RESUMO
Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D-/-ESCs) as expected. Surprisingly, D-/-ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D-/-ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D-/-ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D-/-ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D-/-ESCs. Treatment of D-/-ESCs with a PKR inhibitor and IFNß-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs.
Assuntos
RNA Helicases DEAD-box/genética , Interferon Tipo I/genética , Interferon gama/genética , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Ribonuclease III/genética , eIF-2 Quinase/genética , Animais , Antivirais/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/efeitos dos fármacos , RNA de Cadeia Dupla/genética , Retroelementos/genética , eIF-2 Quinase/antagonistas & inibidoresRESUMO
Despite technological advances in cancer treatment, the survival rate of patients with head and neck cancer (HNC) has not improved significantly. Many studies have shown that endoplasmic reticulum (ER) stress-related signals are associated with mitochondrial damage and that these signals determine whether cells maintain homeostasis or activate cell death programs. The unfolded protein response (UPR) is regulated by ER membrane proteins such as double-stranded RNA-activated protein kinase R(PKR)-like ER kinase (PERK), which directly activate transcription of chaperones or genes that function in redox homeostasis, protein secretion, or cell death programs. In this study, we focused on the role of mitophagy and ER stress-mediated cell death induced by DIM-C-pPhtBu in HNC cancer. We found that DIM-C-pPhtBu, a compound that activates ER stress in many cancers, induced lysosomal dysfunction, excessive mitophagy, and cell death in HNC cells. Moreover, DIM-C-pPhtBu strongly inhibited HNC progression in a xenograft model by altering mitophagy related protein expression. Taken together, the results demonstrate that DIM-C-pPhtBu induces excessive mitophagy and eventually UPR-mediated cell death in HNC cells, suggesting that new anti-cancer drugs could be developed based on the connection between mitophagy and cancer cell death.
Assuntos
Morte Celular , Lisossomos/metabolismo , Mitofagia , Resposta a Proteínas não Dobradas , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Ethanol (EtOH) neurotoxicity can result in devastating effects on brain and behavior by disrupting homeostatic signaling cascades and inducing cell death. One such mechanism involves double-stranded RNA activated protein kinase (PKR), a primary regulator of protein translation and cell viability in the presence of a virus or other external stimuli. EtOH-mediated up-regulation of interferon-gamma (IFN-γ; the oxidative stress-inducible regulator of PKR), PKR, and its target, p53, are still being fully elucidated. METHODS: Using Western blot analysis, immunofluorescence, and linear regression analyses, changes in the IFN-γ-PKR-p53 pathway following chronic EtOH treatment in the frontal cortex of rodents were examined. The role of PKR on cell viability was also assessed in EtOH-treated cells using PKR overexpression vector and PKR inhibitor (PKRI). RESULTS: In rats chronically fed EtOH, PKR, phosphorylated PKR (p-PKR), IFN-γ, and p53 were significantly increased following chronic EtOH exposure. Linear regression revealed a significant correlation between IFN-γ and p-PKR protein levels, as well as p-PKR expression and age of EtOH exposure. Overexpression of PKR resulted in greater cell death, while use of PKRI enhanced cell viability in EtOH-treated cells. CONCLUSIONS: Chronic EtOH exposure activates the IFN-γ-PKR-p53 pathway in the frontal cortex of rodents. p-PKR expression is greater in brains of rodents exposed to EtOH at earlier ages compared to later life, suggesting a mechanism by which young brains could be more susceptible to EtOH-related brain injury. PKR and p-PKR were also colocalized in neurons and astrocytes of rats. This study provides additional insight into biochemical mechanisms underlying alcohol use disorder related neuropathology and warrants further investigation of PKR as a potential pharmacotherapeutic target to combat EtOH-related neurotoxicity, loss of protein translation and brain injury.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Interferon gama/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , eIF-2 Quinase/metabolismo , Idade de Início , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Masculino , Córtex Pré-Frontal/metabolismo , Distribuição Aleatória , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Sepsis, which refers to a systemic inflammatory response syndrome resulting from a microbial infection, represents the leading cause of death in intensive care units. The pathogenesis of sepsis remains poorly understood although it is attributable to dysregulated immune responses orchestrated by innate immune cells that are sequentially released early (e.g., tumor necrosis factor(TNF), interleukin-1(IL-1), and interferon-γ(IFN-γ)) and late (e.g., high mobility group box 1(HMGB1)) pro-inflammatory mediators. As a ubiquitous nuclear protein, HMGB1 can be passively released from pathologically damaged cells, thereby converging infection and injury on commonly dysregulated inflammatory responses. We review evidence that supports extracellular HMGB1 as a late mediator of inflammatory diseases and discuss the potential of several Chinese herbal components as HMGB1-targeting therapies. We propose that it is important to develop strategies for specifically attenuating injury-elicited inflammatory responses without compromising the infection-mediated innate immunity for the clinical management of sepsis and other inflammatory diseases.
RESUMO
Peripheral artery disease (PAD) is a common clinical problem, and its pathophysiological mechanisms are incompletely understood. Double-stranded RNA-activated protein kinase (PKR) is a ubiquitously expressed serine/threonine protein kinase. Although PKR has been reported in antivirus and the immune system, the role of PKR in vascular function, especially in angiogenesis, is still unclear. PKR(-/-) mice were used in our experiments. Blood flow recovery was significantly delayed in PKR(-/-) vs. WT mice (Laser Doppler detection, n = 9, P < 0.01), accompanied by 34% reduced CD31-positive stain in ischemic muscle 28 days after procedure (immunohistochemistry, n = 9, P < 0.05). PKR expression decreased in the first 12 h and increased to peak at 24 h in human umbilical vein endothelial cells (HUVECs) in response to hypoxia (Western blot analyses, n = 3, P < 0.05). Accordingly, phospho-PKR expression increased in HUVECs 24 h after treatment with hypoxia (Western blot analyses, n = 3, P < 0.05). Inhibition of PKR (siRNA transfection) reduced microtubule formation (Matrigel tube formation, n = 3, vs. control siRNA, P < 0.05) and migration (wound healing, n = 3, vs. control siRNA, P < 0.05) by 33 and 59%, respectively. Vascular endothelial growth factor (VEGF) expression in ischemic muscle from PKR(-/-) mice was significantly decreased by 54% 1 day after procedure (n = 3, P < 0.05, vs. WT) and by 63% 7 days after procedure (n = 3, P < 0.01, vs. WT), respectively. At the same time, VEGF expression in HUVECs decreased by 21% (n = 3, P < 0.05, PKR siRNA vs. control siRNA). These findings demonstrate that PKR mediates angiogenesis through a VEGF pathway, which may form the basis for future intervention of PAD.
Assuntos
Neovascularização Fisiológica/genética , Fator A de Crescimento do Endotélio Vascular/fisiologia , eIF-2 Quinase/genética , Animais , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Doença Arterial Periférica/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , eIF-2 Quinase/antagonistas & inibidoresRESUMO
New negative regulators of interferon (IFN) signaling, preferably with tissue specificity, are needed to develop therapeutic means to enhance the efficacy of type I IFNs (IFN-α/ß) and reduce their side effects. We conducted cell-based screening for IFN signaling enhancer and discovered that luteolin, a natural flavonoid, sensitized the antiproliferative effect of IFN-α in hepatoma HepG2 cells and cervical carcinoma HeLa cells. Luteolin promoted IFN-ß-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation by enhancing the phosphorylation of Jak1, Tyk2, and STAT1/2, thereby promoting STAT1 accumulation in the nucleus and endogenous IFN-α-regulated gene expression. Of interest, inhibition of phosphodiesterase (PDE) abolished the effect of IFN-ß and luteolin on STAT1 phosphorylation. Luteolin also increased the cAMP-degrading activity of PDE bound with type I interferon receptor 2 (IFNAR2) and decreased the intracellular cAMP level, indicating that luteolin may act on the JAK/STAT pathway via PDE. Protein kinase A (PKA) was found to negatively regulate IFN-ß-induced JAK/STAT signaling, and its inhibitory effect was counteracted by luteolin. Pull-down and immunoprecipitation assays revealed that type II PKA interacted with IFNAR2 via the receptor for activated C-kinase 1 (RACK-1), and such interaction was inhibited by luteolin. Src homology domain 2 containing tyrosine phosphatase-2 (SHP-2) was further found to mediate the inhibitory effect of PKA on the JAK/STAT pathway. These data suggest that PKA/PDE-mediated cAMP signaling, integrated by RACK-1 to IFNAR2, may negatively regulate IFN signaling through SHP-2. Inhibition of this signaling may provide a new way to sensitize the efficacy of IFN-α/ß.
Assuntos
Interferon-alfa/farmacologia , Interferon beta/farmacologia , Janus Quinase 1/metabolismo , Luteolina/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Adjuvantes Imunológicos/farmacologia , Anticorpos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas de Ligação ao GTP , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Fatores Imunológicos/farmacologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/imunologia , Proteínas de Neoplasias , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Receptor de Interferon alfa e beta/química , Receptor de Interferon alfa e beta/metabolismo , Receptores de Quinase C Ativada , Receptores de Superfície Celular , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/antagonistas & inibidores , Fator de Transcrição STAT2/imunologia , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais/efeitos dos fármacos , TYK2 Quinase/imunologia , TYK2 Quinase/metabolismoRESUMO
Glutathione is present in millimolar concentrations in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)-glutathione disulfide (GSSG) redox couple with known equilibrium constant, we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously determined value for [GSH](2):[GSSG] in the ER of 83 mM, this translates into a total glutathione concentration in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ~7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space.
Assuntos
Retículo Endoplasmático/metabolismo , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Citosol/metabolismo , Glutarredoxinas/genética , Células HeLa , Homeostase , HumanosRESUMO
Endoplasmic reticulum (ER) stress is associated with the development of diabetes. The present study sought to investigate the effect of Liraglutide, a glucagon like peptide 1 analogue, on ER stress in ß-cells. We found that Liraglutide protected the pancreatic INS-1 cells from thapsigargin-induced ER stress and the ER stress associated cell apoptosis, mainly by suppressing the PERK and IRE1 pathways. We further tested the effects of Liraglutide in the Akita mouse, an ER-stress induced type 1 diabetes model. After administration of Liraglutide for 8weeks, p-eIF2α and p-JNK were significantly decreased in the pancreas of the Akita mouse, while the treatment showed no significant impact on the levels of insulin of INS-cells. Taken together, our findings suggest that Liraglutide may protect pancreatic cells from ER stress and its related cell death.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Animais , Morte Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Liraglutida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologiaRESUMO
BACKGROUND: The 58-kDa inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (P58IPK) is a cellular protein that is activated during influenza virus infection. Although the function of human P58IPK has been studied for a long time, porcine P58IPK (pP58IPK) has little been studied except for its cloning. OBJECTIVE: In this study, we aimed to investigate the characteristics of the pP58IPK gene, determine its subcellular localization, and find its expression change during H1N1 or H3N2 infection. STUDY DESIGN: First, the sequence and structure of pP58IPK were analyzed. Second, pP58IPK gene was cloned into pEGFP-N1 and pEGFP-C1 vectors, respectively, which were transfected into cells to determine its subcellular localization. Third, Lung tissues of piglets from H1N1 infected, H3N2 infected and control groups were analyzed using histopathology, real-time PCR, and immunohistochemistry. RESULTS: The sequence and structure of pP58IPK was highly similar to the counterpart of human. pP58IPK protein distributed only in the cytoplasm. Lung tissues of piglets infected by H1N1 or H3N2 appeared obvious pathological changes, and the expression of pP58IPK in both mRNA and protein level was up-regulated by approximate 1.5-fold in piglets infected by H1N1 or H3N2 comparing with control piglets. CONCLUSIONS: We analyzed the characteristics of the pP58IPK gene, constructed a phylogenetic tree, determined its subcellular localization, and investigated its expression changes during H1N1 or H3N2 infection. The fundamental data accumulated in this study provides a potential medical model for investigating the function of P58IPK during influenza A viruses infection.