RESUMO
Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.
Assuntos
Glucose , Animais , Cricetinae , Proliferação de Células , Células CHO , Cricetulus , Proteínas Recombinantes/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.
Assuntos
Envelhecimento , Encéfalo , Proteínas Serina-Treonina Quinases , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box , Animais , Camundongos , Envelhecimento/genética , Encéfalo/metabolismo , Estresse do Retículo Endoplasmático/genética , Proteínas Serina-Treonina Quinases/genética , Proteômica , Transdução de Sinais/fisiologia , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
The unfolded protein response (UPR) is a complex network of intracellular pathways that transmits signals from ER lumen and/or ER bilayer to the nuclear compartment in order to activate gene transcription. UPR is activated by the loss of ER capacities, known as ER stress, and occurs to restore ER properties. In this regard, glycerolipid (GL) synthesis activation contributes to ER membrane homeostasis and IRE1α-XBP1, one UPR pathway, has a main role in lipogenic genes transcription. Herein, we describe the strategy and methodology used to evaluate whether IRE1α-XBP1 pathway regulates lipid metabolism in renal epithelial cells subjected to hyperosmolar environment. XBP1s activity was hindered by blocking IRE1α RNAse activity and by impeding its expression; under these conditions, we determined GL synthesis and lipogenic enzymes expression.
Assuntos
Endorribonucleases , Proteínas Serina-Treonina Quinases , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Lipídeos , Proteínas Serina-Treonina Quinases/genética , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
BACKGROUND AND AIM: The endoplasmic reticulum (ER) stress response and the unfolded protein response (UPR) are essential cellular mechanisms to ensure the proper functioning of ER in adverse conditions. However, activation of these pathways has also been associated with insulin resistance and cell death in pathological conditions such as diabetes mellitus. In the present study, we investigated whether stromal cell-derived factor 2 (SDF2)-an ER stress-responsive factor-is related to ER response in placental cells exposed to maternal gestational diabetes mellitus (GDM) or to a hyperglycaemic in vitro condition. OBJECTIVE: The study aimed to investigate the role of SDF2 in BeWo cells , a trophoblast cell line originating from choriocarcinoma , and in placental tissue under hyperglycaemic conditions. METHODS: Protein levels of SDF2 and UPR factors, glucose-related protein 78 (GRP78) and eukaryotic initiation factor 2 alpha (elF2 alpha) were evaluated in the placentae of pregnant women diagnosed with GDM and treated by diet-control (insulin was added when necessary). The mRNA expression of SDF2 and UPR factors CHOP and sXBP1 were assessed in cultured BeWo cells challenged with glucose and treated with or without insulin. RESULTS: SDF2 expression was increased in the placentae of GDM women treated with diet. However, its values were similar to those of normoglycemic controls when the GDM women were treated with insulin and diet. BeWo cells cultured with high glucose and insulin showed decreased SDF2 expression, while high glucose increased CHOP and sXBP1 expression, which was then significantly reverted with insulin treatment. CONCLUSION: Our findings extend the understanding of ER stress and SDF2 expression in placentae exposed to hyperglycaemia, highlighting the relevance of insulin in reducing the levels of ER stress factors in placental cells. Understanding the effect of ER stress partners such as SDF2 on signalling pathways involved in gestation, complicated by hyperglycaemia, is pivotal for basic biomedical research and may lead to new therapeutic possibilities.
Assuntos
Glicemia/metabolismo , Diabetes Gestacional/metabolismo , Estresse do Retículo Endoplasmático , Proteínas/metabolismo , Trofoblastos/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Estudos Transversais , Diabetes Gestacional/sangue , Diabetes Gestacional/patologia , Diabetes Gestacional/terapia , Dieta Saudável , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Proteínas de Choque Térmico/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Gravidez , Proteínas/genética , Transdução de Sinais , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/patologia , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Syzygium cumini is used worldwide for the treatment of metabolic syndrome-associated outcomes. Previously, we described the antihypertriglyceridemic effect of the hydroethanolic extract of S. cumini leaf (HESc) in monosodium L-glutamate- (MSG-) induced obese rats. This study sought to investigate the molecular mechanisms underlying the antihypertriglyceridemic effect of HESc in MSG-obese rats. Newborn male Wistar rats were injected subcutaneously with MSG (4.0 g/kg/day, obese group) or saline 1.25% (1.0 mL/kg/day, lean group), from 2nd through 10th postnatal day. At 8 weeks old, obese rats started to be orally treated with HESc (0.5 or 1.0 g/kg/day, n = 7) or saline 0.9% (1 mL/kg/day, n = 7). Lean rats received saline solution (1 mL/kg/day, n = 7). Upon 8-week treatment, animals were euthanized for blood and tissue collection. Another set of adult nonobese Wistar rats was used for the assessment of HESc acute effects on Triton WR1339-induced hypertriglyceridemia. HESc reduced weight gain, as well as adipose tissue fat pads, without altering food intake of obese rats. HESc restored fasting serum glucose, triglycerides, total cholesterol, and free fatty acids, as well as insulin sensitivity, to levels similar to lean rats. Additionally, HESc halved the triglyceride content into very low-density lipoprotein particles, as well as healed liver steatosis, in obese rats. Hepatic protein expression of the endoplasmic reticulum chaperone GRP94 was decreased by HESc, which also downregulated the hepatic triglyceride secretion pathway by reducing the splicing of X-box binding protein 1 (XBP-1s), as well as protein disulfide isomerase (PDI) and microsomal triglyceride transfer protein (MTP) translational levels. This action was further corroborated by the acute inhibitory effect of HESc on triglyceride accumulation on Triton WR1339-treated rats. Our data support the downregulation of the XBP-1s/PDI/MTP axis in the liver of MSG-obese rats as a novel feasible mechanism for the antihypertriglyceridemic effect promoted by the polyphenolic phytocomplex present in S. cumini leaf.
Assuntos
Regulação para Baixo , Hipertrigliceridemia/tratamento farmacológico , Fígado/metabolismo , Obesidade/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Transdução de Sinais/efeitos dos fármacos , Syzygium/química , Tecido Adiposo/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Transporte/metabolismo , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fígado Gorduroso/sangue , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/fisiopatologia , Glicolipídeos/sangue , Hipertrigliceridemia/sangue , Hipertrigliceridemia/fisiopatologia , Lipoproteínas VLDL/sangue , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiopatologia , Masculino , Obesidade/sangue , Obesidade/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Polifenóis/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Ratos Wistar , Glutamato de Sódio , Triglicerídeos/sangue , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
NEW FINDINGS: What is the central question of this study? Is there sexual dimorphism in the occurrence of hepatic endoplasmic reticulum stress? What is the main finding and its importance? The transition from prepubertal to the adult age is associated with an increase in the unfolded protein response markers in the liver of male rats, which is probably due to an increase in serum testosterone levels. ABSTRACT: Male rodents present a higher predisposition to obesity and insulin resistance than females. These disorders have been associated with endoplasmic reticulum (ER) stress. To investigate a possible sexual dimorphism in the hepatic occurrence of ER stress, we evaluated the expression of ER stress markers in the livers of male and female rats in two phases of sexual development. In the first experimental model, male and female prepubertal and adult Wistar rats were used. Adult males presented higher body mass and greater mass of the adipose tissue and liver than adult females. Prepubertal animals presented no differences in these parameters between males and females. Despite this finding, the hepatic expression levels of Bip, Ire1α and Xbp1s mRNA were lower in prepubertal males than in females, while in adult animals, they did not differ between sexes. In the second experimental model, we anticipated the sexually mature phase by daily injections of testosterone propionate for 10 days in prepubertal males or by daily injections of oestradiol benzoate for 7 days in prepubertal females. Oestradiol administration in prepubertal females did not change any of the parameters evaluated. Testosterone administration to prepubertal males led to a higher body mass and greater expression of Bip, Ire1α, Atf4 and Xbp1s in the liver. These findings suggest that the increased ER stress predisposition observed in males during puberty is due to an increase in testosterone levels, indicating that ER stress is sexually dimorphic before puberty due to the lack of testosterone in males.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Hormônios Esteroides Gonadais/farmacologia , Fígado/metabolismo , Animais , Endorribonucleases/metabolismo , Estradiol/farmacologia , Feminino , Glucose/metabolismo , Proteínas de Choque Térmico/metabolismo , Fígado/efeitos dos fármacos , Masculino , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Caracteres Sexuais , Maturidade Sexual , Testosterona/farmacologia , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Increasing evidence shows that the endoplasmic reticulum (ER) stress is an early event that injures pancreatic acinar cells and contributes to the pathogenesis of acute pancreatitis. In the present work we sought to establish whether atrial natriuretic peptide (ANP) alleviated ER stress in rats with cerulein-induced pancreatitis. The major components of the unfolded protein response (UPR) and their downstream effectors were assessed by immunoblotting or fluorimetry and the ultrastructure of ER evaluated by electron transmission microscopy. Cross-talk with autophagy was evaluated by beclin-1 expression. ANP reduced binding immunoglobulin protein (Bip) expression (UPR major controller) which under non-stress conditions keeps inactive the stress sensor proteins: protein kinase-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating transcription factor 6 (ATF6). Although ANP did not change PERK expression it decreased p-eIF2α and enhanced downstream effector CHOP, suggesting that ANP stimulates ER-dependent apoptosis. In accordance, ANP also decreased Bcl2 expression and enhanced proapoptotic proteins Bax and Bak. The atrial peptide enhanced ATF6 expression and although it did not affect IRE1/sXBP1 signaling, it increased caspase-2 activity, also involved in ER-dependent apoptosis. Furthermore, ANP decreased beclin-1 expression. The ultrastructure of the RE revealed decreased swelling and conserved ribosomes in the presence of ANP. Present findings support that ANP alleviates ER stress in acute pancreatitis by modulating the three branches of the UPR and stimulates ER-dependent apoptosis. Gaining insights into the modulation of ER stress may help to develop specific therapeutic strategies for acute pancreatitis and/or medical interventions at risk of its developing like endoscopic retrograde cholangiopancreatography.
Assuntos
Fator Natriurético Atrial/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Pancreatite/patologia , Fator 6 Ativador da Transcrição/metabolismo , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Caspase 12/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Pâncreas/ultraestrutura , Pancreatite/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , eIF-2 Quinase/metabolismoRESUMO
StarD7 is a lipid binding protein involved in the delivery of phosphatidylcholine to the mitochondria whose promoter is activated by Wnt/ß-catenin signaling. Although the majority of glucose enters glycolysis, ~ 2-5% of it can be metabolized via the hexosamine biosynthetic pathway (HBP). Considering that HBP has been implicated in the regulation of ß-catenin we explored if changes in glucose levels modulate StarD7 expression by the HBP in trophoblast cells. We found an increase in StarD7 as well as in ß-catenin expression following high-glucose (25 mM) treatment in JEG-3 cells; these effects were abolished in the presence of HBP inhibitors. Moreover, since HBP is able to promote unfolded protein response (UPR) the protein levels of GRP78, Ire1α, calnexin, p-eIF2α and total eIF2α as well as XBP1 mRNA was measured. Our results indicate that a diminution in glucose concentration leads to a decrease in StarD7 expression and an increase in the UPR markers: GRP78 and Ire1α. Conversely, an increase in glucose is associated to high StarD7 levels and low GRP78 expression, phospho-eIF2α and XBP1 splicing, although Ire1α remains high when cells are restored to high glucose. Taken together these findings indicate that glucose modulates StarD7 and ß-catenin expression through the HBP associated to UPR, suggesting the existence of a link between UPR and HBP in trophoblast cells. This is the first study reporting the effects of glucose on StarD7 in trophoblast cells. These data highlight the importance to explore the role of StarD7 in placenta disorders related to nutrient availability.
Assuntos
Proteínas de Transporte/metabolismo , Hexosaminas/metabolismo , Processamento Alternativo/genética , Vias Biossintéticas , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/metabolismo , Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Resposta a Proteínas não Dobradas , Via de Sinalização Wnt , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
To relieve endoplasmic reticulum (ER) stress, IRE1 splices XBP1 messenger RNA (mRNA) or engages regulated IRE1-dependent decay (RIDD) of other mRNAs. Upon XBP1 deficiency, IRE1 switches to perform RIDD. We examined IRE1 in XBP1-deficient B cells and discovered that IRE1 undergoes phosphorylation at S729. We generated an anti-phospho-S729 antibody to investigate such phosphorylation. Compared with pharmacological ER stress inducers or Toll-like receptor ligands, the bacterial subtilase cytotoxin has an unusual capability in causing rapid and strong phosphorylation at S729 and triggering B cells to express spliced XBP1. To assess the function of S729 in IRE1, we generated S729A knock-in mice and found S729 is critically important for lipopolysaccharide-stimulated plasmablasts to respond to additional ER stress and for antibody production in response to immunization. We further crossed mice carrying an S729A mutation or ΔIRE1 (missing the kinase domain) with B cell-specific XBP1-deficient mice to trigger RIDD and discovered a critical role for S729 in regulating RIDD in B cells.
Assuntos
Formação de Anticorpos , Linfócitos B/metabolismo , Imunização , Proteínas de Membrana/metabolismo , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA , Sequência de Aminoácidos , Animais , Ditiotreitol/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lipopolissacarídeos , Proteínas de Membrana/química , Camundongos Endogâmicos C57BL , Modelos Animais , Modelos Biológicos , Fosforilação , Proteínas Serina-Treonina Quinases/química , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.