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1.
Invest Ophthalmol Vis Sci ; 58(11): 4662-4669, 2017 09 01.
Article in English | MEDLINE | ID: mdl-28903152

ABSTRACT

Purpose: The CXC receptor 4 (CXCR4) is required for various physiologic and pathologic processes in the eye, including stem cell trafficking, neuronal development, immune responses, and ocular neovascularization. Here, we used the rat retina models to determine the mechanisms driving CXCR4 transcription. Methods: The expression pattern of CXCR4 and nuclear respiratory factor-1 (NRF-1) were profiled in the rat retina during the course of development. Chromatin immunoprecipitation (CHiP) assay determined the transcriptional mechanism of CXCR4 in rat retina. A rat model of oxygen-induced retinopathy (OIR) that mimics retinal ischemia-reperfusion injury was established. Under either normoxic or hypoxic conditions, CXCR4 and NRF-1 expression in rat retinas was tracked by RT-PCR and Western analysis. Immunofluorescence staining localized CXCR4 and NRF-1. Results: Both CXCR4 and NRF-1 were highly expressed in the neonatal rat retina, down-regulated in parallel, and silenced in fully developed retinas (1 month of age). ChIP assays revealed that NRF-1 was required for CXCR4 promoter activity in rat retinas. In the OIR rat model, retinal hypoxia induced up-regulation of CXCR4 and NRF-1 concurrently. Conclusions: Our findings suggest that NRF-1 regulates the expression of CXCR4 in normal retinal development and in pathologic processes of retinal hypoxia and neovascularization.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Nuclear Respiratory Factor 1/physiology , Receptors, CXCR4/genetics , Reperfusion Injury/genetics , Retina/metabolism , Retinal Neovascularization/genetics , Transcriptional Activation/physiology , Animals , Animals, Newborn , Blotting, Western , Disease Models, Animal , Down-Regulation , Fluorescent Antibody Technique, Indirect , Oxygen/toxicity , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Receptors, CXCR4/metabolism , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Retinal Neovascularization/metabolism , Retinal Neovascularization/pathology , Up-Regulation
2.
Biochem Biophys Res Commun ; 484(1): 176-183, 2017 02 26.
Article in English | MEDLINE | ID: mdl-28088524

ABSTRACT

The transcription factor Nrf1 (NFE2L1) maintains protein homeostasis (proteostasis) by regulating the gene expression of proteasome subunits in response to proteasome inhibition. The deletion of the Nrf1 gene in neural stem/progenitor cells causes severe neurodegeneration due to the accumulation of ubiquitinated proteins in Purkinje cells and motor neurons (Nrf1 NKO mice). However, the molecular mechanisms governing this neurodegenerative process remain unclear. We demonstrate herein that the loss of Nrf1 leads to the reduced gene expression of the deubiquitinating enzymes (DUBs) but not proteasome subunits in Nrf1 NKO mice between P7 and P18. First, we show that K48-linked polyubiquitinated proteins accumulate in Nrf1-deficient Purkinje cells and cerebral cortex neurons. Nevertheless, loss of Nrf1 does not alter the expression and proteolytic activity of proteasome. A significantly reduced expression of deubiquitinating enzymes was also demonstrated in Nrf1-deficient cerebellar tissue using microarray analysis. The genome database further reveals species-conserved ARE, a Nrf1 recognition element, in the regulatory region of certain DUB genes. Furthermore, we show that Nrf1 can activate Usp9x gene expression related to neurodegeneration. Altogether these findings suggest that neurodegeneration in Nrf1 NKO mice may stem from the dysfunction of the ubiquitin-mediated regulation of neuronal proteins.


Subject(s)
Cerebellum/enzymology , Deubiquitinating Enzymes/genetics , Homeostasis/physiology , Neural Stem Cells/metabolism , Nuclear Respiratory Factor 1/physiology , Animals , Cerebellum/pathology , Deubiquitinating Enzymes/metabolism , Gene Expression Regulation, Enzymologic , Mice , Mice, Knockout , Neural Stem Cells/cytology , Neural Stem Cells/enzymology , Nuclear Respiratory Factor 1/genetics
3.
Biochem J ; 473(8): 961-1000, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-27060105

ABSTRACT

The consensuscis-regulatory AP-1 (activator protein-1)-like AREs (antioxidant-response elements) and/or EpREs (electrophile-response elements) allow for differential recruitment of Nrf1 [NF-E2 (nuclear factor-erythroid 2)-related factor 1], Nrf2 and Nrf3, together with each of their heterodimeric partners (e.g. sMaf, c-Jun, JunD or c-Fos), to regulate different sets of cognate genes. Among them, NF-E2 p45 and Nrf3 are subject to tissue-specific expression in haemopoietic and placental cell lineages respectively. By contrast, Nrf1 and Nrf2 are two important transcription factors expressed ubiquitously in various vertebrate tissues and hence may elicit putative combinational or competitive functions. Nevertheless, they have de facto distinct biological activities because knockout of their genes in mice leads to distinguishable phenotypes. Of note, Nrf2 is dispensable during development and growth, albeit it is accepted as a master regulator of antioxidant, detoxification and cytoprotective genes against cellular stress. Relative to the water-soluble Nrf2, less attention has hitherto been drawn to the membrane-bound Nrf1, even though it has been shown to be indispensable for embryonic development and organ integrity. The biological discrepancy between Nrf1 and Nrf2 is determined by differences in both their primary structures and topovectorial subcellular locations, in which they are subjected to distinct post-translational processing so as to mediate differential expression of ARE-driven cytoprotective genes. In the present review, we focus on the molecular and cellular basis for Nrf1 and its isoforms, which together exert its essential functions for maintaining cellular homoeostasis, normal organ development and growth during life processes. Conversely, dysfunction of Nrf1 results in spontaneous development of non-alcoholic steatohepatitis, hepatoma, diabetes and neurodegenerative diseases in animal models.


Subject(s)
Cell Membrane/physiology , Homeostasis/physiology , Nuclear Respiratory Factor 1/physiology , Organogenesis/physiology , Animals , Humans , Signal Transduction/physiology , Transcription Factors/genetics , Transcription Factors/metabolism
4.
Mol Cell Biol ; 34(20): 3800-16, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25092871

ABSTRACT

Liver-specific Nrf1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific Nrf1 knockout mouse line using animals harboring an Nrf1(flox) allele and a rat CYP1A1-Cre transgene (Nrf1(flox/flox)::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (Nrf1(flox/flox)::CYP1A1-Cre+3MC mice) resulted in loss of hepatic Nrf1 expression. The livers of mice lacking Nrf1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the Nrf1(flox/flox)::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of Nrf1(flox/flox)::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x(c)(-), was significantly upregulated in livers of Nrf1(flox/flox)::CYP1A1-Cre+3MC mice, suggesting that Nrf1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, Nrf1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, Nrf1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, Nrf1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.


Subject(s)
Amino Acid Transport Systems, Acidic/metabolism , Fatty Acid Desaturases/genetics , Gene Expression Regulation, Enzymologic , Lipid Metabolism , Nuclear Respiratory Factor 1/physiology , Amino Acid Transport System y+/genetics , Amino Acid Transport System y+/metabolism , Amino Acid Transport Systems, Acidic/genetics , Animals , Cell Line , Cystine/metabolism , Cytochrome P-450 CYP1A1/genetics , Fatty Acid Desaturases/metabolism , Female , Glutathione/metabolism , Hepatocytes/metabolism , LDL-Receptor Related Proteins/genetics , LDL-Receptor Related Proteins/metabolism , Liver/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Binding , Rats , Receptors, Lipoprotein/genetics , Receptors, Lipoprotein/metabolism , Response Elements , Transcriptional Activation , Triglycerides/metabolism
5.
Exp Cell Res ; 322(1): 39-50, 2014 Mar 10.
Article in English | MEDLINE | ID: mdl-24462598

ABSTRACT

Recent evidences indicated Nrf2 is more potent than Nrf1 in the activation of antioxidant genes. However, the roles of Nrf proteins in the regulation of copper-responsive transcription have not been well addressed. We took the toxicogenomic approach and the present network and Gene Ontology analyses results showed that Nrf1 and Nrf2 are distinctively involved in copper-responsive transcriptional regulation in HepG2 transcriptome. Cells deficient in either Nrf1 or Nrf2 were more susceptible to copper exposure than wild type cells. Nrf1 and Nrf2 null cells were transfected with the luciferase reporters containing either ARE(s) or a combination of ARE(s) and MREs, and then treated with copper. In Nrf2-null (Nrf2(-/-)) cells, copper did not activate transcription of reporter genes, whereas Nrf1 deficiency did not affect copper-inducible activation. Ectopic expression of Nrf2 restored copper-inducible transcription in Nrf2(-/-) cells. However, the changes in the intrinsic mRNA levels of MT-1 in Nrf null cells following copper treatment showed that Nrf1 and Nrf2 equally contributed to MT-1 activation after 4h, while Nrf1involved more than Nrf2 following 24h exposure. These results suggest that while Nrf2 is crucial for MRE/ARE-mediated transcription in response to copper, Nrf1 may activate MT-1 expression by a mechanism different from that Nrf2 employs.


Subject(s)
Copper/pharmacology , Gene Expression Regulation/drug effects , NF-E2-Related Factor 2/physiology , Nuclear Respiratory Factor 1/physiology , Animals , Cells, Cultured , Gene Expression Profiling , Gene Regulatory Networks , Hep G2 Cells , Humans , Metallothionein/genetics , Metallothionein/metabolism , Mice , Mice, Knockout , NF-E2-Related Factor 2/genetics , Nuclear Respiratory Factor 1/genetics , Transcription, Genetic/drug effects
6.
Sci Rep ; 3: 2006, 2013.
Article in English | MEDLINE | ID: mdl-23774320

ABSTRACT

The integral membrane-bound Nrf1 transcription factor fulfils important functions in maintaining cellular homeostasis and organ integrity, but how it is controlled vectorially is unknown. Herein, creative use of Gal4-based reporter assays with protease protection assays (GRAPPA), and double fluorescence protease protection (dFPP), reveals that the membrane-topogenic vectorial behaviour of Nrf1 dictates its post-translational modification and transactivation activity. Nrf1 is integrated within endoplasmic reticulum (ER) membranes through its NHB1-associated TM1 in cooperation with other semihydrophobic amphipathic regions. The transactivation domains (TADs) of Nrf1, including its Asn/Ser/Thr-rich (NST) glycodomain, are transiently translocated into the ER lumen, where it is glycosylated in the presence of glucose to become a 120-kDa isoform. Thereafter, the NST-adjoining TADs are partially repartitioned out of membranes into the cyto/nucleoplasmic side, where Nrf1 is subject to deglycosylation and/or proteolysis to generate 95-kDa and 85-kDa isoforms. Therefore, the vectorial process of Nrf1 controls its target gene expression.


Subject(s)
Nuclear Respiratory Factor 1/physiology , Protein Biosynthesis , Transcriptional Activation , Animals , Mice , Nuclear Respiratory Factor 1/genetics
7.
Biochim Biophys Acta ; 1833(1): 48-58, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23085505

ABSTRACT

Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level.


Subject(s)
Energy Metabolism/genetics , GA-Binding Protein Transcription Factor/physiology , Nuclear Respiratory Factor 1/physiology , Receptors, N-Methyl-D-Aspartate/genetics , Synaptic Transmission/genetics , Animals , Cells, Cultured , Energy Metabolism/physiology , GA-Binding Protein Transcription Factor/genetics , GA-Binding Protein Transcription Factor/metabolism , Gene Expression Regulation , Humans , Mice , Models, Biological , Neurons/metabolism , Neurons/physiology , Nuclear Respiratory Factor 1/genetics , Nuclear Respiratory Factor 1/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction/genetics , Synaptic Transmission/physiology
8.
Adv Exp Med Biol ; 748: 283-304, 2012.
Article in English | MEDLINE | ID: mdl-22729863

ABSTRACT

Cytochrome c oxidase is the terminal enzyme of the mitochondrial electron transport chain, without which oxidative metabolism cannot be carried to completion. It is one of only four unique, bigenomic proteins in mammalian cells. The holoenzyme is made up of three mitochondrial-encoded and ten nuclear-encoded subunits in a 1:1 stoichiometry. The ten nuclear subunit genes are located in nine different chromosomes. The coordinated regulation of such a multisubunit, multichromosomal, bigenomic enzyme poses a challenge. It is especially so for neurons, whose mitochondria are widely distributed in extensive dendritic and axonal processes, resulting in the separation of the mitochondrial from the nuclear genome by great distances. Neuronal activity dictates COX activity that reflects protein amount, which, in turn, is regulated at the transcriptional level. All 13 COX transcripts are up- and downregulated by neuronal activity. The ten nuclear COX transcripts and those for Tfam and Tfbms important for mitochondrial COX transcripts are transcribed in the same transcription factory. Bigenomic regulation of all 13 transcripts is mediated by nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2). NRF-1, in addition, also regulates critical neurochemicals of glutamatergic synaptic transmission, thereby ensuring the tight coupling of energy metabolism and neuronal activity at the molecular level in neurons.


Subject(s)
Electron Transport Complex IV/physiology , Energy Metabolism , Neurons/physiology , Animals , GA-Binding Protein Transcription Factor/physiology , Glutamic Acid/physiology , Humans , Neurons/enzymology , Nuclear Respiratory Factor 1/physiology , Transcription, Genetic
9.
J Biol Chem ; 287(22): 18788-95, 2012 May 25.
Article in English | MEDLINE | ID: mdl-22500024

ABSTRACT

Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.


Subject(s)
Cell Survival , DNA Repair , Glutathione/metabolism , Homeostasis , Nuclear Proteins/physiology , Nuclear Respiratory Factor 1/physiology , RNA-Binding Proteins/physiology , Transcription Factors/physiology , Humans
10.
J Am Heart Assoc ; 1(6): e003855, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23316324

ABSTRACT

BACKGROUND: Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)-regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. METHODS AND RESULTS: We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi-infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)-regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. CONCLUSIONS: The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi-infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for replication and gene expression in Chagas disease.


Subject(s)
Chagas Disease/physiopathology , DNA Replication/physiology , DNA, Mitochondrial/physiology , Mitochondrial Turnover/physiology , Trypanosoma cruzi , Blotting, Western , Cells, Cultured , Chagas Disease/genetics , Chagas Disease/metabolism , DNA, Mitochondrial/metabolism , Gene Expression Regulation , Humans , Immunohistochemistry , Microscopy, Fluorescence , Mitochondrial Diseases/genetics , Mitochondrial Diseases/physiopathology , Myocytes, Cardiac/physiology , Myocytes, Cardiac/ultrastructure , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/physiology , Nuclear Respiratory Factor 1/genetics , Nuclear Respiratory Factor 1/metabolism , Nuclear Respiratory Factor 1/physiology , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors/physiology
11.
Basic Clin Pharmacol Toxicol ; 109(2): 115-22, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21401886

ABSTRACT

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.


Subject(s)
Benzo(a)pyrene/toxicity , Bronchi/drug effects , Mitochondria/drug effects , Nuclear Respiratory Factor 1/physiology , Adenosine Triphosphate/analysis , Apoptosis/drug effects , Bronchi/cytology , Cell Proliferation/drug effects , Cells, Cultured , DNA-Binding Proteins/analysis , Epithelial Cells/drug effects , Humans , Membrane Potential, Mitochondrial/drug effects , Mitochondria/physiology , Mitochondrial Proteins/analysis , Nuclear Respiratory Factor 1/analysis , Nuclear Respiratory Factor 1/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Transcription Factors/analysis
13.
J Neurochem ; 115(3): 676-83, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21064266

ABSTRACT

Neuronal activity and energy metabolism are tightly coupled processes. Recently, we found that nuclear respiratory factor 1 co-regulates all subunits of cytochrome c oxidase (COX, representing oxidative energy metabolism) and glutamatergic neurochemicals, including NR1 (Grin1) and NR2B (Grin2b) of NMDA receptors, GluR2 (Gria2) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and neuronal nitric oxide synthase (Nos1). Moreover, all 10 nuclear-encoded COX subunit genes and three transcription factor genes for the three mitochondrial-encoded COX subunits are transcribed in the same transcription factory. The goal of the present study was to test our hypothesis that genomic loci for Grin1, Grin2b, Gria2, and Nos1 interact with those for COX at the transcriptional level. By means of chromosome conformation capture, interactions were found among all of these genes in neurons, but not in C2C12 muscle cells. COX subunit genes also did not interact with neurochemical genes not regulated by nuclear respiratory factor 1, nor with genes for calreticulin, a non-mitochondrial protein. Depolarizing stimulation up-regulated interaction frequencies between COX and neurochemical genes, whereas impulse blockade with tetrodotoxin or inhibition of COX with KCN down-regulated them in neurons. Thus, an efficient mechanism is in place for coordinating the transcriptional coupling of energy metabolism and glutamatergic neurotransmission at the molecular level in neurons.


Subject(s)
Chromosomes/genetics , Electron Transport Complex IV/genetics , Gene Expression Regulation, Enzymologic/physiology , Glutamates/physiology , Neurons/physiology , Synaptic Transmission/physiology , Animals , Cell Line, Tumor , Cells, Cultured , Computer Simulation , Electron Transport Complex IV/metabolism , Energy Metabolism/physiology , Gene Expression Regulation, Enzymologic/drug effects , Mice , Neurons/drug effects , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type I , Nuclear Respiratory Factor 1/genetics , Nuclear Respiratory Factor 1/physiology , Potassium Chloride/pharmacology , Potassium Cyanide/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/genetics , Reverse Transcriptase Polymerase Chain Reaction , Synaptic Transmission/drug effects , Tetrodotoxin/pharmacology
14.
Arch Biochem Biophys ; 492(1-2): 74-81, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19772854

ABSTRACT

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.


Subject(s)
Foam Cells/pathology , Macrophages, Peritoneal/pathology , Mitochondria/pathology , Triglycerides/toxicity , Animals , Cell Line , Cells, Cultured , DNA, Mitochondrial/antagonists & inhibitors , DNA, Mitochondrial/biosynthesis , Down-Regulation/physiology , Foam Cells/metabolism , Lipid Metabolism/physiology , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Nuclear Respiratory Factor 1/antagonists & inhibitors , Nuclear Respiratory Factor 1/physiology , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/toxicity , Glycine max , Transcription Factors/antagonists & inhibitors , Transcription Factors/biosynthesis
15.
Endocrinology ; 150(7): 3197-206, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19282378

ABSTRACT

Human skin is constantly exposed to UV light, the most ubiquitous environmental stressor. Here, we investigated the expression and regulation of Nrf1-3, transcription factors crucially involved in protection against oxidative stress in human skin cells in vitro, ex vivo, and in situ. In particular, we examined whether alpha-MSH, a UV-induced peptide, is capable of modulating Nrf2 and Nrf-dependent gene expression. Nrf1, -2, and -3 were found to be expressed in various cutaneous cell types in vitro. Surprisingly, UVB irradiation at physiological doses (10 mJ/cm(2)) reduced Nrf2 and Nrf-dependent gene expression in normal keratinocytes and melanocytes in vitro as well as ex vivo in skin organ cultures. alpha-MSH alone significantly increased Nrf2 as well as Nrf-dependent heme oxygenase-1, gamma-glutamylcysteine-synthetase, and glutathione-S-transferase Pi gene expression in both keratinocytes and melanocytes. This effect of alpha-MSH occurred at physiological doses and was due to transcriptional induction, mimicked by the artificial cAMP inducer forskolin, and blocked by protein kinase A pathway inhibition. In silico promoter analysis of Nrf2 further identified several putative binding sites for activator protein 1 and cAMP response element-binding protein, transcription factors typically activated by alpha-MSH. Importantly, alpha-MSH prevented or even overcompensated the UVB-induced suppression of Nrf2 and Nrf-dependent genes not only in normal keratinocytes and melanocytes in vitro but also in skin organ cultures. These findings, for the first time, show regulation of Nrf2 and Nrf-dependent genes by alpha-MSH. Our data also highlight a novel facet in the cytoprotective and antioxidative effector mechanisms of alpha-MSH and perhaps of related melanocortin peptides.


Subject(s)
NF-E2-Related Factor 2/physiology , Nuclear Respiratory Factor 1/physiology , Skin/radiation effects , Ultraviolet Rays , alpha-MSH/pharmacology , Basic-Leucine Zipper Transcription Factors/biosynthesis , Humans , Immunohistochemistry , Keratinocytes/drug effects , Keratinocytes/metabolism , Melanocytes/drug effects , Melanocytes/metabolism , Organ Culture Techniques , Skin/metabolism , Up-Regulation
16.
Gene ; 410(1): 197-206, 2008 Feb 29.
Article in English | MEDLINE | ID: mdl-18234454

ABSTRACT

Ubiquitously expressed micro- and m-calpain are cysteine proteases with broad functions in cell spreading, migration, proliferation, apoptosis, and in tumor invasion. They are heterodimers, with a distinct large 80-kDa catalytic, and a common small 28-kDa regulatory subunit (Capn4/CAPNS1). CAPNS1 is required to maintain stability and activity of both calpains. Despite its biological importance, the transcriptional regulation of this gene has not been studied, and the CAPNS1 promoter has not yet been characterized. In this study, we identified the main transcriptional start site, and cloned and characterized the ~2.0 kb upstream region of the CAPNS1 gene. Deletion analysis identified the core promoter located within region -187/+174. Site-directed mutagenesis, EMSA- and supershift analysis identified Sp1-, NRF-1-, and AP-1-binding elements within the CAPNS1 core promoter. Binding of NRF-1, Sp1 and AP-1 to the natural core promoter was confirmed by chromatin immunoprecipitation (ChIP). Site-directed mutagenesis at the NRF-1 site in HeLa and MCF7 cells substantially reduced core promoter activity by 70%, whereas mutation of the AP-1-binding and Sp1-binding site reduced promoter activity by 50% and 30%, respectively. Double mutation of the NRF-1 and the AP-1 site reduced promoter activity by 90%. In Drosophila SL2 cells, ectopic expression of NRF-1 led to a significant induction of CAPNS1 promoter activity. Furthermore, an siRNA against NRF-1 substantially reduced promoter activity in HeLa cells, which was paralleled by a significant downregulation of CAPNS1 mRNA. These results reveal that especially NRF-1, along with AP-1 and, to a minor extent, an Sp1 site, is essential for human CAPNS1 promoter activity and gene expression.


Subject(s)
Calpain/genetics , Nuclear Respiratory Factor 1/physiology , Promoter Regions, Genetic , Transcription Factor AP-1/physiology , Base Sequence , DNA , DNA Primers , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA Interference
17.
Br J Sports Med ; 42(5): 361-6, 2008 May.
Article in English | MEDLINE | ID: mdl-18184751

ABSTRACT

BACKGROUND: Nuclear respiratory factor 1 (NRF-1) is a critical component of the energy-sensing mechanism in mammalian cells, and translates physiological signals (particularly those induced by exercise) into increased capacity for mitochondrial biogenesis and oxidative phosphorylation. OBJECTIVE: To study the possible association between rs2402970, rs6949152 and rs10500120 NRF-1 genotypes and several phenotypes indicative of maximum (VO(2max)) and submaximum aerobic capacity (ventilatory threshold (VT) and metabolic cost of submaximum running at 12 km/hour (running economy; RE)) both at baseline and in response to a 18-week endurance training programme in young Chinese men of Han origin (n = 102; 19 (SD 1) years). RESULTS: For rs2402970, a significant genotype effect was seen for VT (p = 0.004) and RE (p = 0.027). For rs6949152, a significant interaction (genotypextraining) effect (p = 0.047) was found for VT. CONCLUSIONS: There is an association between NRF-1 genotypes (rs2402970 and rs6949152 polymorphisms) and the baseline and/or training response of human aerobic capacity. More research is needed to corroborate our data in other ethnic groups with lower fitness levels at the pre-training state (particularly Caucasians) and to identify the molecular mechanisms involved in the genotype-phenotype associations we found.


Subject(s)
Nuclear Respiratory Factor 1/genetics , Physical Endurance/physiology , Physical Fitness/physiology , Running/physiology , Adolescent , Adult , China , Genotype , Humans , Male , Nuclear Respiratory Factor 1/physiology , Oxygen Consumption , Phenotype
18.
Biochem J ; 408(2): 161-72, 2007 Dec 01.
Article in English | MEDLINE | ID: mdl-17705787

ABSTRACT

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is negatively controlled by its NTD (N-terminal domain) that lies between amino acids 1 and 124. This domain contains a leucine-rich sequence, called NHB1 (N-terminal homology box 1; residues 11-30), which tethers Nrf1 to the ER (endoplasmic reticulum). Electrophoresis resolved Nrf1 into two major bands of approx. 95 and 120 kDa. The 120-kDa Nrf1 form represents a glycosylated protein that was present exclusively in the ER and was converted into a substantially smaller polypeptide upon digestion with either peptide:N-glycosidase F or endoglycosidase H. By contrast, the 95-kDa Nrf1 form did not appear to be glycosylated and was present primarily in the nucleus. NHB1 and its adjacent residues conform to the classic tripartite signal peptide sequence, comprising n-, h- and c-regions. The h-region (residues 11-22), but neither the n-region (residues 1-10) nor the c-region (residues 23-30), is required to direct Nrf1 to the ER. Targeting Nrf1 to the ER is necessary to generate the 120-kDa glycosylated protein. The n-region and c-region are required for correct membrane orientation of Nrf1, as deletion of residues 2-10 or 23-30 greatly increased its association with the ER and the extent to which it was glycosylated. The NHB1 does not contain a signal peptidase cleavage site, indicating that it serves as an ER anchor sequence. Wild-type Nrf1 is glycosylated through its Asn/Ser/Thr-rich domain, between amino acids 296 and 403, and this modification was not observed in an Nrf1(Delta299-400) mutant. Glycosylation of Nrf1 was not necessary to retain it in the ER.


Subject(s)
Asparagine/metabolism , Endoplasmic Reticulum/metabolism , Nuclear Respiratory Factor 1/physiology , Sequence Homology, Amino Acid , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Endoplasmic Reticulum/chemistry , Glycosylation , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/physiology , Mice , Molecular Sequence Data , Nuclear Respiratory Factor 1/chemistry , Peptide Fragments/chemistry , Peptide Fragments/physiology , Protein Binding/physiology
19.
J Biol Chem ; 282(34): 24670-8, 2007 Aug 24.
Article in English | MEDLINE | ID: mdl-17609210

ABSTRACT

Oxidative stress-responsive transcription is regulated in part through cis-active sequences known as antioxidant response elements (ARE). Activation through the ARE involves members of the CNC-subfamily of basic leucine zipper proteins including Nrf1 and Nrf2. In particular, Nrf2 has been shown to coordinate induction of genes encoding antioxidant and phase 2 metabolizing enzymes in response to stimulation with electrophilic compounds and exposure to xenobiotics. Here we show that the 65-kDa isoform of the Nrf1 gene functions as a repressor of Nrf2. Transient expression of p65Nrf1 suppressed Nrf2-mediated activation of ARE-dependent reporter genes in cells. Induction of endogenous ARE-genes is blocked in Hepa1c1c7 cells stably expressing p65Nrf1 leading to increased cell death. Consistent with these findings, electrophilic activation of ARE-gene expression is augmented by loss of p65Nrf1 function in Nrf1(-/-) fibroblasts, and the protective effects of oxidative preconditioning and ARE-gene expression are blocked in Nrf1(-/-) cells stably expressing p65Nrf1. Gel shift experiments demonstrated that p65Nrf1 binds the antioxidant response element as a heterodimer with small-Maf protein. Immunoprecipitation studies demonstrated that p65Nrf1 competes with Nrf2 for interaction with small-Maf protein and binding to the antioxidant response element in vivo. Together, these results demonstrate that p65Nrf1 has the potential to play an important role in modulating the response to oxidative stress by functioning as a transdominant repressor of Nrf2-mediated activation of ARE-dependent gene transcription.


Subject(s)
Antioxidants/metabolism , Gene Expression Regulation , NF-E2-Related Factor 2/metabolism , Nuclear Respiratory Factor 1/physiology , Animals , COS Cells , Cell Line , Cell Line, Tumor , Cell Survival , Chlorocebus aethiops , Genes, Dominant , HeLa Cells , Humans , Nuclear Respiratory Factor 1/chemistry , Oxidative Stress , Protein Isoforms , Retroviridae , Transcription, Genetic
20.
J Biol Chem ; 282(30): 22052-61, 2007 Jul 27.
Article in English | MEDLINE | ID: mdl-17510056

ABSTRACT

We recently found that deletion of the gulonolactone oxidase gene, which is involved in the synthesis of ascorbic acid (AA), was responsible for the fracture phenotype in spontaneous fracture mice. To explore the molecular mechanisms by which AA regulates osteoblast differentiation, we examined the effect of AA on osterix expression via Nrf1 (NF-E2-related factor-1) binding to antioxidant-responsive element (ARE) in bone marrow stromal (BMS) cells. AA treatment caused a 6-fold increase in osterix expression in mutant BMS cells at 24 h, which was unaffected by pretreatment with protein synthesis inhibitor. Sequence analyses of mouse osterix promoter revealed a putative ARE located at -1762 to -1733 upstream of the transcription start site to which Nrf potentially binds. A gel mobility shift assay revealed that nuclear proteins from AA-treated BMS cells bound to radiolabeled ARE much more strongly than nuclear extracts from AA-untreated cells. A chromatin immunoprecipitation assay with Nrf1 antibody confirmed the interaction of Nrf1 with the mouse osterix promoter. A reporter assay demonstrated that the promoter activity of mouse osterix containing an ARE was stimulated 4-fold by a 48-h treatment with AA in spontaneous fracture BMS cells. Treatment of mutant BMS cells with AA resulted in a 3.9-fold increase in the nuclear accumulation of Nrf1. Transfection of mutant BMS cells with Nrf1 small interfering RNA decreased Nrf1 protein by 4.5-fold, blocked AA induction of osterix expression, and impaired BMS cell differentiation. Our data provided the first experimental evidence that AA modulated osterix expression via a novel mechanism involving Nrf1 nuclear translocation and Nrf1 binding to ARE to activate genes critical for cell differentiation.


Subject(s)
Ascorbic Acid/pharmacology , Bone and Bones/physiology , Nuclear Respiratory Factor 1/physiology , Stromal Cells/physiology , Transcription Factors/genetics , Animals , Antioxidants/pharmacology , Bone and Bones/drug effects , Gene Expression Regulation , Mice , Mice, Inbred BALB C , Nuclear Respiratory Factor 1/genetics , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sp7 Transcription Factor , Stromal Cells/drug effects
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