RESUMEN
The single Nrf1 gene has capability to be differentially transcripted alongside with alternative mRNA-splicing and subsequent translation through different initiation signals so as to yield distinct lengths of polypeptide isoforms. Amongst them, three of the most representatives are Nrf1α, Nrf1ß and Nrf1γ, but the putative specific contribution of each isoform to regulating ARE-driven target genes remains unknown. To address this, we have herein established three cell lines on the base of the Flp-In T-REx system, which are allowed for the tetracycline-inducibly stable expression of Nrf1α, Nrf1ß and Nrf1γ. Consequently, the RNA-Sequencing results have demonstrated that a vast majority of differentially expressed genes (i.e. >90% DEGs detected) were dominantly up-regulated by Nrf1α and/or Nrf1ß following induction by tetracycline. By contrast, the other DEGs regulated by Nrf1γ were far less than those regulated by Nrf1α/ß (i.e. ~11% of Nrf1α and ~7% of Nrf1ß). However, further transcriptomic analysis revealed that the tetracycline-induced expression of Nrf1γ significantly increased the percentage of down-regulated genes in total DEGs. These statistical data were further validated by quantitative real-time PCR. The experimental results indicate that distinct Nrf1 isoforms make diverse and even opposing contributions to regulating different subsets of target genes, such as those encoding 26S proteasomal subunits and others involved in various biological processes and functions. Collectively, Nrf1γ acts as a major dominant-negative inhibitor competitively against Nrf1α/ß activity, such that a number of DEGs regulated by Nrf1α/ß are counteracted by Nrf1γ.
Asunto(s)
Factor 1 Relacionado con NF-E2/genética , Factor 1 Relacionado con NF-E2/metabolismo , Factor 1 Relacionado con NF-E2/fisiología , Línea Celular , Perfilación de la Expresión Génica/métodos , Humanos , Isoformas de Proteínas , Análisis de Secuencia de ARN/métodos , Activación TranscripcionalRESUMEN
Nrf1 (also referred to as NFE2L1) is a member of the CNC-bZIP family of transcription factors that are characterized by a highly conserved CNC-domain, and a basic-leucine zipper domain required for dimerization and DNA binding. Nrf1 is ubiquitously expressed across tissue and cell types as various isoforms, and is induced by stress signals from a broad spectrum of stimuli. Evidence indicates that Nrf1 plays an important role in regulating a range of cellular functions including oxidative stress response, differentiation, inflammatory response, metabolism, and maintaining proteostasis. Thus, Nrf1 has been implicated in the pathogenesis of various disease processes including cancer development, and degenerative and metabolic disorders. This review summarizes our current understanding of Nrf1 and the molecular mechanism underlying its regulation and action in different cellular functions.
Asunto(s)
Regulación de la Expresión Génica , Factor 1 Relacionado con NF-E2/fisiología , Animales , Homeostasis/genética , Humanos , Factor 1 Relacionado con NF-E2/química , Factor 1 Relacionado con NF-E2/genética , Conformación ProteicaRESUMEN
The endoplasmic reticulum (ER) lumen is chemically complex and crowded with polypeptides in different stages of assembly. ER quality control monitors chaperone-assisted protein folding, stochastic errors and off-pathway intermediates. In acute conditions, potentially toxic polypeptides overflow the capacity of the chaperone system and lead to ER stress. Activation of the unfolded protein response (UPR) following ER stress buys time for non-native polypeptides to refold or be eliminated; otherwise cell death occurs. The clearance routes for deleterious proteins are endoplasmic reticulum-associated degradation (ERAD) and ER stress-activated autophagy. The ERAD pathway is a chaperone and proteasome-mediated polypeptide degradation, while autophagy applies to wider range of substances. ER stress signal transduction recruits diverse molecules and pathways upon UPR induction to compensate stress condition. NF-E2-related factor 1 (Nrf1) and Nrf2 are two transcription factors mostly known by their induction through an antioxidant response; they can also be activated by UPR machinery. Discovery of diverse molecules downstream of Nrf1 and Nrf2 has expanded our understanding of the biological impacts of these transcription factors beyond classic antioxidant activation. In this review, we summarize our current understanding of mutual relationships between Nrf1, Nrf2, and ER stress clearance mechanisms and highlight the crosstalk of specific molecules mediating these correlations.
Asunto(s)
Estrés del Retículo Endoplásmico , Factor 1 Relacionado con NF-E2/fisiología , Factor 2 Relacionado con NF-E2/fisiología , Animales , Autofagia , Degradación Asociada con el Retículo Endoplásmico , Humanos , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
The ubiquitin-proteasome system is important in maintaining protein homeostasis. NFE2-related factor 1 (Nrf1), a transcription factor in the cap 'n' collar basic-leucine zipper family, regulates expression of cytoprotective genes. It was previously shown that liver-specific knockout of Nrf1 (Nrf1LKO) leads to hepatic cell death, steatohepatitis and cancer. However, the mechanisms underlying these pathologies are not clear. Here, we report that Nrf1 is critical for proteasome gene expression in the liver. Liver-specific knockout of Nrf1 results in impaired basal and induced expression of proteasome genes, and diminished proteasome activity in hepatocytes. In addition, our findings demonstrated that endoplasmic reticulum stress signaling pathway was also activated in Nrf1LKO livers. Inhibition of proteasome activity leads to endoplasmic reticulum stress in Nrf1-deficient hepatocytes, prompting the development of steatosis in the liver. Our results indicate that Nrf1 plays an integral role in the maintenance of proteasome function in hepatocytes and in the prevention of liver steatosis development. Moreover, these results highlight an association between proteasome dysfunction, endoplasmic reticulum stress and steatosis.
Asunto(s)
Estrés del Retículo Endoplásmico , Hígado Graso/metabolismo , Hepatocitos/enzimología , Factor 1 Relacionado con NF-E2/fisiología , Complejo de la Endopetidasa Proteasomal/genética , Animales , Ácidos Borónicos , Bortezomib , Hígado Graso/inducido químicamente , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hígado/metabolismo , Ratones , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , PirazinasRESUMEN
Nuclear factor erythroid-derived 2-related factor 1 (Nrf1) regulates cellular stress response genes, and has also been suggested to play a role in other cellular processes. We previously demonstrated that hepatocyte-specific deletion of Nrf1 in mice resulted in spontaneous apoptosis, inflammation, and development of liver tumors. Here, we showed that both fibroblasts derived from Nrf1 null mouse embryos and fibroblasts expressing a conditional Nrf1 allele showed increased micronuclei and formation of abnormal nuclei. Lentiviral shRNA-mediated knockdown of Nrf1 in SAOS-2 cells also resulted in increased micronuclei, abnormal mitosis and multi-nucleated cells. Metaphase analyses showed increased aneuploidy in Nrf1(-/-) embryonic fibroblasts. Nuclear defects in Nrf1-deficient cells were associated with decreased expression of various genes encoding kinetochore and mitotic checkpoint proteins. Our findings suggest that Nrf1 may play a role in maintaining genomic integrity, and that Nrf1 dysregulation may induce tumorigenesis.
Asunto(s)
Núcleo Celular/patología , Neoplasias del Colon/patología , Fibroblastos/patología , Inestabilidad Genómica , Micronúcleos con Defecto Cromosómico , Subunidad p45 del Factor de Transcripción NF-E2/fisiología , Factor 1 Relacionado con NF-E2/fisiología , Osteosarcoma/patología , Aneuploidia , Animales , Apoptosis , Western Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Núcleo Celular/genética , Proliferación Celular , Células Cultivadas , Neoplasias del Colon/genética , Fibroblastos/metabolismo , Humanos , Cinetocoros/metabolismo , Ratones , Ratones Noqueados , Subunidad p45 del Factor de Transcripción NF-E2/antagonistas & inhibidores , Subunidad p45 del Factor de Transcripción NF-E2/genética , Factor 1 Relacionado con NF-E2/antagonistas & inhibidores , Factor 1 Relacionado con NF-E2/genética , Osteosarcoma/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We studied the implication of focal adhesion kinase (FAK) in cardiac mitochondrial biogenesis induced by mechanical stress. Prolonged stretching (2-12 h) of neonatal rat ventricular myocytes (NRVM) upregulated the main components of mitochondrial transcription cascade [peroxisome proliferator-activated receptor coactivator-1 (PGC-1α), nuclear respiratory factor (NRF-1), and mitochondrial transcription factor A]. Concomitantly, prolonged stretching enhanced mitochondrial biogenesis [copy number of mitochondrial DNA (mtDNA), content of the subunit IV of cytochrome oxidase, and mitochondrial staining-green fluorescence intensity of Mitotracker green] and induced the hypertrophic growth (cell size and atrial natriuretic peptide transcripts) of NRVM. Furthermore, the stretching of NRVM enhanced phosphorylation, nuclear localization, and association of FAK with PGC-1α. Recombinant FAK COOH-terminal, but not the NH(2)-terminal or kinase domain, precipitated PGC-1α from nuclear extracts of NRVM. Depletion of FAK by RNA interference suppressed the upregulation of PGC-1α and NRF-1 and markedly attenuated the enhanced mitochondrial biogenesis and hypertrophic growth of stretched NRVM. In the context of energy metabolism, FAK depletion became manifest by a reduction of ATP levels in stretched NRVM. Complementary studies in adult mice left ventricle demonstrated that pressure overload upregulated PGC-1α, NRF-1, and mtDNA. In vivo FAK silencing transiently attenuated the upregulation of PGC-1α, NRF-1, and mtDNA, as well as the left ventricular hypertrophy induced by pressure overload. In conclusion, activation of FAK signaling seems to be important for conferring enhanced mitochondrial biogenesis coupled to the hypertrophic growth of cardiomyocytes in response to mechanical stress, via control of mitochondrial transcription cascade.
Asunto(s)
Quinasa 1 de Adhesión Focal/metabolismo , Mitocondrias Cardíacas/enzimología , Miocitos Cardíacos/enzimología , Estrés Mecánico , Animales , Animales Recién Nacidos , Células Cultivadas , ADN Mitocondrial/metabolismo , ADN Mitocondrial/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Complejo IV de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/fisiología , Ratones , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Miocitos Cardíacos/fisiología , Factor 1 Relacionado con NF-E2/metabolismo , Factor 1 Relacionado con NF-E2/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Ratas , Ratas Wistar , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Regulación hacia ArribaRESUMEN
In rat liver RL-34 cells, endogenous Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is localized in the ER (endoplasmic reticulum) where it exists as a glycosylated protein. Electron microscopy has demonstrated that ectopic Nrf1 in COS-1 cells is located in the ER and the NE (nuclear envelope). Subcellular fractionation, together with a membrane proteinase protection assay, revealed that Nrf1 is an integral membrane protein with both luminal and cytoplasmic domains. The N-terminal 65 residues of Nrf1 direct its integration into the ER and NE membranes and tether it to a Triton X-100-resistant membrane microdomain that is associated with lipid rafts. The activity of Nrf1 was increased by the electrophile tBHQ (t-butyl hydroquinone) probably through an N-terminal domain-dependent process. We found that the NST (Asn/Ser/Thr-rich) domain, along with AD1 (acidic domain 1), contributes positively to the transactivation activity of full-length Nrf1. Furthermore, the NST domain contains seven putative -Asn-Xaa-Ser/Thr- glycosylation sites and, when glycosylation was prevented by replacing all of the seven asparagine residues with either glutamine (Nrf1(1-7xN/Q)) or aspartic acid (Nrf1(1-7xN/D)), the former multiple point mutant possessed less activity than the wild-type factor, whereas the latter mutant exhibited substantially greater activity. Lastly, the ER stressors tunicamycin, thapsigargin and Brefeldin A were found to inhibit basal Nrf1 activity by approximately 25%, and almost completely prevented induction of Nrf1-mediated transactivation by tBHQ. Collectively, these results suggest that the activity of Nrf1 critically depends on its topology within the ER, and that this is modulated by redox stressors, as well as by its glycosylation status.
Asunto(s)
Retículo Endoplásmico/efectos de los fármacos , Hidroquinonas/farmacología , Factor 1 Relacionado con NF-E2/fisiología , Membrana Nuclear/metabolismo , Estrés Fisiológico/fisiología , Activación Transcripcional/efectos de los fármacos , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Brefeldino A/farmacología , Células COS , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Glicosilación , Factor 1 Relacionado con NF-E2/metabolismo , Oxidación-Reducción/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Estrés Fisiológico/efectos de los fármacos , Tapsigargina/farmacología , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Tunicamicina/farmacologíaRESUMEN
Mitochondria have their own DNA (mtDNA) and hence biogenesis of mitochondria requires a coordination of nuclear and mtDNA, both of which encode for mitochondria proteins. Our understanding of the molecular control of mitochondria biogenesis has increased in recent years, providing key signatures of the process. To determine whether or not a tissue or an organ of human or animal origin is undergoing mitochondria biogenesis, multiple parameters should be analyzed. First and foremost is visualization and measurement of mitochondria mass/volume in histological sections using fluorescent mitochondria dyes and light microscopy or transmission electron microscopy to yield quantitative results. To confirm or extend these types of analysis, biochemical markers of mitochondria biogenesis should also be included, including assessment of mtDNA copy number, steady-state levels of biogenesis-related transcription factors (e.g. mitochondria transcription factor A, mitochondrial transcription specificity factors, nuclear respiratory factors 1 and 2, and peroxisome proliferator activated receptor gamma coactivator-1-alpha), mtDNA-encoded transcripts and proteins, and rates of mitochondria translation. These techniques are described in isolation and in the context of transgenic and dietary animal models that have been used as tools to study the regulation of mitochondria biogenesis and its role in disease pathology.