nrf2 mitochondria

1: Arch Biochem Biophys. 2006 Oct 1;454(1):7-15. Epub 2006 Aug 23. Related Articles, Links
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Nrf2 regulates an adaptive response protecting against oxidative damage following diquat-mediated formation of superoxide anion.

Osburn WO, Wakabayashi N, Misra V, Nilles T, Biswal S, Trush MA, Kensler TW.

Johns Hopkins University Bloomberg School of Public Health, Department of Environmental Health Sciences, Baltimore, MD, USA.

Mouse embryonic fibroblasts derived from Nrf2-/- mice (N0) and Nrf2+/+ mice (WT) have been used to characterize both basal and diquat (DQ)-induced oxidative stress levels and to examine Nrf2 activation during exposure to DQ-generated superoxide anion. Microarray analysis revealed that N0 cells have similar constitutive mRNA expression of genes responsible for the direct metabolism of reactive oxygen species but decreased expression of genes responsible for the production of reducing equivalents, repair of oxidized proteins and defense against lipid peroxidation, compared to WT cells. Nonetheless, the basal levels of ROS flux and oxidative damage biomarkers in WT and N0 cells were not different. Diquat dibromide (DQ), a non-electrophilic redox cycling bipyridylium herbicide, was used to generate intracellular superoxide anion. Isolated mitochondria from both cell lines exposed to DQ produced equivalent amounts of ROS, indicating a similar cellular capacity to generate ROS. However, N0 cells exposed to DQ for 24-h exhibited markedly decreased cell viability and aconitase activity as well as increased lipid peroxidation and glutathione oxidation, relative to WT cells. 2′,7′-Dichlorofluorescein fluorescence was not increased in WT and N0 cells after 30-min of DQ exposure. However, increased levels of ROS were detected in N0 cells but not WT cells after 13-h of DQ treatment. Additionally, total glutathione concentrations increased in WT, but not N0 cells following a 24-h exposure to DQ. DQ exposure resulted in activation of an antioxidant response element-luciferase reporter gene, as well as induction of Nrf2-regulated genes in WT, but not N0 cells. Thus the enhanced sensitivity of N0 cells does not reflect basal differences in antioxidative capacity, but rather an impaired ability to mount an adaptive response to sustained oxidative stress.

PMID: 16962985 [PubMed – in process]

2: Arterioscler Thromb Vasc Biol. 2006 Sep;26(9):2027-34. Epub 2006 Jul 13. Related Articles, Links
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Protection of human vascular smooth muscle cells from H2O2-induced apoptosis through functional codependence between HO-1 and AKT.

Brunt KR, Fenrich KK, Kiani G, Tse MY, Pang SC, Ward CA, Melo LG.

Department of Physiology, Queen’s University, 18 Stuart St, Kingston, Ontario, K7L 4S7. Canada.

OBJECTIVE: Oxidative stress (OS) induces smooth muscle cell apoptosis in the atherosclerotic plaque, leading to plaque instability and rupture. Heme oxygenase-1 (HO-1) exerts cytoprotective effects in the vessel wall. Recent evidence suggests that PKB/Akt may modulate HO-1 activity. This study examined the role of Akt in mediating the cytoprotective effects of HO-1 in OS-induced apoptosis of human aortic smooth muscle cells (HASMCs). METHODS AND RESULTS: HASMCs were transduced with retroviral vectors expressing HO-1, Akt, or GFP and exposed to H2O2. Cell viability was assessed by MTT assay. OS was determined by CM-H2DCFDA fluorescence, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), caspase-3 activity, and Bcl-2/Bad levels. Mitochondrial membrane potential (delta psi(m)) was assessed by fluorescence-activated cell sorter (FACS) using JC-1. HO-1 reduced H2O2-induced OS and apoptosis. Akt knockdown removed the protective effect of HO-1 on delta psi(m) during exposure to H2O2. Conversely, HO-1 knockdown removed the protective effect of Akt on delta psi(m). Inhibition of PI3K-Akt reduced induction of HO-1 protein expression by H2O2 and blocked its anti-apoptotic effects. The Akt-mediated upregulation of HO-1 was dependent on activation of HO-1 promoter by Nrf2. CONCLUSIONS: HO-1 and Akt exert codependent cytoprotective effects against OS-induced apoptosis in HASMCs. These findings may have implications for the design of novel therapeutic strategies for plaque stabilization.

PMID: 16840713 [PubMed – indexed for MEDLINE]

3: Toxicol Sci. 2006 Mar;90(1):120-32. Epub 2005 Dec 1. Related Articles, Links
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Resveratrol protects against 4-hydroxynonenal-induced apoptosis by blocking JNK and c-JUN/AP-1 signaling.

Kutuk O, Poli G, Basaga H.

Biological Sciences and Bioengineering Program, Sabanci University, 34956 Orhanli, Tuzla Istanbul, Turkey.

In the present study we have studied the effect of resveratrol in signal transduction mechanisms leading to apoptosis in 3T3 fibroblasts when exposed to 4-hydroxynonenal (HNE). In order to gain insight into the mechanisms of apoptotic response by HNE, we followed MAP kinase and caspase activation pathways; HNE induced early activation of JNK and p38 proteins but downregulated the basal activity of ERK (1/2). We were also able to demonstrate HNE-induced release of cytochrome c from mitochondria, caspase-9, and caspase-3 activation. Resveratrol effectively prevented HNE-induced JNK and caspase activation, and hence apoptosis. Activation of AP-1 along with increased c-Jun and phospho-c-Jun levels could be inhibited by pretreatment of cells with resveratrol. Moreover, Nrf2 downregulation by HNE could also be blocked by resveratrol. Overexpression of dominant negative c-Jun and JNK1 in 3T3 fibroblasts prevented HNE-induced apoptosis, which indicates a role for JNK-c-Jun/AP-1 pathway. In light of the JNK-dependent induction of c-Jun/AP-1 activation and the protective role of resveratrol, these data may show a critical potential role for JNK in the cellular response against toxic products of lipid peroxidation. In this respect, resveratrol acting through MAP kinase pathways and specifically on JNK could have a role other than acting as an antioxidant-quenching reactive oxygen intermediate.

PMID: 16322078 [PubMed – indexed for MEDLINE]

4: Invest Ophthalmol Vis Sci. 2005 Nov;46(11):4311-9. Related Articles, Links
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Hepatocyte growth factor protects RPE cells from apoptosis induced by glutathione depletion.

Jin M, Yaung J, Kannan R, He S, Ryan SJ, Hinton DR.

Arnold and Mabel Beckman Macular Research Center, Los Angeles, California, USA.

PURPOSE: To study the mechanism of the protective effect of hepatocyte growth factor (HGF) in oxidative injury to RPE cells induced by glutathione (GSH) depletion. METHODS: RPE cells were treated with HGF for 24 hours (20 ng/mL) and then were treated with DL-buthionine-(S,R)-sulfoximine (BSO) for an additional 24 hours. Cell death, apoptosis, and GSH levels were measured. Levels of intracellular reactive oxygen species (ROS) and their cellular localization were assessed by confocal microscopy. Expression of Bcl-2 and release of cytochrome c from mitochondria were quantified. The effect of BSO on caspase-3 activation and expression was determined. Gene expression of key enzymes of GSH metabolism by real-time PCR and regulation and translocation of the transcription factor NF-E2-related factor (Nrf2) by BSO were examined. RESULTS: Treatment with BSO-induced apoptosis in RPE caused a significant decrease in intracellular GSH and in GSH/GSSG ratios. Marked increases in lipid peroxidase (LPO), ROS, and mitochondrial cytochrome c release and a decrease in Bcl-2 expression were observed. Elevated GSH/GSSG ratio (especially in mitochondria), decreased LPO and ROS, attenuation of apoptosis, and partial restoration of Bcl-2 expression were found in the HGF-pretreated cells. BSO activated caspase-3, and this effect was significantly blocked by HGF. Both HGF and BSO induced anti-oxidant gene expression. Nrf2 translocated to the nuclear region after treatment with BSO, whereas HGF did not induce such translocation. CONCLUSIONS: The protective effect of HGF may be attributed in part to the elevation of mitochondrial GSH. BSO and HGF act in concert to enhance GSH-related gene expression in stressed RPE cells.

PMID: 16249513 [PubMed – indexed for MEDLINE]

5: Free Radic Biol Med. 2005 Jul 15;39(2):227-36. Epub 2005 Apr 7. Related Articles, Links
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Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2.

Anwar AA, Li FY, Leake DS, Ishii T, Mann GE, Siow RC.

Cardiovascular Division, GKT Schools of Biomedical Sciences and Medicine, King’s College, University of London, Guy’s Hospital Campus, London SE1 1UL, UK.

Oxidized low-density lipoproteins (LDL) play a central role in atherogenesis and induce expression of the antioxidant stress protein heme oxygenase 1 (HO-1). In the present study we investigated induction of HO-1 and adaptive increases in reduced glutathione (GSH) in human aortic smooth muscle cells (SMC) in response to moderately oxidized LDL (moxLDL, 100 microg protein/ml, 24 h), a species containing high levels of lipid hydroperoxides. Expression and activity of HO-1 and GSH levels were elevated to a greater extent by moxLDL than highly oxidized LDL but unaffected by native or acetylated LDL. Inhibitors of protein kinase C (PKC) or mitogen-activated protein kinases (MAPK) p38(MAPK) and MEK or c-jun-NH2-terminal kinase (JNK) significantly attenuated induction of HO-1. Phosphorylation of p38(MAPK), extracellular signal-regulated kinase (ERK1/2), or JNK and nuclear translocation of the transcription factor Nrf2 were enhanced following acute exposure of SMC to moxLDL (100 microg protein/ml, 1-2 h). Pretreatment of SMC with the antioxidant vitamin C (100 microM, 24 h) attenuated the induction of HO-1 by moxLDL. Native and oxidized LDL did not alter basal levels of intracellular ATP, mitochondrial dehydrogenase activity, or expression of the lectin-like oxidized LDL receptor (LOX-1) in SMC. These findings demonstrate for the first time that activation of PKC, p38(MAPK), JNK, ERK1/2, and Nrf2 by oxidized LDL in human SMC leads to HO-1 induction, constituting an adaptive response against oxidative injury that can be ameliorated by vitamin C.

PMID: 15964514 [PubMed – indexed for MEDLINE]

6: J Biol Chem. 2005 Jun 17;280(24):22925-36. Epub 2005 Apr 19. Related Articles, Links
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Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo.

Shih AY, Imbeault S, Barakauskas V, Erb H, Jiang L, Li P, Murphy TH.

Kinsmen Laboratory, Department of Psychiatry, Brain Research Center, Vancouver, Canada.

NF-E2 related factor (Nrf2) controls a pleiotropic cellular defense, where multiple antioxidant/detoxification pathways are up-regulated in unison. Although small molecule inducers of Nrf2 activity have been reported to protect neurons in vitro, whether similar pathways can be accessed in vivo is not known. We have investigated whether in vivo toxicity of the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP) can be attenuated by constitutive and inducible Nrf2 activity. The absence of Nrf2 function in Nrf2(-/-) mice resulted in 3-NP hypersensitivity that became apparent with time and increasing dose, causing motor deficits and striatal lesions on a more rapid time scale than identically treated Nrf2(+/+) and Nrf2(+/-) controls. Striatal succinate dehydrogenase activity, the target of 3-NP, was inhibited to the same extent in all genotypes by a single acute dose of 3-NP, suggesting that brain concentrations of 3-NP were similar. Dietary supplementation with the Nrf2 inducer tert-butylhydroquinone attenuated 3-NP toxicity in Nrf2(+/-) mice, but not Nrf2(-/-), confirming the Nrf2-specific action of the inducer in vivo. Increased Nrf2 activity alone was sufficient to protect animals from 3-NP toxicity because intrastriatal adenovirus-mediated Nrf2 overexpression significantly reduced lesion size compared with green fluorescent protein overexpressing controls. In cultured astrocytes, 3-NP was found to increase Nrf2 activity leading to antioxidant response element-dependent gene expression providing a potential mechanism for the increased sensitivity of Nrf2(-/-) animals to 3-NP toxicity in vivo. We conclude that Nrf2 may underlie a feedback system limiting oxidative load during chronic metabolic stress.

PMID: 15840590 [PubMed – indexed for MEDLINE]

7: Mamm Genome. 2003 Jan;14(1):1-6. Related Articles, Links
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Characterization of the mouse genes for mitochondrial transcription factors B1 and B2.

Rantanen A, Gaspari M, Falkenberg M, Gustafsson CM, Larsson NG.

Department of Medical Nutrition, Karolinska Institutet, NOVUM, Huddinge Hospital, S-14186 Huddinge, Sweden.

We have recently fully reconstituted the basal human mitochondrial transcription machinery in a pure in vitro system. Surprisingly, we found two different transcription factors (TFB1M and TFB2M) that each interact with mitochondrial RNA polymerase in human mitochondria, whereas there is only one such factor in budding yeast mitochondria. This unexpected finding raised important questions concerning the regulation of mitochondrial transcription in mammals in general and in other metazoans. We have now further analyzed putative homologs to TFB1M and TFB2M in different species. We mapped the mouse homologs, Tfb1m and Tfb2m, by linkage analysis to mouse Chr 17 and Chr 1, respectively. These regions display conserved linkage synteny with human Chr 6 and Chr 1, where TFB1M and TFB2M map. The intron-exon arrangements of Tfb1m and TFB1M and of Tfb2m and TFB2M were identical, and the promoter regions had similar predicted recognition elements for transcriptional factors NRF2 and Sp1. Northern blot analyses showed that Tfb1m and Tfb2m were ubiquitously expressed and had expression patterns that were very similar to the previously reported expression patterns for TFB1M and TFB2M. These findings show that Tfb1m and Tfb2m indeed are orthologs to TFB1M and TFB2M. Bioinformatic analyses indicated that most metazoans have two TFBM genes, since putative homologs to both TFB1M and TFB2M were found in D. melanogaster. Our data thus suggest that a duplication event of the TFBM gene in early metazoan evolution has permitted a more flexible regulation of mtDNA transcription, possibly in response to the complex physiological demands of multicellular organisms.

PMID: 12532263 [PubMed – indexed for MEDLINE]

8: Biochem Pharmacol. 2002 Sep;64(5-6):765-70. Related Articles, Links
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Erratum in:

  • Biochem Pharmacol 2002 Nov 15;64(10):1547.

Antioxidants and oxidants regulated signal transduction pathways.

Owuor ED, Kong AN.

Department of Pharmaceutics, Ernest Mario School of Pharmacy, and the Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.

Many drugs and xenobiotics induce signal transduction events leading to gene expression of either pharmacologically beneficial effects, or unwanted side effects such as cytotoxicity which can compromise drug therapy. Using dietary chemopreventive compounds (isothiocyanates and green tea polyphenols), which are effective against various chemically-induced carcinogenesis models in animals studies, we studied the signal transduction events and gene expression profiles. These compounds have typically generated cellular “oxidative stress” and modulated gene expression including phase II detoxifying enzymes GST and QR as well as cellular defensive enzymes, heme oxygenase 1 (HO-1) and GST via the antioxidant/electrophile response element (ARE/EpRE). Members of the bZIP transcription factor, Nrf2 which heterodimerizes with Maf G/K, were found to bind to ARE, and transcriptionally activate ARE. Additionally the mitogen-activated protein kinases (MAPK; ERK, JNK and p38) were differentially activated by these compounds, and involved in the transcriptional activation of ARE-mediated reporter gene. Transfection studies with various cDNA encoding for wild-type of MAPK and Nrf2 showed synergistic response during co-transfection and to these agents. However, by increasing the concentrations of these xenobiotics, caspase activities and apoptosis were observed which were preceded by mitochondria damage and cytochrome c mitochondria release. Further, increased concentrations led to rapid cell necrosis. [corrected] Thus, we have proposed a model, that at low concentrations, these compounds activate MAPK pathway leading to activation of Nrf2 and ARE with subsequent induction of phase II and other defensive genes which protect cells against toxic insults thereby enhancing cell survival, a beneficial homeostatic response. At higher concentrations, these agents activate the caspase pathways, leading to apoptosis, a potential cytotoxic effect if it occurred in normal cells. The studies of these signaling pathways may yield important insights into the pharmacodynamic and toxicodynamic effects of drugs and xenobiotics during pharmaceutical drug discovery and development.

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PMID: 12213568 [PubMed – indexed for MEDLINE]

9: Prog Nucleic Acid Res Mol Biol. 1998;61:309-44. Related Articles, Links
Structural organization and transcription regulation of nuclear genes encoding the mammalian cytochrome c oxidase complex.

Lenka N, Vijayasarathy C, Mullick J, Avadhani NG.

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.

Cytochrome c Oxidase (COX) is the terminal component of the bacterial as well as the mitochondrial respiratory chain complex that catalyzes the conversion of redox energy to ATP. In eukaryotes, the oligomeric enzyme is bound to mitochondrial innermembrane with subunits ranging from 7 to 13. Thus, its biosynthesis involves a coordinate interplay between nuclear and mitochondrial genomes. The largest subunits, I, II, and III, which represent the catalytic core of the enzyme, are encoded by the mitochondrial DNA and are synthesized within the mitochondria. The rest of the smaller subunits implicated in the regulatory function are encoded on the nuclear DNA and imported into mitochondria following their synthesis in the cytosol. Some of the nuclear coded subunits are expressed in tissue and developmental specific isologs. The ubiquitous subunits IV, Va, Vb, VIb, VIc, VIIb, VIIc, and VIII (L) are detected in all the tissues, although the mRNA levels for the individual subunits vary in different tissues. The tissue specific isologs VIa (H), VIIa (H), and VIII (H) are exclusive to heart and skeletal muscle. cDNA sequence analysis of nuclear coded subunits reveals 60 to 90% conservation among species both at the amino acid and nucleotide level, with the exception of subunit VIII, which exhibits 40 to 80% interspecies homology. Functional genes for COX subunits IV, Vb, VIa ‘L’ & ‘H’, VIIa ‘L’ & ‘H’, VIIc and VIII (H) from different mammalian species and their 5′ flanking putative promoter regions have been sequenced and extensively characterized. The size of the genes range from 2 to 10 kb in length. Although the number of introns and exons are identical between different species for a given gene, the size varies across the species. A majority of COX genes investigated, with the exception of muscle-specific COXVIII(H) gene, lack the canonical ‘TATAA’ sequence and contain GC-rich sequences at the immediate upstream region of transcription start site(s). In this respect, the promoter structure of COX genes resemble those of many house-keeping genes. The ubiquitous COX genes show extensive 5′ heterogeneity with multiple transcription initiation sites that bind to both general as well as specialized transcription factors such as YY1 and GABP (NRF2/ets). The transcription activity of the promoter in most of the ubiquitous genes is regulated by factors binding to the 5′ upstream Sp1, NRF1, GABP (NRF2), and YY1 sites. Additionally, the murine COXVb promoter contains a negative regulatory region that encompasses the binding motifs with partial or full consensus to YY1, GTG, CArG, and ets. Interestingly, the muscle-specific COX genes contain a number of striated muscle-specific regulatory motifs such as E box, CArG, and MEF2 at the proximal promoter regions. While the regulation of COXVIa (H) gene involves factors binding to both MEF2 and E box in a skeletal muscle-specific fashion, the COXVIII (H) gene is regulated by factors binding to two tandomly duplicated E boxes in both skeletal and cardiac myocytes. The cardiac-specific factor has been suggested to be a novel bHLH protein. Mammalian COX genes provide a valuable system to study mechanisms of coordinated regulation of nuclear and mitochondrial genes. The presence of conserved sequence motifs common to several of the nuclear genes, which encode mitochondrial proteins, suggest a possible regulatory function by common physiological factors like heme/O2/carbon source. Thus, a well-orchestrated regulatory control and cross talks between the nuclear and mitochondrial genomes in response to changes in the mitochondrial metabolic conditions are key factors in the overall regulation of mitochondrial biogenesis.

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PMID: 9752724 [PubMed – indexed for MEDLINE]


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