RESUMEN
Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αß) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor-induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum-associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.
Asunto(s)
Inmunidad Innata/inmunología , Interleucinas/inmunología , eIF-2 Quinasa/inmunología , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , eIF-2 Quinasa/deficienciaRESUMEN
Ischemic cardiomyopathy is associated with an increased risk of sudden death, activation of the unfolded protein response (UPR), and reductions in multiple cardiac ion channels. When activated, the protein kinase-like ER kinase (PERK) branch of the UPR reduces protein translation and abundance. We hypothesized that PERK inhibition could prevent ion channel downregulation and reduce arrhythmia risk after myocardial infarct (MI). MI induced in mice by coronary artery ligation resulted in reduced ion channel levels, ventricular tachycardia (VT), and prolonged corrected intervals between the Q and T waves on the ECGs (QTc). Protein levels of major cardiac ion channels were decreased. MI cardiomyocytes showed significantly prolonged action potential duration and decreased maximum upstroke velocity. Cardiac-specific PERK KO reduced electrical remodeling in response to MI, with shortened QTc intervals, fewer VT episodes, and higher survival rates. Pharmacological PERK inhibition had similar effects. In conclusion, we found that activated PERK during MI contributed to arrhythmia risk by the downregulation of select cardiac ion channels. PERK inhibition prevented these changes and reduced arrhythmia risk. These results suggest that ion channel downregulation during MI is a fundamental arrhythmia mechanism and that maintenance of ion channel levels is antiarrhythmic.
Asunto(s)
Arritmias Cardíacas/prevención & control , Infarto del Miocardio/complicaciones , Infarto del Miocardio/metabolismo , Respuesta de Proteína Desplegada/fisiología , eIF-2 Quinasa/antagonistas & inhibidores , Adenina/análogos & derivados , Adenina/farmacología , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/metabolismo , Regulación hacia Abajo , Femenino , Factores de Riesgo de Enfermedad Cardiaca , Humanos , Indoles/farmacología , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Cardiovasculares , Inhibidores de Proteínas Quinasas/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genéticaRESUMEN
The architecture of cristae provides a spatial mitochondrial organization that contains functional respiratory complexes. Several protein components including OPA1 and MICOS complex subunits organize cristae structure, but upstream regulatory mechanisms are largely unknown. Here, in vivo and in vitro reconstitution experiments show that the endoplasmic reticulum (ER) kinase PERK promotes cristae formation by increasing TOM70-assisted mitochondrial import of MIC19, a critical subunit of the MICOS complex. Cold stress or ß-adrenergic stimulation activates PERK that phosphorylates O-linked N-acetylglucosamine transferase (OGT). Phosphorylated OGT glycosylates TOM70 on Ser94, enhancing MIC19 protein import into mitochondria and promoting cristae formation and respiration. In addition, PERK-activated OGT O-GlcNAcylates and attenuates CK2α activity, which mediates TOM70 Ser94 phosphorylation and decreases MIC19 mitochondrial protein import. We have identified a cold-stress inter-organelle PERK-OGT-TOM70 axis that increases cell respiration through mitochondrial protein import and subsequent cristae formation. These studies have significant implications in cellular bioenergetics and adaptations to stress conditions.
Asunto(s)
Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , eIF-2 Quinasa/metabolismo , Adipocitos Marrones/citología , Adipocitos Marrones/efectos de los fármacos , Adipocitos Marrones/metabolismo , Animales , Quinasa de la Caseína II/metabolismo , Frío , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Glicosilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/genética , Proteínas Mitocondriales/genética , N-Acetilglucosaminiltransferasas/genética , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , ARN Guía de Kinetoplastida/metabolismo , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genéticaRESUMEN
The primary mechanisms supporting immunoregulatory polarization of myeloid cells upon infiltration into tumors remain largely unexplored. Elucidation of these signals could enable better strategies to restore protective anti-tumor immunity. Here, we investigated the role of the intrinsic activation of the PKR-like endoplasmic reticulum (ER) kinase (PERK) in the immunoinhibitory actions of tumor-associated myeloid-derived suppressor cells (tumor-MDSCs). PERK signaling increased in tumor-MDSCs, and its deletion transformed MDSCs into myeloid cells that activated CD8+ T cell-mediated immunity against cancer. Tumor-MDSCs lacking PERK exhibited disrupted NRF2-driven antioxidant capacity and impaired mitochondrial respiratory homeostasis. Moreover, reduced NRF2 signaling in PERK-deficient MDSCs elicited cytosolic mitochondrial DNA elevation and, consequently, STING-dependent expression of anti-tumor type I interferon. Reactivation of NRF2 signaling, conditional deletion of STING, or blockade of type I interferon receptor I restored the immunoinhibitory potential of PERK-ablated MDSCs. Our findings demonstrate the pivotal role of PERK in tumor-MDSC functionality and unveil strategies to reprogram immunosuppressive myelopoiesis in tumors to boost cancer immunotherapy.
Asunto(s)
Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Epitelial de Ovario/inmunología , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/inmunología , Proteínas de la Membrana/inmunología , Neoplasias Cutáneas/inmunología , eIF-2 Quinasa/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Femenino , Humanos , Terapia de Inmunosupresión , Interferón-alfa/genética , Interferón-alfa/inmunología , Interferón beta/genética , Interferón beta/inmunología , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/inmunología , Mitocondrias/metabolismo , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/patología , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/inmunología , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Respuesta de Proteína Desplegada/inmunología , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genéticaRESUMEN
Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Células Dendríticas/inmunología , Estrés del Retículo Endoplásmico/inmunología , Activación de Linfocitos , Células T Asesinas Naturales/inmunología , Animales , Presentación de Antígeno , Antígenos CD1d/biosíntesis , Antígenos CD1d/inmunología , Autoantígenos/inmunología , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Citoesqueleto/ultraestructura , Endosomas/inmunología , Glicoesfingolípidos/inmunología , Glicoesfingolípidos/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Lípidos/inmunología , Lisosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Células THP-1 , Tapsigargina/farmacología , Respuesta de Proteína Desplegada/inmunología , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/fisiologíaRESUMEN
MoHrip2, identified from Magnaporthe oryzae as an elicitor, can activate plant defense responses either in the form of recombinant protein in vitro or ectopic expressed protein in rice. However, its intrinsic function in the infective interaction of M. oryzae-rice is largely unknown. Here, we found that mohrip2 expression was significantly induced at stages of fungal penetration and colonization. Meanwhile, the induced MoHrip2 mainly accumulated in the rice apoplast by outlining the entire invasive hyphae during infection, and its secretion was via the conventional endoplasmic reticulum (ER)-to-Golgi pathway, demonstrating the nature of MoHrip2 as an apoplastic effector. What's more, the disease facilitating function of MoHrip2 was revealed by the significantly compromised virulence of Δmohrip2 mutants on rice seedlings and even on the wounded rice leaves. Inoculations of these mutant strains on rice leaf sheaths showed a reduction in penetration and subsequent expansion of fungal growth, which is probably due to activated host immunity including the expression of certain defense-related genes and the production of certain phytoalexins. Altogether, these results demonstrated the necessity of MoHrip2 in suppression of host immunity and the full virulence of M. oryzae.
Asunto(s)
Interacciones Huésped-Patógeno , Evasión Inmune , Magnaporthe/patogenicidad , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Factores de Virulencia/metabolismo , eIF-2 Quinasa/metabolismo , Eliminación de Gen , Magnaporthe/enzimología , Oryza/inmunología , Virulencia , Factores de Virulencia/deficiencia , eIF-2 Quinasa/deficienciaRESUMEN
Brain lesions in Alzheimer's disease (AD) include amyloid plaques made of Aß peptides and neurofibrillary tangles composed of hyperphosphorylated tau protein with synaptic and neuronal loss and neuroinflammation. Aß oligomers can trigger tau phosphorylation and neuronal alterations through activation of neuronal kinases leading to progressive cognitive decline. PKR is a ubiquitous pro-apoptotic serine/threonine kinase, and levels of activated PKR are increased in AD brains and AD CSF. In addition, PKR regulates negatively memory formation in mice. To assess the role of PKR in an AD in vivo model, we crossed 5xFAD transgenic mice with PKR knockout (PKRKO) mice and we explored the contribution of PKR on cognition and brain lesions in the 5xFAD mouse model of AD as well as in neuron-microglia co-cultures exposed to the innate immunity activator lipopolysaccharide (LPS). Nine-month-old double-mutant mice revealed significantly improved memory consolidation with the new object location test, starmaze test, and elevated plus maze test as compared to 5xFAD mice. Brain amyloid accumulation and BACE1 levels were statistically decreased in double-mutant mice. Apoptosis, neurodegeneration markers, and synaptic alterations were significantly reduced in double-mutant mice as well as neuroinflammation markers such as microglial load and brain cytokine levels. Using cocultures, we found that PKR in neurons was essential for LPS microglia-induced neuronal death. Our results demonstrate the clear involvement of PKR in abnormal spatial memory and brain lesions in the 5xFAD model and underline its interest as a target for neuroprotection in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Memoria Espacial , eIF-2 Quinasa/metabolismo , Enfermedad de Alzheimer/patología , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/patología , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , eIF-2 Quinasa/deficienciaRESUMEN
Metabolic disorders including obesity and type 2 diabetes are known to be associated with chronic inflammation and are obvious risk factors for Alzheimer's disease. Recent evidences concerning obesity and diabetes suggest that the metabolic inflammasome ("metaflammasome") mediates chronic inflammation. The double-stranded RNA-dependent protein kinase (PKR) is a central component of the metaflammasome. In wild type (WT) and PKR-/- mice, blood glucose, insulin and lipid levels and the brain expression of the phosphorylated components of the metaflammasome-PKR, JNK, IRS1 and IKKbeta-were studied after the induction of obesity by a high fat diet (HFD). The results showed significant increased levels of activated brain metaflammasome proteins in exposed WT mice but the changes were not significant in PKR-/- mice. In addition, gain weight was observed in WT mice and also in PKR-/- mice exposed to HFD. Increased blood insulin level was more accentuated in PKR -/- mice. The modulation of PKR activity could be an appropriate therapeutic approach, aimed at reducing abnormal brain metabolism and inflammation linked to metabolic disorders in order to reduce the risk of neurodegeneration.
Asunto(s)
Glucemia/metabolismo , Encéfalo/metabolismo , Dieta Alta en Grasa/efectos adversos , Insulina/sangre , Obesidad/metabolismo , eIF-2 Quinasa/genética , Animales , Encéfalo/patología , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inflamación , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/genética , Obesidad/patología , Transducción de Señal , Triglicéridos/sangre , Aumento de Peso/genética , eIF-2 Quinasa/deficienciaRESUMEN
As chondrocytes are highly secretory and they experience a variety of stresses, physiological unfolded protein response (UPR) signalling is essential for extracellular matrix (ECM) secretion and chondrogenesis. In the three branches of the UPR pathway, PERK governs the translational attenuation and transcriptional upregulation of amino acid and redox metabolism and induction of apoptosis. It was previously demonstrated that a defect of the PERK branch of the UPR signalling pathway causes the accumulation of unfolded proteins, leading to cell death without perturbing endoplasmic reticulum (ER)-to-Golgi transport in pancreatic ß cells. However, little is known about the role of PERK in chondrocytes. In this study, we found that PERK signalling is activated in chondrocytes, and inhibition of PERK reduces collagen secretion despite causing excessive collagen synthesis in the ER. Perk -/- mice displayed reduced collagen in articular cartilage but no differences in chondrocyte proliferation or apoptosis compared to the findings in wild-type mice. PERK inhibition increases misfolded protein levels in the ER, which largely hinder ER-to-Golgi transport. These results suggest that the translational control mediated by PERK is a critical determinant of ECM secretion in chondrocytes.
Asunto(s)
Condrocitos/metabolismo , Colágeno/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Línea Celular , Ratones , Ratones Noqueados , eIF-2 Quinasa/deficienciaRESUMEN
In humans, the pathogenesis of diabetes is characterised by two major pancreatic ß cell defects: a reduction in ß cell mass and the failure of ß cells to produce enough insulin. Over the past two decades, multiple studies involving cell cultures, animal models and human subjects have established the importance of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) in the adaptive functional capacity of pancreatic ß cells during embryonic development and into adulthood. In this review, we will highlight major findings identifying PERK as a crucial player in ß cell physiology and in diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Epífisis/anomalías , Factor 2 Eucariótico de Iniciación/genética , Homeostasis/genética , Células Secretoras de Insulina/enzimología , Osteocondrodisplasias/genética , eIF-2 Quinasa/genética , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/inmunología , Adulto , Animales , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/patología , Epífisis/enzimología , Epífisis/inmunología , Epífisis/patología , Factor 2 Eucariótico de Iniciación/inmunología , Regulación del Desarrollo de la Expresión Génica , Homeostasis/inmunología , Humanos , Recién Nacido , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/patología , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/inmunología , Osteocondrodisplasias/enzimología , Osteocondrodisplasias/inmunología , Osteocondrodisplasias/patología , Transducción de Señal , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/inmunologíaRESUMEN
While lentiviral expression vectors are widely used in many facets of molecular biology, due to their ability to stably express heterologous genes in both dividing and non-dividing cells, they suffer from the disadvantage that introns inserted into the vector genome are generally rapidly lost by splicing in packaging cell lines. The presence of an intron, if achievable, has the potential to facilitate the expression of transgene cDNAs, as splicing has been extensively shown to facilitate mRNA biogenesis and function. Moreover, if a stable intron could be introduced into a lentiviral vector, this could greatly facilitate the expression of microRNAs (miRNAs), and especially miRNA clusters, as the introduction of pri-miRNA stems into the exonic region of a lentiviral vector can strongly reduce both vector titer and the expression of any miRNA-linked indicator gene due to cleavage of the vector RNA genome by cellular Drosha. Here, we describe a novel lentiviral vector design in which transgenes and/or miRNAs are expressed using an antisense-orientated, inducible promoter driving an expression cassette bearing a functional intron. We demonstrate that this lentiviral vector, called pTREX, is able to express higher levels of both transgenes and pri-miRNA clusters when compared with a closely similar conventional lentiviral vector.
Asunto(s)
Ingeniería Genética/métodos , Intrones , Lentivirus/genética , MicroARNs/genética , Empalme del ARN , Línea Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Exones , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Insulina/genética , Insulina/metabolismo , Lentivirus/metabolismo , MicroARNs/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Regiones Promotoras Genéticas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Transgenes , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genéticaRESUMEN
While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of viral short interfering RNAs (siRNAs) that can control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two cofactors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be mounted in human somatic cells, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of human Dicer (hDcr) and PKR. We observed significant production of â¼21-nt long siRNAs, derived from a cotransfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. Surprisingly, dDcr2 was able to produce siRNAs even in the absence of Loqs-PD, which is thought to be required for dsRNA cleavage by dDcr2. This result may be explained by our finding that dDcr2 is able to bind the human Loqs-PD homolog TRBP when expressed in human cells in the absence of Loqs-PD. We conclude that it is possible to at least partially rescue the ability of mammalian somatic cells to express functional siRNAs using gene products of invertebrate origin.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , ARN Helicasas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Ribonucleasa III/genética , Animales , Ingeniería Celular , Línea Celular , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Prueba de Complementación Genética , Humanos , Coactivadores de Receptor Nuclear/genética , Coactivadores de Receptor Nuclear/metabolismo , Unión Proteica , ARN Helicasas/metabolismo , ARN Interferente Pequeño/biosíntesis , Proteínas de Unión al ARN/metabolismo , Complejo Silenciador Inducido por ARN/biosíntesis , Complejo Silenciador Inducido por ARN/genética , Ribonucleasa III/deficiencia , Ribonucleasa III/metabolismo , Transgenes , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genéticaRESUMEN
The sensing of double-stranded RNA (dsRNA) in the liver is important for antiviral defenses but can also contribute to sterile inflammation during liver injury. Hepatocytes are often the target of viral infection and are easily injured by inflammatory insults. Here we sought to establish the pathways involved in the production of type I interferons (IFN-I) in response to extracellular poly(I:C), a dsRNA mimetic, in hepatocytes. This was of interest because hepatocytes are long-lived and, unlike most immune cells that readily die after activation with dsRNA, are not viewed as cells with robust antimicrobial capacity. We found that poly(I:C) leads to rapid up-regulation of inducible nitric oxide synthase (iNOS), double-stranded RNA-dependent protein kinase (PKR), and Src. The production of IFN-ß was dependent on iNOS, PKR, and Src and partially dependent on TLR3/Trif. iNOS and Src up-regulation was partially dependent on TLR3/Trif but entirely dependent on PKR. The phosphorylation of TLR3 on tyrosine 759 was shown to increase in parallel to IFN-ß production in an iNOS- and Src-dependent manner, and Src was found to directly interact with TLR3 in the endosomal compartment of poly(I:C)-treated cells. Furthermore, we identified a robust NO/cGMP/PKG-dependent feedforward pathway for the amplification of iNOS expression. These data identify iNOS/NO as an integral component of IFN-ß production in response to dsRNA in hepatocytes in a pathway that involves the coordinated activities of TLR3/Trif and PKR.
Asunto(s)
Hepatocitos/inmunología , Hepatocitos/metabolismo , Interferón beta/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Bicatenario/inmunología , ARN Bicatenario/farmacología , Receptor Toll-Like 3/metabolismo , eIF-2 Quinasa/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Hepatocitos/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Fosforilación/efectos de los fármacos , Poli I-C/farmacología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 3/deficiencia , Receptor Toll-Like 3/genética , Tirosina/química , Regulación hacia Arriba/efectos de los fármacos , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genética , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genéticaRESUMEN
In peripheral nerves, P0 glycoprotein accounts for more than 20% of myelin protein content. P0 is synthesized by Schwann cells, processed in the endoplasmic reticulum (ER) and enters the secretory pathway. However, the mutant P0 with S63 deleted (P0S63del) accumulates in the ER lumen and induces a demyelinating neuropathy in Charcot-Marie-Tooth disease type 1B (CMT1B)-S63del mice. Accumulation of P0S63del in the ER triggers a persistent unfolded protein response. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER stress sensor that phosphorylates eukaryotic initiation factor 2 alpha (eIF2alpha) in order to attenuate protein synthesis. We have shown that increasing phosphophorylated-eIF2alpha (P-eIF2alpha) is a potent therapeutic strategy, improving myelination and motor function in S63del mice. Here, we explore the converse experiment:Perkhaploinsufficiency reduces P-eIF2alpha in S63del nerves as expected, but surprisingly, ameliorates, rather than worsens S63del neuropathy. Motor performance and myelin abnormalities improved in S63del//Perk+/- compared with S63del mice. These data suggest that mechanisms other than protein translation might be involved in CMT1B/S63del neuropathy. In addition,Perkdeficiency in other cells may contribute to demyelination in a non-Schwann-cell autonomous manner.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Mutación/genética , eIF-2 Quinasa/deficiencia , Factores de Edad , Animales , Animales Recién Nacidos , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Embrión de Mamíferos , Ganglios Espinales/citología , Regulación de la Expresión Génica/genética , Inmunoprecipitación , Ratones , Ratones Transgénicos , Proteína Básica de Mielina/metabolismo , Proteína P0 de la Mielina/genética , Proteína P0 de la Mielina/metabolismo , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/genética , Neuronas/efectos de los fármacos , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Nervio Ciático/metabolismo , Nervio Ciático/patología , eIF-2 Quinasa/genéticaRESUMEN
To prevent excess accumulation of unfolded proteins in endoplasmic reticulum (ER), eukaryotic cells have signaling pathways from the ER to the cytosol or nucleus. These processes are known as the endoplasmic reticulum stress (ERS) response. Protein kinase R like endoplasmic reticulum kinase (PERK) is a major transducer of the ERS response and it directly phosphorylate α-subunit of eukaryotic initiation factor 2 (eIF2α), resulting in translational attenuation. Phosphorylated eIF2α specifically promoted the translation of the activating transcription factor 4 (ATF4). ATF4 is a known important transcription factor which plays a pivotal role in osteoblast differentiation and bone formation. Furthermore, ATF4 is a downstream target of PERK. Studies have shown that PERK-eIF2α-ATF4 signal pathway mediated by ERS was involved in osteoblastic differentiation of osteoblasts. We have known that orthodontic tooth movement is a process of periodontal ligament cells (PDLCs) osteodifferentiation and alveolar bone remodeling under mechanical force. However, the involvement of PERK-eIF2α-ATF4 signal pathway mediated by ERS in osteogenic differentiation of PDLCs under mechanical force has not been unclear. In our study, we applied the cyclic mechanical force at 10% elongation with 0.5Hz to mimic occlusal force, and explored whether PERK-eIF2α-ATF4 signaling pathway mediated by ERS involved in osteogenic differentiation of PDLCs under mechanical force. Firstly, cyclic mechanical force will induce ERS and intensify several osteoblast marker genes (ATF4, OCN, and BSP). Next, we found that PERK overexpression increased eIF2α phosphorylation and expression of ATF4, furthermore induced BSP, OCN expression, thus it will promote osteodifferentiation of hPDLCs; mechanical force could promote this effect. However, PERK(-/-) cells showed the opposite changes, which will inhibit osteodifferentiation of hPDLCs. Taken together, our study proved that PERK-eIF2α-ATF4 signaling pathway mediated by ERS involved in osteoblast differentiation of PDLCs under cyclic mechanical force.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Diferenciación Celular , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/metabolismo , Ligamento Periodontal/patología , Transducción de Señal , Estrés Mecánico , eIF-2 Quinasa/metabolismo , Adolescente , Diferenciación Celular/genética , Niño , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis/genética , eIF-2 Quinasa/deficienciaRESUMEN
Murine cytomegalovirus (MCMV) proteins m142 and m143 are essential for viral replication. They bind double-stranded RNA and prevent protein kinase R-induced protein synthesis shutoff. Whether the two viral proteins have additional functions such as their homologs in human cytomegalovirus do remained unknown. We show that MCMV m142 and m143 knockout mutants attain organ titers equivalent to those attained by wild-type MCMV in Pkr knockout mice, suggesting that these viral proteins do not encode additional PKR-independent functions relevant for pathogenesis in vivo.
Asunto(s)
Muromegalovirus/fisiología , Mutación , Proteínas Virales/genética , Replicación Viral , eIF-2 Quinasa/deficiencia , Animales , Ratones Noqueados , Muromegalovirus/genética , Carga ViralRESUMEN
We report the first detailed examination of the brain of a patient with Wolcott-Rallison syndrome. Wolcott-Rallison syndrome is an extremely rare clinical manifestation of a lack of protein kinase R-like endoplasmic reticulum kinase (PERK) function caused by mutations in the PERK gene EIF2AK3. Protein kinase R-like endoplasmic reticulum kinase is thought to play a significant pathogenetic role in several neurodegenerative diseases, including Alzheimer disease, other tauopathies, and Parkinson disease. The brain of a male patient aged 4 years 7 months showed pathologic and immunohistochemical evidence that the absence of PERK for several years is sufficient to induce early changes reminiscent of various neurodegenerative conditions. These include neurofibrillary tangles (as in progressive supranuclear palsy), FUS-immunopositive and p62-immunopositive neurons, and reactive glial changes. We also detected an increased amount of p62-positive puncta coimmunostaining for LC3 and ubiquitin, suggesting changes in autophagic flux. Studying a human brain with absent PERK function presents the opportunity to assess the long-term consequences of nonfunctioning of PERK in the presence of all of the compensatory mechanisms that are normally active in a living human, thereby confirming the importance of PERK for autophagy in the brain and for neurodegeneration.
Asunto(s)
Encéfalo/patología , Diabetes Mellitus Tipo 1/patología , Epífisis/anomalías , Degeneración Nerviosa/patología , Osteocondrodisplasias/patología , eIF-2 Quinasa/deficiencia , Preescolar , Diabetes Mellitus Tipo 1/genética , Epífisis/patología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Masculino , Degeneración Nerviosa/genética , Osteocondrodisplasias/genéticaRESUMEN
Brain thiamine homeostasis has an important role in energy metabolism and displays reduced activity in Alzheimer's disease (AD). Thiamine deficiency (TD) induces regionally specific neuronal death in the animal and human brains associated with a mild chronic impairment of oxidative metabolism. These features make the TD model amenable to investigate the cellular mechanisms of neurodegeneration. Once activated by various cellular stresses, including oxidative stress, PKR acts as a pro-apoptotic kinase and negatively controls the protein translation leading to an increase of BACE1 translation. In this study, we used a mouse TD model to assess the involvement of PKR in neuronal death and the molecular mechanisms of AD. Our results showed that the TD model activates the PKR-eIF2α pathway, increases the BACE1 expression levels of Aß in specific thalamus nuclei and induces motor deficits and neurodegeneration. These effects are reversed by PKR downregulation (using a specific inhibitor or in PKR knockout mice).
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Regulación hacia Abajo , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/patología , Tiamina/metabolismo , eIF-2 Quinasa/metabolismo , Amiloide/metabolismo , Animales , Encéfalo/enzimología , Encéfalo/patología , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Actividad Motora , Degeneración Nerviosa/fisiopatología , Neuronas/metabolismo , Neuronas/patología , Estrés Oxidativo , Transporte de Proteínas , Transducción de Señal , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/deficienciaRESUMEN
Ethanol-induced neuronal loss is closely related to the pathogenesis of fetal alcohol spectrum disorders. The cerebellum is one of the brain areas that are most sensitive to ethanol. The mechanism underlying ethanol neurotoxicity remains unclear. Our previous in vitro studies have shown that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) regulates neuronal apoptosis upon ethanol exposure and ethanol activates PKR through association with its intracellular activator RAX. However, the role of PKR and its interaction with RAX in vivo have not been investigated. In the current study, by utilizing N-PKR-/- mice, C57BL/6J mice with a deficient RAX-binding domain in PKR, we determined the critical role of RAX/PKR association in PKR-regulated ethanol neurotoxicity in the developing cerebellum. Our data indicate that while N-PKR-/- mice have a similar BAC profile as wild-type mice, ethanol induces less brain/body mass reduction as well as cerebellar neuronal loss. In addition, ethanol promotes interleukin-1ß (IL-1ß) secretion, and IL-1ß is a master cytokine regulating inflammatory response. Importantly, ethanol-promoted IL-1ß secretion is inhibited in the developing cerebellum of N-PKR-/- mice. Thus, RAX/PKR interaction and PKR activation regulate ethanol neurotoxicity in the developing cerebellum, which may involve ethanol-induced neuroinflammation. Further, PKR could be a possible target for pharmacological intervention to prevent or treat fetal alcohol spectrum disorder (FASD).
Asunto(s)
Depresores del Sistema Nervioso Central/toxicidad , Cerebelo , Etanol/toxicidad , Síndromes de Neurotoxicidad/etiología , eIF-2 Quinasa/deficiencia , Factores de Edad , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Células Cultivadas , Cerebelo/efectos de los fármacos , Cerebelo/crecimiento & desarrollo , Cerebelo/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Síndromes de Neurotoxicidad/patología , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/genética , Sincalida/farmacología , Factores de Tiempo , eIF-2 Quinasa/genéticaRESUMEN
Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (HSL-C12) as a quorum-sensing molecule to regulate bacterial gene expression. Because HSL-C12 is membrane permeant, multiple cell types in P. aeruginosa-infected airways may be exposed to HSL-C12, especially adjacent to biofilms where local (HSL-C12) may be high. Previous reports showed that HSL-C12 causes both pro- and anti-inflammatory effects. To characterize HSL-C12's pro- and anti-inflammatory effects in host cells, we measured protein synthesis, NF-κB activation, and KC (mouse IL-8) and IL-6 mRNA and protein secretion in wild-type mouse embryonic fibroblasts (MEF). To test the role of the endoplasmic reticulum stress inducer, PERK we compared these responses in PERK(-/-) and PERK-corrected PERK(-/-) MEF. During 4-h treatments of wild-type MEF, HSL-C12 potentially activated NF-κB p65 by preventing the resynthesis of IκB and increased transcription of KC and IL-6 genes (quantitative PCR). HSL-C12 also inhibited secretion of KC and/or IL-6 into the media (ELISA) both in control conditions and also during stimulation by TNF-α. HSL-C12 also activated PERK (as shown by increased phosphorylation of eI-F2α) and inhibited protein synthesis (as measured by incorporation of [(35)S]methionine by MEF). Comparisons of PERK(-/-) and PERK-corrected MEF showed that HSL-C12's effects were explained in part by activation of PERKâphosphorylation of eI-F2αâinhibition of protein synthesisâreduced IκBα productionâactivation of NF-κBâincreased transcription of the KC gene but reduced translation and secretion of KC. HSL-C12 may be an important modulator of early (up to 4 h) inflammatory signaling in P. aeruginosa infections.