RESUMEN

Mitochondrial biogenesis deficits in neuronal cells are associated with the pathological progression of neurodegenerative diseases. Resistin, a secretory adipocytokine, possesses multiple physiological functions in diverse cells and tissues. However, the effects of resistin on mitochondrial biogenesis in neuronal cells are still elusive. In the current study, we found that resistin caused a sustainable decrease in mitochondrial contents, including mitochondrial DNA/nuclear DNA ratio (mtDNA/nDNA), mitochondrial mass, cytochrome b protein expression, and cytochrome c oxidase activity, which were correlated with "loss of mitochondrial function" including reduced mitochondrial respiration rate and ATP production in human SH-SY5Y neuronal cells. Indeed, resistin treatment destroyed the expression of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), a master regulator of mitochondrial biogenesis, as well as its downstream target genes including nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM). Notably, overexpression of PGC-1α could completely rescue mitochondrial biogenesis and mitochondrial deficits induced by resistin. Mechanistically, inhibition of 5'-adenosine monophosphate-activated protein kinase (AMPK) was shown to mediate the inhibitory effects of resistin on mitochondrial biogenesis.

Asunto(s)

Mitocondrias/efectos de los fármacos , Resistina/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Línea Celular , Respiración de la Célula/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Factor Nuclear 1 de Respiración/antagonistas & inhibidores , Factor Nuclear 1 de Respiración/genética , Factor Nuclear 1 de Respiración/metabolismo , Biogénesis de Organelos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/antagonistas & inhibidores , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The proteasome inhibitors carfilzomib (Cfz) and bortezomib (Btz) are used successfully to treat multiple myeloma, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the ß5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of ß1 and ß2 to demonstrate that inhibiting a second site of the proteasome, particularly the ß2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both ß5 and ß2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual ß5 and ß2 inhibitors for the treatment of solid tumors.

Asunto(s)

Antineoplásicos/farmacología , Factor Nuclear 1 de Respiración/antagonistas & inhibidores , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Factor Nuclear 1 de Respiración/metabolismo , Inhibidores de Proteasoma/síntesis química , Inhibidores de Proteasoma/química , Relación Estructura-Actividad , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas

RESUMEN

Hypoxia-inducible factor 1α (HIF-1α) is a master regulator of oxygen homeostasis. Under hypoxia, the active HIF1-α subunits are mainly regulated through increased protein stabilization. Little is known concerning HIF-1α transcriptional regulation. Nuclear respiratory factor 1 (NRF-1) is a DNA-binding transcription factor that regulates mitochondrial biogenesis. In this study, we showed that NRF-1was a repressor of HIF-1α. The cellular depletion of NRF-1 by siRNA targeting leads to increased HIF-1αtranscriptional activity. EMSA, ChIP and luciferase activity allowed the identification of two functional NRF-1 binding sites within HIF-1α promoter. This study therefore identifies NRF-1 as a novel regulator of HIF-1α. © 2016 IUBMB Life, 68(9):748-755, 2016.

Asunto(s)

Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Mitocondrias/genética , Factor Nuclear 1 de Respiración/genética , Activación Transcripcional/genética , Sitios de Unión/genética , Hipoxia de la Célula/genética , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Mitocondrias/metabolismo , Factor Nuclear 1 de Respiración/antagonistas & inhibidores , Oxígeno/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética

RESUMEN

microRNAs (miRNAs) are involved in the various processes of DNA damage repair and play crucial roles in regulating response of tumors to radiation therapy. Here, we used nasopharyngeal carcinoma (NPC) radio-resistant cell lines as models and found that the expression of miR-504 was significantly up-regulated. In contrast, the expression of nuclear respiratory factor 1 (NRF1) and other mitochondrial metabolism factors, including mitochondrial transcription factor A (TFAM) and oxidative phosphorylation (OXPHOS) complex III were down-regulated in these cell lines. At the same time, the Seahorse cell mitochondrial stress test results indicated that the mitochondrial respiratory capacity was impaired in NPC radio-resistant cell lines and in a miR-504 over-expressing cell line. We also conducted dual luciferase reporter assays and verified that miR-504 could directly target NRF1. Additionally, miR-504 could down-regulate the expression of TFAM and OXPHOS complexes I, III, and IV and impaired the mitochondrial respiratory function of NPC cells. Furthermore, serum from NPC patients showed that miR-504 was up-regulated during different weeks of radiotherapy and correlated with tumor, lymph nodes and metastasis (TNM) stages and total tumor volume. The radio-therapeutic effect at three months after radiotherapy was evaluated. Results indicated that patients with high expression of miR-504 exhibited a relatively lower therapeutic effect ratio of complete response (CR), but a higher ratio of partial response (PR), compared to patients with low expression of miR-504. Taken together, these results demonstrated that miR-504 affected the radio-resistance of NPC by down-regulating the expression of NRF1 and disturbing mitochondrial respiratory function. Thus, miR-504 might become a promising biomarker of NPC radio-resistance and targeting miR-504 might improve tumor radiation response.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/radioterapia , Factor Nuclear 1 de Respiración/antagonistas & inhibidores , Tolerancia a Radiación/genética , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Western Blotting , Carcinoma , Proliferación Celular , Humanos , Metástasis Linfática , Ratones , MicroARNs/sangre , NAD/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/sangre , Neoplasias Nasofaríngeas/patología , Estadificación de Neoplasias , Factor Nuclear 1 de Respiración/genética , Factor Nuclear 1 de Respiración/metabolismo , Pronóstico , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF-Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A-Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation.

Asunto(s)

Glucógeno Sintasa Quinasa 3/fisiología , Factor Nuclear 1 de Respiración/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Supervivencia Celular/genética , Células Cultivadas , Regulación hacia Abajo/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/fisiología , Proteína 7 que Contiene Repeticiones F-Box-WD , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Células HEK293 , Humanos , Ratones , Factor Nuclear 1 de Respiración/antagonistas & inhibidores , Factor Nuclear 1 de Respiración/metabolismo , Unión Proteica/fisiología , Proteolisis , Estrés Fisiológico/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología

RESUMEN

In this study, we investigated the role of nuclear respiratory factor-1(NRF-1) in benzo(a)pyrene (BaP)-induced mitochondrial events in human bronchial epithelial cells (16HBE). Cytotoxicity was determined with MTT assay, and apoptosis was measured by flow cytometry. The results showed that BaP inhibited cell proliferation in a dose-dependent manner and induced apoptosis in 16HBE cells. Time-dependent reactive oxygen species (ROS) generation induced by BaP was observed in 16HBE cells. The loss of mitochondrial membrane permeability transition (MPT) was obtained by a laser scanning confocal microscope, and the decreasing ATP level was detected by a Cell-Titer-Glo(®) Luminescent Cell Viability Assay. Results of western blotting assay revealed that both NRF-1 and mitochondrial transcription factor A (mtTFA) decreased in 12-µM BaP-treated cells at both 12 and 24 hr. The results of RT-PCR indicate that NRF-1 and mtTFA mRNA in 16HBE cells were not changed after BaP treatment 12 or 24 hr. Down-regulation of NRF-1 by shRNA further reduced the loss of MPT and increased ROS generation in response to BaP treatment. Therefore, our results demonstrate that NRF-1 is responsible for BaP-induced mitochondrial dysfunction in 16HBE cells and associated with the level of mtTFA protein, loss of MPT and ROS overproduction.

Asunto(s)

Benzo(a)pireno/toxicidad , Bronquios/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Factor Nuclear 1 de Respiración/fisiología , Adenosina Trifosfato/análisis , Apoptosis/efectos de los fármacos , Bronquios/citología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/análisis , Células Epiteliales/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/fisiología , Proteínas Mitocondriales/análisis , Factor Nuclear 1 de Respiración/análisis , Factor Nuclear 1 de Respiración/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/análisis

RESUMEN

The aim of this study was to detect mitochondrial alterations in J774.2 macrophages and mouse peritoneal macrophages (MPM) foam cells. J774.2 and MPM cells were exposed to triacylglycerol (TG) emulsion (1 mg/ml) for induction of fat accumulation. Impairment of mitochondrial function was reflected by reduced cellular ATP production and decreased expression of subunits of mitochondrial complexes I and III. The expression of subunit IV of complex IV remained unchanged, however, the content of its precursor in cells increased. Inhibitors of mitochondrial complexes, rotenone (0.1 microM) and myxothiazol (25 nM), protected the viability in TG-loaded macrophages. The exposure to TG caused downregulation of PPARgamma coactivator (PGC)-1alpha and nuclear respiratory factor (NRF)-1. Activation of peroxisome proliferator-activated receptors attenuated reactive oxygen species production in the foam cells. Treatment with antioxidant N-acetylcysteine (NAC) prevented lipid-mediated mitochondrial and cellular damage. In conclusion, this study demonstrates the important role of mitochondrial biogenesis dysfunction in TG-induced lipotoxicity in macrophages.

Asunto(s)

Células Espumosas/patología , Macrófagos Peritoneales/patología , Mitocondrias/patología , Triglicéridos/toxicidad , Animales , Línea Celular , Células Cultivadas , ADN Mitocondrial/antagonistas & inhibidores , ADN Mitocondrial/biosíntesis , Regulación hacia Abajo/fisiología , Células Espumosas/metabolismo , Metabolismo de los Lípidos/fisiología , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Factor Nuclear 1 de Respiración/antagonistas & inhibidores , Factor Nuclear 1 de Respiración/fisiología , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/toxicidad , Glycine max , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/biosíntesis

RESUMEN

Neuronal activity is highly dependent on energy metabolism; yet, the two processes have traditionally been regarded as independently regulated at the transcriptional level. Recently, we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1) co-regulates an important energy-generating enzyme, cytochrome c oxidase, as well as critical subunits of glutamatergic receptors. The present study tests our hypothesis that the co-regulation extends to the next level of glutamatergic synapses, namely, neuronal nitric oxide synthase, which generates nitric oxide as a downstream signaling molecule. Using in silico analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, promoter mutations, and NRF-1 silencing, we documented that NRF-1 functionally bound to Nos1, but not Nos2 (inducible) and Nos3 (endothelial) gene promoters. Both COX and Nos1 transcripts were up-regulated by depolarizing KCl treatment and down-regulated by TTX-mediated impulse blockade in neurons. However, NRF-1 silencing blocked the up-regulation of both Nos1 and COX induced by KCl depolarization, and over-expression of NRF-1 rescued both Nos1 and COX transcripts down-regulated by TTX. These findings are consistent with our hypothesis that synaptic neuronal transmission and energy metabolism are tightly coupled at the molecular level.

Asunto(s)

Complejo IV de Transporte de Electrones/genética , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa/genética , Factor Nuclear 1 de Respiración/metabolismo , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Células Cultivadas , Cartilla de ADN/genética , Técnicas In Vitro , Ratones , Modelos Neurológicos , Mutagénesis Sitio-Dirigida , Neuronas/efectos de los fármacos , Factor Nuclear 1 de Respiración/antagonistas & inhibidores , Factor Nuclear 1 de Respiración/genética , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tetrodotoxina/farmacología , Transcripción Genética/efectos de los fármacos