransgenesRESUMEN
Our previous work has shown that autophagy plays a pro-survival function in two necrotic cell death models: zVAD-treated L929 cells as well as H(2)O(2)-treated Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (DKO MEF). This study aims to further explore the regulatory role of autophagy in necrosis by examining the functional role of the phosphoinositide-3 kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway. Our initial intriguing finding was that insulin is able to promote necrotic cell death induced by zVAD and MNNG in L929 cells or by H(2)O(2) in DKO MEF cells cultured in full-growth medium. The pro-necrosis function of insulin was further supported by the observations that insulin is capable of abolishing the protective effect of starvation on necrotic cell death induced by zVAD in L929 cells. Next, we demonstrated that insulin acts on the PI3K-Akt-mTOR pathway to promote necrosis as the suppression of the above pathway by either chemical inhibitors (LY294002 and rapamycin) or mTOR knockdown is able to mitigate the pro-death function of insulin. Finally, we provided evidence that the pro-death function of insulin is dependent on its inhibitory effect on autophagy, which serves as an important pro-survival function in necrosis. Taken together, here we provide compelling evidence to show that activation of the PI3K-Akt-mTOR signaling pathway can promote necrotic cell death via suppression of autophagy, at least in the necrosis models defined in our study in which autophagy serves as a pro-survival function. Data from this study not only further underscore the pro-survival function of autophagy in necrotic cell death, but also provide a novel insight into the intricate connections linking the PI3K-Akt-mTOR signaling pathway with cell death via modulation of autophagy.
Asunto(s)
Autofagia , Necrosis/enzimología , Necrosis/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Autofagia/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Técnicas de Silenciamiento del Gen , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Modelos Biológicos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TORRESUMEN
The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.