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BACKGROUND: Cryopreservation is a crucial method for long-term storage and stable allocation of human pluripotent stem cells (hPSCs), which are increasingly being used in various applications. However, preserving hPSCs in cryogenic conditions is challenging due to reduced recovery rates. METHODS: To address this issue, the Arginine-Glycine-Aspartate (RGD) motif was incorporated into a recombinant elastin-like peptide (REP). Human embryonic stem cells (hESCs) were treated with REP containing RGD motif (RGD-REP) during suspension and cryopreservation, and the survival rate was analyzed. The underlying mechanisms were also investigated. RESULTS: The addition of RGD-REP to the cryopreservation solution improved cell survival and pluripotency marker expression. The improvement was confirmed to be due to the activation of the FAK-AKT cascade by RGD-REP binding to hESC surface interin protein, and consequent inhibition of FoxO3a. The inactivation of FoxO3a reduced the expression of apoptosis-related genes, such as BIM, leading to increased survival of PSCs in a suspension state. CONCLUSION: RGD-REP, as a ligand for integrin protein, improves the survival and maintenance of hPSCs during cryopreservation by activating survival signals via the RGD motif. These results have potential implications for improving the efficiency of stem cell usage in both research and therapeutic applications.
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Células Madre Embrionarias Humanas , Células Madre Pluripotentes , Humanos , Células Madre Embrionarias Humanas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Elastina/metabolismo , Criopreservación/métodos , Transducción de Señal , Oligopéptidos/farmacologíaRESUMEN
Background and Objectives: The colonic epithelial layer is a complex structure consisting of multiple cell types that regulate various aspects of colonic physiology, yet the mechanisms underlying epithelial cell differentiation during development remain unclear. Organoids have emerged as a promising model for investigating organogenesis, but achieving organ-like cell configurations within colonic organoids is challenging. Here, we investigated the biological significance of peripheral neurons in the formation of colonic organoids. Methods and Results: Colonic organoids were co-cultured with human embryonic stem cell (hESC)-derived peripheral neurons, resulting in the morphological maturation of columnar epithelial cells, as well as the presence of enterochromaffin cells. Substance P released from immature peripheral neurons played a critical role in the development of colonic epithelial cells. These findings highlight the vital role of inter-organ interactions in organoid development and provide insights into colonic epithelial cell differentiation mechanisms. Conclusions: Our results suggest that the peripheral nervous system may have a significant role in the development of colonic epithelial cells, which could have important implications for future studies of organogenesis and disease modeling.
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Nuclear factor erythroid 2-related factor 2 (Nrf2) provides a cellular defense against oxidative stress by inducing the expression of antioxidant and detoxification enzymes. The calcium antagonist, verapamil, is an FDA-approved drug prescribed for the treatment of hypertension. Here, we show that verapamil acts as a potent Nrf2 activator without causing cytotoxicity, through degradation of Kelch-like ECH-associated protein 1 (Keap1), a Nrf2 repressor. Furthermore, verapamilinduced Keap1 degradation is prominently mediated by a p62-dependent autophagic pathway. Correspondingly, verapamil protects cells from acetaminophen-induced oxidative damage through Nrf2 activation. These results demonstrated the underlying mechanisms for the protective role of verapamil against acetaminophen-induced cytotoxicity. [BMB Reports 2017; 50(2): 91-96].
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Acetaminofén/toxicidad , Antihipertensivos/farmacología , Citoprotección , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Verapamilo/farmacología , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Células Cultivadas , Citoprotección/efectos de los fármacos , Citoprotección/genética , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Ratones , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Proteolisis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Endoplasmic reticulum (ER) stress is triggered by various cellular stresses that disturb protein folding or calcium homeostasis in the ER. To cope with these stresses, ER stress activates the unfolded protein response (UPR) pathway, but unresolved ER stress induces reactive oxygen species (ROS) accumulation leading to apoptotic cell death. However, the mechanisms that underlie protection from ER stress-induced cell death are not clearly defined. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway plays a crucial role in the protection of cells against ROS-mediated oxidative damage. Keap1 acts as a negative regulator of Nrf2 activation. In this study, we investigated the role of the Nrf2-Keap1 pathway in protection from ER stress-induced cell death using tunicamycin (TM) as an ER stress inducer. We found that Nrf2 is an essential protein for the prevention from TM-induced apoptotic cell death and its activation is driven by autophagic Keap1 degradation. Furthermore, ablation of p62, an adapter protein in the autophagy process, attenuates the Keap1 degradation and Nrf2 activation that was induced by TM treatment, and thereby increases susceptibility to apoptotic cell death. Conversely, reinforcement of p62 alleviated TM-induced cell death in p62-deficient cells. Taken together, these results demonstrate that p62 plays an important role in protecting cells from TM-induced cell death through Nrf2 activation.
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Proteínas de Unión al ARN/metabolismo , Proteína Sequestosoma-1/metabolismo , Animales , Apoptosis/fisiología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Muerte Celular/fisiología , Estrés del Retículo Endoplásmico/fisiología , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Tunicamicina/farmacologíaRESUMEN
Oxidative stress is important for the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a chronic disease that ranges from hepatic steatosis to nonalcoholic steatohepatitis (NASH). The nuclear factor erythroid 2-related factor 2-Kelch-like ECH associated protein 1 (Nrf2-Keap1) pathway is essential for cytoprotection against oxidative stress. In this study, we found that oxidative stress or inflammatory biomarkers and TUNEL positive cells were markedly increased in NASH patients compared to normal or simple steatosis. In addition, we identified that the hepatic mRNA levels of Nrf2 target genes such as Nqo-1 and GSTA-1 were significantly increased in NASH patients. Ezetimibe, a drug approved by the Food and Drug Administration for the treatment of hypercholesterolemia, improves NAFLD and alleviates oxidative stress. However, the precise mechanism of its antioxidant function remains largely unknown. We now demonstrate that ezetimibe activates Nrf2-Keap1 pathway which was dependent of autophagy adaptor protein p62, without causing cytotoxicity. Ezetimibe activates AMP-activated protein kinase (AMPK), which in turn phosphorylates p62 (p-S351) via their direct interaction. Correspondingly, Ezetimibe protected liver cells from saturated fatty acid-induced apoptotic cell death through p62-dependent Nrf2 activation. Furthermore, its role as an Nrf2 activator was supported by methione- and choline- deficient (MCD) diet-induced NASH mouse model, showing that ezetimibe decreased the susceptibility of the liver to oxidative injury. These data demonstrate that the molecular mechanisms underlying ezetimibe's antioxidant role in the pathogenesis of NASH.
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Antioxidantes/farmacología , Ezetimiba/farmacología , Proteínas de Transporte de Membrana/genética , Factor 2 Relacionado con NF-E2/genética , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Apoptosis , Dieta/efectos adversos , Regulación de la Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/agonistas , Factor 2 Relacionado con NF-E2/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Estrés Oxidativo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de SeñalRESUMEN
Sestrins (Sesns) protect cells from oxidative stress. The mechanism underlying the antioxidant effect of Sesns has remained unknown, however. The Nrf2-Keap1 pathway provides cellular defense against oxidative stress by controlling the expression of antioxidant enzymes. We now show that Sesn1 and Sesn2 interact with the Nrf2 suppressor Keap1, the autophagy substrate p62, and the ubiquitin ligase Rbx1 and that the antioxidant function of Sesns is mediated through activation of Nrf2 in a manner reliant on p62-dependent autophagic degradation of Keap1. Sesn2 was upregulated in the liver of mice subjected to fasting or subsequent refeeding with a high-carbohydrate, fat-free diet, whereas only refeeding promoted Keap1 degradation and Nrf2 activation, because only refeeding induced p62 expression. Ablation of Sesn2 blocked Keap1 degradation and Nrf2 activation induced by refeeding and thereby increased the susceptibility of the liver to oxidative damage resulting from the acute stimulation of lipogenesis associated with refeeding.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Hígado/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Animales , Autofagia , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Hígado/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares , Estrés Oxidativo , Peroxidasas , Unión Proteica , Factor de Transcripción TFIIH , Transfección , Regulación hacia ArribaRESUMEN
AIMS: To define the mechanisms underlying pyrazole-induced oxidative stress and the protective role of peroxiredoxins (Prxs) and sulfiredoxin (Srx) against such stress. RESULTS: Pyrazole increased Srx expression in the liver of mice in a nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent manner and induced Srx translocation from the cytosol to the endoplasmic reticulum (ER) and mitochondria. Pyrazole also induced the expression of CYP2E1, a primary reactive oxygen species (ROS) source for ethanol-induced liver injury, in ER and mitochondria. However, increased CYP2E1 levels only partially accounted for the pyrazole-mediated induction of Srx, prompting the investigation of CYP2E1-independent ROS generation downstream of pyrazole. Indeed, pyrazole increased ER stress, which is known to elevate mitochondrial ROS. In addition, pyrazole up-regulated CYP2E1 to a greater extent in mitochondria than in ER. Accordingly, among Prxs I to IV, PrxIII, which is localized to mitochondria, was preferentially hyperoxidized in the liver of pyrazole-treated mice. Pyrazole-induced oxidative damage to the liver was greater in PrxIII(-/-) mice than in wild-type mice. Such damage was also increased in Srx(-/-) mice treated with pyrazole, underscoring the role of Srx as the guardian of PrxIII. INNOVATION: The roles of Prxs, Srx, and ER stress have not been previously studied in relation to pyrazole toxicity. CONCLUSION: The concerted action of PrxIII and Srx is important for protection against pyrazole-induced oxidative stress arising from the convergent induction of CYP2E1-derived and ER stress-derived ROS in mitochondria.
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Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Peroxiredoxina III/metabolismo , Pirazoles/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Peroxiredoxina III/genéticaRESUMEN
Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins (Prxs). Having high affinity toward H2O2, 2-Cys Prxs can efficiently reduce H2O2 at low concentration. We previously showed that Prx I is hyperoxidized at a rate of 0.072% per turnover even in the presence of low steady-state levels of H2O2. Here we examine the novel role of Srx in cells exposed to low steady-state levels of H2O2, which can be achieved by using glucose oxidase. Exposure of low steady-state levels of H2O2 (10-20 µm) to A549 or wild-type mouse embryonic fibroblast (MEF) cells does not lead to any significant change in oxidative injury because of the maintenance of balance between H2O2 production and elimination. In contrast, loss-of-function studies using Srx-depleted A549 and Srx-/- MEF cells demonstrate a dramatic increase in extra- and intracellular H2O2, sulfinic 2-Cys Prxs, and apoptosis. Concomitant with hyperoxidation of mitochondrial Prx III, Srx-depleted cells show an activation of mitochondria-mediated apoptotic pathways including mitochondria membrane potential collapse, cytochrome c release, and caspase activation. Furthermore, adenoviral re-expression of Srx in Srx-depleted A549 or Srx-/- MEF cells promotes the reactivation of sulfinic 2-Cys Prxs and results in cellular resistance to apoptosis, with enhanced removal of H2O2. These results indicate that Srx functions as a novel component to maintain the balance between H2O2 production and elimination and then protects cells from apoptosis even in the presence of low steady-state levels of H2O2.
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Peróxido de Hidrógeno/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Peróxido de Hidrógeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/deficiencia , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacosRESUMEN
UNLABELLED: Peroxiredoxins (Prxs) are peroxidases that catalyze the reduction of reactive oxygen species (ROS). The active site cysteine residue of members of the 2-Cys Prx subgroup (Prx I to IV) of Prxs is hyperoxidized to cysteine sulfinic acid (Cys-SO(2) ) during catalysis with concomitant loss of peroxidase activity. Reactivation of the hyperoxidized Prx is catalyzed by sulfiredoxin (Srx). Ethanol consumption induces the accumulation of cytochrome P450 2E1 (CYP2E1), a major contributor to ethanol-induced ROS production in the liver. We now show that chronic ethanol feeding markedly increased the expression of Srx in the liver of mice in a largely Nrf2-dependent manner. Among Prx I to IV, only Prx I was found to be hyperoxidized in the liver of ethanol-fed wildtype mice, and the level of Prx I-SO(2) increased to ≈30% to 50% of total Prx I in the liver of ethanol-fed Srx(-/-) mice. This result suggests that Prx I is the most active 2-Cys Prx in elimination of ROS from the liver of ethanol-fed mice and that, despite the up-regulation of Srx expression by ethanol, the capacity of Srx is not sufficient to counteract the hyperoxidation of Prx I that occurs during ROS reduction. A protease protection assay revealed that a large fraction of Prx I is located together with CYP2E1 at the cytosolic side of the endoplasmic reticulum membrane. The selective role of Prx I in ROS removal is thus likely attributable to the proximity of Prx I and CYP2E1. CONCLUSION: The pivotal functions of Srx and Prx I in protection of the liver in ethanol-fed mice was evident from the severe oxidative damage observed in mice lacking either Srx or Prx I.
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Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Etanol/toxicidad , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Peroxirredoxinas/metabolismo , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Masculino , Ratones , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/deficiencia , Peroxiredoxina III , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The cysteine residue at the active site of peroxiredoxin (Prx) I, Prx II, or Prx III is reversibly hyperoxidized to cysteine sulfinic acid, with concomitant loss of peroxidase activity, during normal catalysis. Sulfiredoxin (Srx) is the enzyme responsible for reversing this hyperoxidation. We now show that the expression of Srx at both the mRNA and protein levels is increased markedly in the lungs of mice exposed to hyperoxia. This hyperoxia-induced expression of Srx was not evident in mice deficient in the transcription factor Nrf2, indicating an essential role for an Nrf2 signaling pathway in this effect. Hyperoxia also elicited the accumulation of the sulfinic form of the mitochondrial enzyme Prx III, but not that of the cytosolic enzymes Prx I or Prx II, in lung tissue. This selective hyperoxidation of Prx III is likely due either to mitochondria being the major site of the hyperoxia-induced production of reactive oxygen species or to the translocation of Srx from the cytosol into mitochondria being rate limiting for the reduction of sulfinic Prx III. Hyperoxia induced the degradation of Prx III in Nrf2-deficient mice but not in wild-type animals, suggesting that, in the absence of a sufficient amount of Srx, sulfinic Prx III is converted to a form that is susceptible to proteolysis.
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Hiperoxia/metabolismo , Pulmón/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/biosíntesis , Peroxirredoxinas/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Masculino , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
COX-2 and its products, including prostaglandin E(2), are involved in many inflammatory processes. Glucosamine (GS) is an amino monosaccharide and has been widely used for alternative regimen of (osteo) arthritis. However, the mechanism of action of GS on COX-2 expression remains unclear. Here we describe a new action mechanism of glucosamine hydrochloride (GS-HCl) to tackle endogenous and agonist-driven COX-2 at protein level. GS-HCl (but not GS sulfate, N-acetyl GS, or galactosamine HCl) resulted in a shift in the molecular mass of COX-2 from 72-74 to 66-70 kDa and concomitant inhibition of prostaglandin E(2) production in a concentration-dependent manner in interleukin (IL)-1beta-treated A549 human lung epithelial cells. Remarkably, GS-HCl-mediated decrease in COX-2 molecular mass was associated with inhibition of COX-2 N-glycosylation during translation, as assessed by the effect of tunicamycin, the protein N-glycosylation inhibitor, or of cycloheximide, the translation inhibitor, on COX-2 modification. Specifically, the effect of low concentration of GS-HCl (1 mM) or of tunicamycin (0.1 microg/ml) to produce the aglycosylated COX-2 was rescued by the proteasomal inhibitor MG132 but not by the lysosomal or caspase inhibitors. However, the proteasomal inhibitors did not show an effect at 5 mM GS-HCl, which produced the aglycosylated or completely deglycosylated form of COX-2. Notably, GS-HCl (5 mM) also facilitated degradation of the higher molecular species of COX-2 in IL-1beta-treated A549 cells that was retarded by MG132. GS-HCl (5 mM) was also able to decrease the molecular mass of endogenous and IL-1beta- or tumor necrosis factor-alpha-driven COX-2 in different human cell lines, including Hep2 (bronchial) and H292 (laryngeal). However, GS-HCl did not affect COX-1 protein expression. These results demonstrate for the first time that GS-HCl inhibits COX-2 activity by preventing COX-2 co-translational N-glycosylation and by facilitating COX-2 protein turnover during translation in a proteasome-dependent manner.
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Cloruros/química , Ciclooxigenasa 2/metabolismo , Glucosamina/química , Glucosamina/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Línea Celular Tumoral , Dinoprostona/metabolismo , Epitelio/metabolismo , Galactosamina/química , Glicosilación , Humanos , Inflamación , Interleucina-1beta/metabolismo , Pulmón/metabolismo , Modelos Biológicos , Osteoartritis/metabolismoRESUMEN
Overexpression of inducible nitric oxide synthase (iNOS) and the resultant overproduction of NO has been implicated in neuronal inflammatory diseases. Leptomycin B (LMB), a metabolite of Streptomyces, has been identified as a specific inhibitor of CRM1 nuclear export receptor. In this study, we evaluated the effect of LMB on lipopolysaccharide (LPS)-induced iNOS expression in BV2 cells, a murine microglial cells and the associated mechanisms. LMB strongly inhibited LPS-induced iNOS protein and mRNA expressions in BV2 cells in which 10 ng/ml of LMB (18 nM) was sufficient to greatly down-regulate iNOS by LPS, suggesting the potency of LMB to inhibit iNOS. The data of iNOS promoter-driven luciferase assay further suggested that the LMB inhibitory effect was in part due to inhibition of iNOS transcription. However, LPS-induced activation of various intracellular signaling proteins, such as nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated kinases (ERKs), p38s, and c-Jun N-terminal kinases (JNKs), whose activations are known to be important for iNOS expression by LPS in BV2 cells, were not affected in the presence of LMB. Together, these results suggest that LMB inhibits iNOS expression in response to LPS in BV2 microglia, and the inhibition seems to be associated with blockage of CRM1-mediated iNOS mRNA nuclear export and also in part transcriptional down-regulation of iNOS, but not through modulation of NF-kappaB and the mitogen-activated protein kinase signaling pathways.