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1.
Nat Commun ; 12(1): 366, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446657

RESUMO

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.


Assuntos
Glicina/metabolismo , Neoplasias/dietoterapia , Serina/biossíntese , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Glicina/análise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Fosfoglicerato Desidrogenase/metabolismo , Serina/análise
2.
Nat Commun ; 11(1): 5594, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154371

RESUMO

The underlying pathological mechanisms of glaucomatous trabecular meshwork (TM) damage and elevation of intraocular pressure (IOP) are poorly understood. Here, we report that the chronic endoplasmic reticulum (ER) stress-induced ATF4-CHOP-GADD34 pathway is activated in TM of human and mouse glaucoma. Expression of ATF4 in TM promotes aberrant protein synthesis and ER client protein load, leading to TM dysfunction and cell death. These events lead to IOP elevation and glaucomatous neurodegeneration. ATF4 interacts with CHOP and this interaction is essential for IOP elevation. Notably, genetic depletion or pharmacological inhibition of ATF4-CHOP-GADD34 pathway prevents TM cell death and rescues mouse models of glaucoma by reducing protein synthesis and ER client protein load in TM cells. Importantly, glaucomatous TM cells exhibit significantly increased protein synthesis along with induction of ATF4-CHOP-GADD34 pathway. These studies indicate a pathological role of ATF4-CHOP-GADD34 pathway in glaucoma and provide a possible treatment for glaucoma by targeting this pathway.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Estresse do Retículo Endoplasmático , Glaucoma de Ângulo Aberto/metabolismo , Biossíntese de Proteínas , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Animais , Humor Aquoso/metabolismo , Morte Celular , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Glaucoma de Ângulo Aberto/tratamento farmacológico , Glaucoma de Ângulo Aberto/patologia , Humanos , Camundongos , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/metabolismo , Hipertensão Ocular/patologia , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Transdução de Sinais , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
3.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.


Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
4.
Nat Commun ; 11(1): 4677, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938929

RESUMO

The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estresse do Retículo Endoplasmático/genética , Fatores de Iniciação em Eucariotos/genética , Biossíntese de Proteínas , Fatores de Transcrição/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Mutação , Fases de Leitura Aberta , Interferência de RNA , Degeneração Retiniana/genética , Fatores de Transcrição/metabolismo
5.
Toxicol Lett ; 331: 178-187, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32569804

RESUMO

Chromium (Cr) (VI) compounds are known to be serious toxic and carcinogenic, but the mechanism is not clear. In our previous study, we found that Cr (VI)-induced ER stress plays an important role in the crosstalk between apoptosis and autophagy, while autophagy was apoptosis-dependent and subsequently prevents apoptosis cell death to keep A549 cells resistant to Cr (VI)-induced toxicity. In this study, we found that Cr (VI) could induce aerobic glycolysis in A549 cells. Both ER stress inhibitor, phenylbutyric acid (4-PBA) and the inhibitor of autophagy, 3-MA, repressed Cr (VI)-induced glycolysis, indicating that both ER stress and autophagy were involved in Cr (VI)-induced glycolysis in A549 cells. Co-treatment of the inhibitor of aerobic glycolysis, 2-DG and Cr (VI) for 24 h increased Cr (VI)-induced cleaved caspase-3, caspase-9 and the number of apoptotic cells, demonstrating that aerobic glycolysis played an important role in attenuating Cr (VI)-induced apoptosis. Furthermore, knockdown of ATF4 by siATF4 significantly decreased Cr (VI)-induced aerobic glycolysis and apoptosis, suggesting that ATF4 was involved in Cr (VI)-induced aerobic glycolysis and its effect of attenuating apoptosis in A549 cells. Taken together, our results demonstrated that autophagy-dependent glycolysis played a role in attenuating Cr (VI)-induced apoptosis. ER stress was involved in facilitating glycolysis, whose induction was mediated by ATF4. These findings open a window for the development of therapeutic interventions to prevent Cr (VI)-induced toxicity.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cromo/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Glicólise/efeitos dos fármacos , Células A549 , Fator 4 Ativador da Transcrição/genética , Apoptose/genética , Autofagia/genética , Estresse do Retículo Endoplasmático/genética , Técnicas de Silenciamento de Genes , Glicólise/genética , Humanos
6.
Mol Cell ; 79(4): 546-560.e7, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32589964

RESUMO

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.


Assuntos
Complexos Multiproteicos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Códon de Iniciação , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Fatores de Iniciação de Peptídeos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Nat Cell Biol ; 22(7): 882-895, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32451439

RESUMO

It is well accepted that cancers co-opt the microenvironment for their growth. However, the molecular mechanisms that underlie cancer-microenvironment interactions are still poorly defined. Here, we show that Rho-associated kinase (ROCK) in the mammary tumour epithelium selectively actuates protein-kinase-R-like endoplasmic reticulum kinase (PERK), causing the recruitment and persistent education of tumour-promoting cancer-associated fibroblasts (CAFs), which are part of the cancer microenvironment. An analysis of tumours from patients and mice reveals that cysteine-rich with EGF-like domains 2 (CRELD2) is the paracrine factor that underlies PERK-mediated CAF education downstream of ROCK. We find that CRELD2 is regulated by PERK-regulated ATF4, and depleting CRELD2 suppressed tumour progression, demonstrating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes the progression of breast cancer, with implications for cancer therapy.


Assuntos
Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Moléculas de Adesão Celular/metabolismo , Reprogramação Celular , Proteínas da Matriz Extracelular/metabolismo , eIF-2 Quinase/metabolismo , Quinases Associadas a rho/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Moléculas de Adesão Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Humanos , Camundongos , Comunicação Parácrina , eIF-2 Quinase/genética , Quinases Associadas a rho/genética
8.
Antioxid Redox Signal ; 33(2): 59-65, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32323565

RESUMO

Human lungs single-cell RNA sequencing data from healthy donors (elderly and young; GEO accession no. GSE122960) were analyzed to isolate and specifically study gene expression in alveolar type II cells. Colocalization of angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 enables severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2) to enter the cells. Expression levels of these genes in the alveolar type II cells of elderly and young patients were comparable and, therefore, do not seem to be responsible for worse outcomes observed in coronavirus disease 2019 (COVID-19) affected elderly. In cells from the elderly, 263 genes were downregulated and 95 upregulated. Superoxide dismutase 3 (SOD3) was identified as the top-ranked gene that was most downregulated in the elderly. Other redox-active genes that were also downregulated in cells from the elderly included activating transcription factor 4 (ATF4) and metallothionein 2A (M2TA). ATF4 is an endoplasmic reticulum stress sensor that defends lungs via induction of heme oxygenase 1. The study of downstream factors known to be induced by ATF4, according to Ingenuity Pathway Analysis™, identified 24 candidates. Twenty-one of these were significantly downregulated in the cells from the elderly. These downregulated candidates were subjected to enrichment using the Reactome Database identifying that in the elderly, the ability to respond to heme deficiency and the ATF4-dependent ability to respond to endoplasmic reticulum stress is significantly compromised. SOD3-based therapeutic strategies have provided beneficial results in treating lung disorders including fibrosis. The findings of this study propose the hypotheses that lung-specific delivery of SOD3/ATF4-related antioxidants will work in synergy with promising antiviral drugs such as remdesivir to further improve COVID-19 outcomes in the elderly.


Assuntos
Fator 4 Ativador da Transcrição/genética , Infecções por Coronavirus/genética , Pulmão/metabolismo , Pneumonia Viral/genética , Superóxido Dismutase/genética , Adulto , Idoso , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Antioxidantes/uso terapêutico , Betacoronavirus/patogenicidade , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Feminino , Regulação da Expressão Gênica/genética , Heme Oxigenase-1/genética , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Metalotioneína/genética , Pessoa de Meia-Idade , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Serina Endopeptidases/genética
9.
Proc Natl Acad Sci U S A ; 117(18): 9932-9941, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312819

RESUMO

Cellular starvation is typically a consequence of tissue injury that disrupts the local blood supply but can also occur where cell populations outgrow the local vasculature, as observed in solid tumors. Cells react to nutrient deprivation by adapting their metabolism, or, if starvation is prolonged, it can result in cell death. Cell starvation also triggers adaptive responses, like angiogenesis, that promote tissue reorganization and repair, but other adaptive responses and their mediators are still poorly characterized. To explore this issue, we analyzed secretomes from glucose-deprived cells, which revealed up-regulation of multiple cytokines and chemokines, including IL-6 and IL-8, in response to starvation stress. Starvation-induced cytokines were cell type-dependent, and they were also released from primary epithelial cells. Most cytokines were up-regulated in a manner dependent on NF-κB and the transcription factor of the integrated stress response ATF4, which bound directly to the IL-8 promoter. Furthermore, glutamine deprivation, as well as the antimetabolic drugs 2-deoxyglucose and metformin, also promoted the release of IL-6 and IL-8. Finally, some of the factors released from starved cells induced chemotaxis of B cells, macrophages, and neutrophils, suggesting that nutrient deprivation in the tumor environment can serve as an initiator of tumor inflammation.


Assuntos
Inflamação/genética , Interleucina-6/genética , Interleucina-8/genética , Neoplasias/metabolismo , Estresse Fisiológico/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Antimetabólitos/farmacologia , Morte Celular/efeitos dos fármacos , Desoxiglucose/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Glucose/metabolismo , Glutamina/metabolismo , Células HeLa , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Metformina/farmacologia , NF-kappa B/genética , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Inanição/genética , Inanição/metabolismo , Estresse Fisiológico/imunologia
10.
Life Sci ; 252: 117654, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32277979

RESUMO

BACKGROUND: Septic encephalopathy, the most frequent complication of sepsis, is orchestrated by a complex interplay of signals that leads to high mortality rates among intensive care unit patients. However, the role of the vascular endothelial growth factor receptor-2 (VEGFR2) in endoplasmic reticulum stress response (ERSR), during septic encephalopathy, is still elusive. AIM: This study was aimed to examine the effect of an in-house designed/synthesized VEGFR2 antagonist, named WAG4S, on septic encephalopathy using cecal ligation and perforation (CLP). MAIN METHODS: Rats were intraperitoneally injected with WAG-4S (1 mg/kg/d) for 7 days post-CLP. KEY FINDINGS: In septic animals, VEGFR2 antagonism declined the expression of cortical p-VEGFR2 and p-mammalian target of rapamycin complex-1 (p-mTORC1). It also worsened the behavioral and histopathological alterations beyond CLP. However, and contrary to CLP, WAG-4S decreased the p-protein kinase R-like ER kinase (p-PERK) and eukaryotic initiation factor-2α (p-eIF2α) expression. Moreover, VEGFR2 blockade upregulated the mRNA expression of activating transcription factor-4 (ATF4), binding immunoglobulin protein/glucose-regulated protein-78 (Bip/GRP78), growth arrest and DNA damage-34 (GADD34) and spliced X-box binding protein-1 (XBP1s) above CLP. Similarly, it boosted inositol requiring enzyme-1α (IRE1α) activation and redox imbalance. In the same context, WAG-4S augmented the protein levels of CLP-induced ERSR apoptotic markers, namely C/EBP homologous protein (CHOP/GADD153), c-jun N-terminal kinase (JNK) and caspase-3. SIGNIFICANCE: In conclusion, the PERK/eIF2α axis inhibition, during septic encephalopathy, is VEGFR2-independent, whereas the activated IRE1α/XBP1s/CHOP/JNK/caspase-3 cue promotes the ERSR execution module through VEGFR2 inhibition. This has turned VEGFR2 into a potential therapeutic target for ameliorating such an ailment.


Assuntos
Encefalopatias/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Sepse/complicações , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Animais , Encefalopatias/etiologia , Encefalopatias/prevenção & controle , Modelos Animais de Doenças , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sepse/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , eIF-2 Quinase/metabolismo
12.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 165-170, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32027271

RESUMO

OBJECTIVE: To explore the expression of Blimp1, ATF4 and CHOP in bone marrow mononuclear cells from patients with multiple myeloma as well as the effect of aspirin on their expression. METHODS: Sixty untreated patients with multiple myeloma and 30 patients with relatively normal bone marrow were selected. Mononuclear cells from the bone marrow fluid were separated using Ficoll separation solution. CD138+ plasma cells were sorted by immunomagnetic beads method. RT-PCR was used to detect the expression levels of Blimp1, ATF4 and CHOP mRNA in U266 cells cultured in vitro. The cells were divided into blank control group, negative control group (no-loaded virus transfection) and positive experimental group [LV-Blimp1-RNAi (40051-2) transfection] by lentivirus transfection. RT-PCR was used to detect the expression of Blimp1, ATF4 and CHOP mRNA in cells of different groups. U266 cells were stimulated in vitro with different concentrations of aspirin solution (0, 0.5 mmol/L, 2.5 mmol/L, 5.0 mmol/L) for 24, 48 h and 72 h, respectively. The ability of cell proliferation in different groups was measured by CCK-8. U266 cells were stimulated with different concentrations of aspirin for 48 hours. And the mRNA expression of Blimp1, ATF4 and CHOP was detected by RT-PCR. RESULTS: Compared with plasma cells in normal group, the expression of Blimp1 mRNA in CD138+ plasma cells of MM patients significantly increased (8.040±1.878), and the mRNA expression levels of ATF4 and CHOP significantly decreased (0.735±0.089; 0.837±0.062) (P<0.05). U266 cells were cultured in vitro. Compared with the blank control group and the negative control group, the mRNA expression level of Blimp in the positive experimental group was significantly down-regulated after infection with LV-Blimp1-RNAi (40051-2) lentiviral expression vector (0.637±0.021). ATF4 and CHOP mRNA expression levels were significantly increased (1.452 ± 0.027; 1.721 ± 0.038) (P<0.05). The proliferation of U266 cells decreased after stimulation with aspirin. In the range of (0.5-5) mmol/L, aspirin could significantly inhibit the proliferation of U266 cells. The inhibition effect of aspirin was increased along with prolongation of time and increase of concentrations. After aspirin stimulation of different concentrations for 48 hours, the expression level of Blimp1 in U266 cells decreased with increasing of drug concentration, while the expression levels of ATF4 and CHOP increased with increasing of drug concentration. CONCLUSION: Inhibition of Blimp1 expression in multiple myeloma cells can promote the expression of ATF4 and CHOP. Aspirin can inhibit the proliferation activity of myeloma cells by down-regulating Blimp1 expression in myeloma cells and up-regulating ATF4 and CHOP expression, therefore plays an anti-tumor rote.


Assuntos
Fator 4 Ativador da Transcrição/genética , Mieloma Múltiplo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose , Aspirina , Linhagem Celular Tumoral , Proliferação de Células , Ciclofosfamida , Doxorrubicina , Humanos , Mieloma Múltiplo/genética , Prednisona , Vincristina
13.
Respir Med ; 161: 105852, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32056726

RESUMO

OBJECTIVE: The aim of the study was to investigate the mechanism and effect of FBXL10 in myocardial ischemia reperfusion injury in vivo and in vitro. METHODS: The myocardial ischemia reperfusion (I/R) model was established by 30 min of coronary occlusion followed by 2 h of reperfusion in rats. Western blot and TUNEL assay were used to measure the apoptosis during I/R. The expression levels of endoplasmic reticulum related proteins in myocardial tissues and H9c2 cells were detected by immunohistochemistry staining and immunofluorescence staining. Flow cytometry and CCK-8 were used to detect the apoptosis and viability of H9c2 cells. RESULTS: The results revealed that FBXL10 significantly reduced myocardial infarction, improved the pathological morphology of myocardium, markedly reduced inflammatory response in the myocardial ischemia reperfusion rats. Moreover the expressions of endoplasmic reticulum stress key proteins were caused by I/R were suppressed significantly by FBXL10 treatment, including CHOP, GRP78, ATF4 and p-PERK. Additionally FBXL10 inhibited the expression of endoplasmic reticulum stress key proteins in H/R H9c2 cells. Furthermore, FBXL10 reduced the levels of apoptotic cells and inflammatory response compared with I/R and H/R group. CONCLUSION: Taken together, we found that FBXL10 could attenuate I/R injury through inhibiting endoplasmic reticulum stress (ERs).


Assuntos
Estresse do Retículo Endoplasmático/genética , Proteínas F-Box/fisiologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/genética , Células Cultivadas , Expressão Gênica/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Ratos Sprague-Dawley , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
14.
Cell Commun Signal ; 18(1): 12, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31987044

RESUMO

BACKGROUND: Cell death triggered by unmitigated endoplasmic reticulum (ER) stress plays an important role in physiology and disease, but the death-inducing signaling mechanisms are incompletely understood. To gain more insight into these mechanisms, the ER stressor thapsigargin (Tg) is an instrumental experimental tool. Additionally, Tg forms the basis for analog prodrugs designed for cell killing in targeted cancer therapy. Tg induces apoptosis via the unfolded protein response (UPR), but how apoptosis is initiated, and how individual effects of the various UPR components are integrated, is unclear. Furthermore, the role of autophagy and autophagy-related (ATG) proteins remains elusive. METHODS: To systematically address these key questions, we analyzed the effects of Tg and therapeutically relevant Tg analogs in two human cancer cell lines of different origin (LNCaP prostate- and HCT116 colon cancer cells), using RNAi and inhibitory drugs to target death receptors, UPR components and ATG proteins, in combination with measurements of cell death by fluorescence imaging and propidium iodide staining, as well as real-time RT-PCR and western blotting to monitor caspase activity, expression of ATG proteins, UPR components, and downstream ER stress signaling. RESULTS: In both cell lines, Tg-induced cell death depended on death receptor 5 and caspase-8. Optimal cytotoxicity involved a non-autophagic function of MAP1LC3B upstream of procaspase-8 cleavage. PERK, ATF4 and CHOP were required for Tg-induced cell death, but surprisingly acted in parallel rather than as a linear pathway; ATF4 and CHOP were independently required for Tg-mediated upregulation of death receptor 5 and MAP1LC3B proteins, whereas PERK acted via other pathways. Interestingly, IRE1 contributed to Tg-induced cell death in a cell type-specific manner. This was linked to an XBP1-dependent activation of c-Jun N-terminal kinase, which was pro-apoptotic in LNCaP but not HCT116 cells. Molecular requirements for cell death induction by therapy-relevant Tg analogs were identical to those observed with Tg. CONCLUSIONS: Together, our results provide a new, integrated understanding of UPR signaling mechanisms and downstream mediators that induce cell death upon Tg-triggered, unmitigated ER stress. Video Abstract.


Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Tapsigargina/metabolismo , Resposta a Proteínas não Dobradas , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Autofagia , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
15.
Am J Reprod Immunol ; 83(1): e13186, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31483910

RESUMO

PROBLEM: Polycystic ovary syndrome (PCOS) is associated with endoplasmic reticulum (ER) stress and pro-inflammatory condition. The aim of the present study was to evaluate the effect of resveratrol treatment on pro-inflammatory and ER stress markers in patients with PCOS. METHOD OF STUDY: Cumulus cells were obtained from 40 patients with PCOS who were divided into two groups: placebo and resveratrol treatment (receiving 800 mg/d for 40 days) groups. Blood samples were obtained from all patients before and after the procedure to evaluate interleukin (IL)-6, IL-1ß, IL-18, TNF-α, NF-κB, and C-reactive protein (CRP). Total RNA was extracted from cumulus cells, and cDNA was synthesized by reverse transcription. Expressions of five genes in ER stress response pathway (ATF4, ATF6, CHOP, GRP78, and XBP1s) were assessed with quantitative real-time PCR. Statistical analysis was performed with Student's t test. RESULTS: After treatment with resveratrol, it was found that serum levels of IL-6, IL-1ß, TNF-α, IL-18, NF-κB, and CRP decreased in the treatment group. In addition, gene expression results showed that the expression levels of ATF4 (P < .05) and ATF6 (P < .001) significantly increased in the resveratrol treatment group, while the expression levels of CHOP, GRP78, and XBP1 (P < .001 for all) significantly decreased. CONCLUSION: Results demonstrated that resveratrol has anti-inflammatory effects through the suppression of NF-κB and NF-κB-regulated gene products. On the other hand, resveratrol can modulate ER stress in granulosa cells (GCs) by altering the expression of genes involved in unfolding protein response (UPR) process. Our findings suggest that ER stress is a potential therapeutic target for patients with PCOS.


Assuntos
Anti-Inflamatórios/farmacologia , Síndrome do Ovário Policístico , Resveratrol/farmacologia , Fator 4 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/genética , Adolescente , Adulto , Anti-Inflamatórios/uso terapêutico , Proteína C-Reativa/análise , Células Cultivadas , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Citocinas/sangue , Método Duplo-Cego , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Feminino , Proteínas de Choque Térmico/genética , Humanos , NF-kappa B/sangue , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/genética , Resveratrol/uso terapêutico , Fator de Transcrição CHOP/genética , Proteína 1 de Ligação a X-Box/genética , Adulto Jovem
16.
Virology ; 539: 1-10, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31605941

RESUMO

Endoplasmic reticulum (ER) stress is associated with numerous mammalian diseases, especially viral diseases. Porcine parvovirus (PPV) is the causative agent of reproductive failure in swine. Here, we observed that the PPV infection of porcine kidney 15 and porcine testis cells resulted in the activation of ER stress sensors mediated by protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 and activating transcription factor 6 (ATF6). ER stress activation obviously blocked PPV replication. Depletion of proteins, such as PERK, eukaryotic initiation factor 2, and ATF4, by small interfering RNA significantly enhanced PPV replication. Moreover, the pro-apoptotic factor C/EBP homologous protein was identified a key factor in the inhibition of PPV replication. These data demonstrate that PPV infection activates ER stress through the PERK signaling pathway and that ER stress inhibits further PPV replication by promoting apoptosis.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Infecções por Parvoviridae/virologia , Parvovirus Suíno/fisiologia , Transdução de Sinais , Replicação Viral , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/patologia , Parvovirus Suíno/metabolismo , Suínos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genética
17.
J Clin Invest ; 130(3): 1217-1232, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31770110

RESUMO

The drug efflux pump ABCB1 is a key driver of chemoresistance, and high expression predicts treatment failure in acute myeloid leukemia (AML). In this study, we identified and functionally validated the network of enhancers that controls expression of ABCB1. We show that exposure of leukemia cells to daunorubicin activated an integrated stress response-like transcriptional program to induce ABCB1 through remodeling and activation of an ATF4-bound, stress-responsive enhancer. Protracted stress primed enhancers for rapid increases in activity following re-exposure of cells to daunorubicin, providing an epigenetic memory of prior drug treatment. In primary human AML, exposure of fresh blast cells to daunorubicin activated the stress-responsive enhancer and led to dose-dependent induction of ABCB1. Dynamic induction of ABCB1 by diverse stressors, including chemotherapy, facilitated escape of leukemia cells from targeted third-generation ABCB1 inhibition, providing an explanation for the failure of ABCB1 inhibitors in clinical trials. Stress-induced upregulation of ABCB1 was mitigated by combined use of the pharmacologic inhibitors U0126 and ISRIB, which inhibit stress signaling and have potential for use as adjuvants to enhance the activity of ABCB1 inhibitors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Elementos Facilitadores Genéticos , Leucemia Mieloide Aguda , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Acetamidas/farmacologia , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Butadienos/farmacologia , Cicloexilaminas/farmacologia , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Nitrilos/farmacologia , Regulação para Cima/efeitos dos fármacos
18.
J Biol Chem ; 295(1): 237-249, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31792031

RESUMO

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.


Assuntos
Trifosfato de Adenosina/metabolismo , MicroRNAs/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Apoptose , Creatina Quinase Forma BB/genética , Creatina Quinase Forma BB/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , MicroRNAs/genética , eIF-2 Quinase/genética
19.
J Nutr ; 150(5): 1022-1030, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31875479

RESUMO

BACKGROUND: The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. OBJECTIVE: The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. METHODS: Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. RESULTS: Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. CONCLUSIONS: The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Leucina/administração & dosagem , Leucina/deficiência , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Peroxidases/metabolismo , Fatores de Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidases/genética , Fatores de Transcrição/genética
20.
Cell Death Dis ; 10(12): 959, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862913

RESUMO

Autophagy, an intracellular system of degrading damaged organelles and misfolded proteins, is essential for cancer cell survival. Despite the progress made towards understanding the mechanism, identification of novel autophagy regulators presents a major obstacle in developing anticancer therapies. Here, we examine the association between the TOR signaling pathway regulator-like (TIPRL) protein and autophagy in malignant transformation of tumors. We show that TIPRL upregulation in non-small cell lung cancer (NSCLC) potentiated autophagy activity and enabled autophagic clearance of metabolic and cellular stress, conferring a survival advantage to cancer cells. Importantly, the interaction of TIPRL with eukaryotic initiation factor 2α (eIF2α) led to eIF2α phosphorylation and activation of the eIF2α-ATF4 pathway, thereby inducing autophagy. Conversely, TIPRL depletion increased apoptosis by reducing autophagic clearance, which was markedly enhanced in TIPRL-depleted A549 xenografts treated with 2-deoxy-D-glucose. Overall, the study indicated that TIPRL is a potential regulator of autophagy and an important drug target for lung cancer therapy.


Assuntos
Fator 4 Ativador da Transcrição/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Fator de Iniciação 2 em Eucariotos/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Células A549 , Animais , Apoptose , Autofagia/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Sobrevivência Celular , Estresse do Retículo Endoplasmático , Feminino , Xenoenxertos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Fosforilação , Transdução de Sinais , Esferoides Celulares/patologia
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