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1.  Nrf2 is essential for the expression of lipocalin–prostaglandin D synthase induced by prostaglandin D2 
Free radical biology & medicine  2013;65:1134-1142.
Nrf2 is a transcription factor that protects against inflammatory diseases, but the underlying mechanism of this effect remains unclear. Here, we report that Nrf2 uses lipocalin–prostaglandin D synthase (L-PGDS) as a mechanism for suppressing inflammation. Exogenously added prostaglandin D2 (PGD2) induced L-PGDS expression in bone-marrow-derived macrophages (BMDMs), suggesting a positive feedback loop between L-PGDS expression and PGD2. Unlike lipopolysaccharide (LPS)-induced L-PGDS expression, PGD2-mediated expression was independent of MAPK, PU.1, or TLR4. Sequence analysis located a putative Nrf2 binding site in the murine L-PGDS promoter, to which Nrf2 bound when treated with PGD2. Chemical activation, or overexpression, of Nrf2 was sufficient to induce L-PGDS expression in macrophages, BMDMs, or lungs of Nrf2-knockout (KO) mice, but treatment with PGD2 failed to do so, suggesting a pivotal role for Nrf2 in the expression of L-PGDS. Consistent with this, expression of Nrf2 in the lungs of Nrf2-KO mice was sufficient to induce the expression of L-PGDS and to reduce neutrophilic lung inflammation elicited by LPS. Furthermore, expression of L-PGDS in mouse lungs decreased neutrophilic infiltration, ameliorating lung inflammation in mice. Together, our results show that Nrf2, activated by PGD2, induced L-PGDS expression, resulting in decreased inflammation. We suggest that the positive feedback induction of L-PGDS by PGD2 is part of the mechanism by which Nrf2 regulates inflammation.
doi:10.1016/j.freeradbiomed.2013.08.192
PMCID: PMC3972891  PMID: 24029383
Nrf2; Lung inflammation; Lipocalin-prostaglandin D synthase; Prostaglandin D2; Gene expression; Free radicals
2.  Emerging Role of Nrf2 in Adipocytes and Adipose Biology123 
Advances in Nutrition  2013;4(1):62-66.
Maintenance of a balanced redox state within the cell is of critical importance to a wide variety of biological systems. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a critical regulator of key aspects of the antioxidant defense pathway and has long been a subject of interest regarding conditions of chronic stress such as inflammation and cancer. Recent data have emerged demonstrating that oxidative stress and Nrf2 also play critical roles in the biology of adipose tissue. This review examines data identifying the roles of Nrf2 and oxidative stress in the biological process of adipose cell differentiation as well as the implications of Nrf2 modulation on obesity. Working to understand the complex interplay among Nrf2, oxidative stress, and adipose biology could lead to a variety of possible treatments for obesity and other related disorders.
doi:10.3945/an.112.003103
PMCID: PMC3648740  PMID: 23319124
3.  Deficiency in the nuclear related factor erythroid-2 transcription factor (Nrf1) leads to genetic instability 
The FEBS journal  2012;279(22):10.1111/febs.12005.
Nuclear factor erythroid-derived 2-related factor 1 (Nrf1) regulates cellular stress response genes, and has also been implicated to have 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 bearing a conditional Nrf1 allele exhibited 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 multinucleated 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 serve a function in maintaining genomic integrity, and Nrf1 dysregulation can induce tumorigenesis.
doi:10.1111/febs.12005
PMCID: PMC3835192  PMID: 22971132
4.  Increased Susceptibility of Nrf2 Knockout Mice to Colitis-Associated Colorectal Cancer 
The nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in protecting various tissues against inflammation, which is a potential risk factor for colorectal and other cancers. Our previously published mouse model work showed that Nrf2 helps protect against dextran sulfate sodium (DSS)–induced colitis/inflammation, and others have shown that Nrf2 helps protect against inflammation-associated colorectal carcinogenesis (aberrant crypt foci). The present study extended these important earlier findings by exploring the role of Nrf2 in colitis-associated colorectal cancer in a mouse model involving azoxymethane/DSS–induced colorectal carcinogenesis in Nrf2 knockout mice. Azoxymethane/DSS–treated Nrf2 knockout mice had increased incidence, multiplicity, and size of all colorectal tumors, including adenomas, versus treated wild-type (WT) mice, and the proportion of tumors that were adenocarcinoma was much higher in knockout (80%) versus WT (29%) mice. Compared with WT mice, knockout mice also had increased markers of inflammation in tumor tissue (cyclooxygenase-2 and 5-lipoxygenase expressions and prostaglandin E2 and leukotriene B4 levels) and in inflamed colonic mucosa (nitrotyrosine expression), supporting the association of knockout mouse tumor formation with inflammation. The phase II detoxifying/antioxidant enzymes NAD(P)H-quinone reductase 1 and UDP-glucurosyltransferase 1A1 were elevated in the normal mucosa of WT, but not Nrf 2 knockout, mice treated with azoxymethane/DSS. Our findings show that Nrf2 plays a critical role in protecting against inflammation-associated colorectal cancer.
doi:10.1158/1940-6207.CAPR-08-0028
PMCID: PMC3580177  PMID: 19138955
5.  Nuclear factor erythroid-derived factor 2-related factor 2 regulates transcription of CCAAT/enhancer-binding protein β during adipogenesis 
Free radical biology & medicine  2011;52(2):462-472.
Nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper transcription factor that is involved in the cellular adaptive response to oxidative stress. Our previous study reported that targeted disruption of the Nrf2 gene in mice decreases adipose tissue mass and protects against obesity induced by a high-fat diet. Deficiency of Nrf2 in preadipocytes and mouse embryonic fibroblasts led to impaired adipogenesis. Consistent with these findings, the current study found that lack of Nrf2 in primary cultured mouse preadipocytes and 3T3-L1 cells hampered adipogenic differentiation induced by hormonal cocktails. Stable knockdown of Nrf2 in 3T3-L1 cells blocked the enhanced adipogenesis caused by deficiency of kelch-like ECH-associated protein 1 (Keap1), a Cul3-adapter protein that allows for Nrf2 to be ubiquinated and degraded by the 26S protesome complex. In addition, increased production of reactive oxygen species (ROS) and activation of Nrf2 occurred at the very early stage upon adipogenic hormonal challenge in 3T3-L1 cells, followed by an immediate induction of CCAAT/enhancer-binding protein β (C/EBPβ). Knockdown of Nrf2 led to reduced expression of C/EBPβ induced by adipogenic hormonal cocktails, chemical Nrf2 activators or Keap1 silencing. Cebpβ promoter-driven reporter assays and chromatin immunoprecipitation suggested that Nrf2 associates with a consensus antioxidant response element (ARE) binding site in the promoter of the Cebpβ gene during adipogenesis and upregulates its expression. These findings demonstrate a novel role of Nrf2 beyond xenobiotic detoxification and antioxidant response, and suggest that Nrf2 is one of the transcription factors that control the early events of adipogenesis by regulating expression of Cebpβ.
doi:10.1016/j.freeradbiomed.2011.10.453
PMCID: PMC3307524  PMID: 22138520
Nrf2; C/EBPβ; Adipogenesis
6.  Characterization of Nrf1b, a Novel Isoform of the Nuclear Factor-Erythroid-2 Related Transcription Factor-1 That Activates Antioxidant Response Element-Regulated Genes 
PLoS ONE  2012;7(10):e48404.
Nuclear factor E2-related factor 1 (Nrf1) is a basic leucine zipper transcription factor that plays an important role in the activation of cytoprotective genes through the antioxidant response elements. The previously characterized long isoform of Nrf1 (Nrf1a) is targeted to the endoplasmic reticulum and accumulates in the nucleus in response to activating signals. Here we characterized a novel Nrf1 protein isoform (Nrf1b) generated through an alternative promoter and first exon that lacks the ER targeting domain of Nrf1a. The 5′-flanking region of Nrf1b directed high levels of luciferase reporter expression in cells. RT-PCR and Western blotting showed Nrf1b is widely expressed in various cell lines and mouse tissues. Immunoblot analysis of subcellular fractions and imaging of green fluorescence protein (GFP)-tagged Nrf1b demonstrate Nrf1b is constitutively localized to the nucleus. Nrf1b can activate GAL4-dependent transcription when fused to the heterologous GAL4 DNA-binding domain. Gel-shift and coimmunoprecipitation experiments demonstrate that Nrf1b forms a complex with MafG, and expression of Nrf1b activates the expression of antioxidant response element containing reporters and genes in cells. These results suggest Nrf1b is targeted to the nucleus where it activates ARE-driven genes and may play a role in modulating antioxidant response elements.
doi:10.1371/journal.pone.0048404
PMCID: PMC3483171  PMID: 23144760
7.  Knockout of the transcription factor Nrf2 disrupts spermatogenesis in an age-dependent manner 
Free radical biology & medicine  2010;49(9):1368-1379.
Oxidative stress occurs when generation of reactive oxygen species (ROS) overwhelms antioxidant defenses. Oxidative stress has been associated with male infertility. The transcription factor Nuclear Factor-Erythroid 2-Related Factor 2 (NRF2) regulates basal and inducible transcription of genes encoding enzymes important for protection against ROS. We hypothesized that deletion of the Nrf2 gene causes testicular and epididymal oxidative stress, which disrupts spermatogenesis. Our results show that male Nrf2−/− mice have decreased fertility compared to wild type and heterozygous littermates, due to accumulating seminiferous tubule damage with increasing age. Testicular sperm head counts, epididymal sperm counts, and epididymal sperm motility in 2 month old Nrf2−/− males did not differ from wild type littermates; however, by age 6 months, Nrf2−/− males had 44% lower testicular sperm head counts, 65% lower epididymal sperm counts, and 66% lower epididymal sperm motility than wild type males. Two to 4 month old Nrf2−/− males had elevated levels of testicular and epididymal lipid peroxidation and testicular germ cell apoptosis, and decreased levels of antioxidants compared to wild type males. These results provide evidence that oxidative stress has deleterious effects on the testis and epididymis and demonstrate a critical role for the transcription factor NRF2 in preventing oxidative disruption of spermatogenesis.
doi:10.1016/j.freeradbiomed.2010.07.019
PMCID: PMC2948056  PMID: 20692336
oxidative stress; spermatogenesis; NRF2; testis; reproduction
8.  Role of Nrf1 in antioxidant response element-mediated gene expression and beyond 
Oxidative stress plays an important part in the pathogenesis of a variety of diseases. The ability to mount an efficient response against the continuous threat posed by exogenous and endogenous oxidants is essential for cellular homeostasis and survival. Oxidative stress activates transcription of a variety of antioxidant genes through cis-acting sequence known as antioxidant response element (ARE). Members of the Cap-N-Collar family of transcription factors, including Nrf1 and Nrf2, have been identified that bind ARE. Nrf1 and Nrf2 are expressed in a wide range of tissues and cell types, and both bind the ARE as heterodimers with small-Maf proteins. Numerous studies indicate a pivotal role of Nrf2 in ARE function. Herein, we review data derived from cell-based studies and knockout mice in an attempt to define the role and regulation of Nrf1 in oxidative stress response and other functions.
doi:10.1016/j.taap.2009.07.034
PMCID: PMC2837788  PMID: 19665035
9.  Long Isoforms of NRF1 Contribute to Arsenic-Induced Antioxidant Response in Human Keratinocytes 
Background
Human exposure to inorganic arsenic (iAs), a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer. Nuclear factor–erythroid 2–related factor 1 (NRF1, also called NFE2L1) plays a critical role in regulating the expression of many antioxidant response element (ARE)-dependent genes.
Objectives
We investigated the role of NRF1 in arsenic-induced antioxidant response and cytotoxicity in human keratinocytes.
Results
In cultured human keratinocyte HaCaT cells, inorganic arsenite (iAs3+) enhanced the protein accumulation of long isoforms (120–140 kDa) of NRF1 in a dose- and time-dependent fashion. These isoforms accumulated mainly in the nuclei of HaCaT cells. Selective deficiency of NRF1 by lentiviral short-hairpin RNAs in HaCaT cells [NRF1-knockdown (KD)] led to decreased expression of γ-glutamate cysteine ligase catalytic subunit (GCLC) and regulatory subunit (GCLM) and a reduced level of intracellular glutathione. In response to acute iAs3+ exposure, induction of some ARE-dependent genes, including NAD(P)H:quinone oxidoreductase 1 (NQO1), GCLC, and GCLM, was significantly attenuated in NRF1-KD cells. However, the iAs3-induced expression of heme oxygenase 1 (HMOX-1) was unaltered by silencing NRF1, suggesting that HMOX-1 is not regulated by NRF1. In addition, the lack of NRF1 in HaCaT cells did not disturb iAs3+-induced NRF2 accumulation but noticeably decreased Kelch-like ECH-associated protein 1 (KEAP1) levels under basal and iAs3+-exposed conditions, suggesting a potential interaction between NRF1 and KEAP1. Consistent with the critical role of NRF1 in the transcriptional regulation of some ARE-bearing genes, knockdown of NRF1 significantly increased iAs3+-induced cytotoxicity and apoptosis.
Conclusions
Here, we demonstrate for the first time that long isoforms of NRF1 contribute to arsenic-induced antioxidant response in human keratinocytes and protect the cells from acute arsenic cytotoxicity.
doi:10.1289/ehp.1002304
PMCID: PMC3018500  PMID: 20805060
apoptosis; arsenic; cytotoxicity; KEAP1; keratinocyte; NRF1; NRF2; oxidative stress
10.  Transcription factor Nrf1 mediates the proteasome recovery pathway after proteasome inhibition in mammalian cells 
Molecular cell  2010;38(1):17-28.
SUMMARY
In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here we report that Nuclear factor erythroid derived 2-related factor 1 (Nrf1), a transcription factor of the cap ‘n’ collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1-/- MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101 and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.
doi:10.1016/j.molcel.2010.02.029
PMCID: PMC2874685  PMID: 20385086
11.  Nrf2 protects against As(III)-induced damage in mouse liver and bladder 
Arsenic compounds are classified as toxicants and human carcinogens. Environmental exposure to arsenic imposes a big health issue worldwide. Arsenic elicits its toxic efforts through many mechanisms, including generation of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls expression of a main cellular antioxidant response, which is required for neutralizing ROS and thus defending cells from exogenous insults. Previously, we demonstrated a protective role of Nrf2 against arsenic-induced toxicity using a cell culture model. In this report, we present evidence that Nrf2 protects against liver and bladder injury in response to six-weeks of arsenic exposure in a mouse model. Nrf2−/− mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2+/+ mice. Furthermore, Nrf2−/− mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. These results indicate a protective role of Nrf2 against arsenic toxicity in vivo. Hence, this work demonstrates the feasibility of using dietary compounds that target activation of the Nrf2 signaling pathway to alleviate arsenic-induced damage.
doi:10.1016/j.taap.2009.06.010
PMCID: PMC2739886  PMID: 19538980
12.  Distinct Nrf1/2-independent mechanisms mediate As3+-induced glutamate-cysteine ligase subunit gene expression in murine hepatocytes 
Free radical biology & medicine  2009;46(12):1614-1625.
Trivalent arsenite (As3+) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and cytotoxic effects of As3+. Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. In this study, we demonstrate that As3+ coordinately upregulates Gclc and Gclm mRNA levels in a murine hepatocyte cell line resulting in increased GCL subunit protein expression, holoenzyme formation and activity. As3+ increased the rate of transcription of both the Gclm and Gclc genes and induced the post-transcriptional stabilization of Gclm mRNA. The antioxidant N-acetylcysteine abolished As3+-induced Gclc expression and attenuated induction of Gclm. As3+ induction of Gclc and Gclm was also differentially regulated by the MAPK signaling pathways and occurred independent of the Nrf1/2 transcription factors. These findings demonstrate that distinct transcriptional and post-transcriptional mechanisms mediate the coordinate induction of the Gclc and Gclm subunits of GCL in response to As3+ and highlight the potential importance of the GSH antioxidant defense system in regulating As3+-induced responses in hepatocytes.
doi:10.1016/j.freeradbiomed.2009.03.016
PMCID: PMC2748780  PMID: 19328227
arsenite; arsenic; glutamate cysteine ligase; GCL; GCLC; GCLM; glutathione; GSH; hepatocyte; Nrf2; gene transcription
13.  Induction of Mrp3 and Mrp4 transporters during acetaminophen hepatotoxicity is dependent on Nrf2 
The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity, and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1–4 (Mrp1-4). It was hypothesized that induction of hepatic Mrp1-4 expression following APAP is Nrf2-dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 hrs after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1-4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP, but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals.
doi:10.1016/j.taap.2007.08.022
PMCID: PMC2214834  PMID: 17935745
Nuclear factor-E2-related factor 2; Nrf2; acetaminophen; APAP; hepatotoxicity; multidrug resistance-associated proteins; Mrp3; Mrp4
14.  Toxicogenomics of Endoplasmic Reticulum stress inducer Tunicamycin in the Small Intestine and Liver of Nrf2 Knockout and C57BL/6J Mice 
Toxicology letters  2006;168(1):21-39.
This objective of this study was to investigate the toxicogenomics and the spatial regulation of global gene expression profiles elicited by Endoplasmic Reticulum (ER) stress inducer Tunicamycin (TM) in mouse small intestine and liver as well as to identify TM-modulated Nuclear Factor-E2-related factor 2 (Nrf2)–dependent genes. Gene expression profiles were analyzed using 45,000 Affymetrix mouse genome 430 2.0 array and GeneSpring 7.2 software. Microarray results were validated by quantitative real-time reverse transcription-PCR analyses. Clusters of genes that were either induced or suppressed more than two fold by TM treatment compared with vehicle in C57BL/6J/Nrf2(−/−; knockout)and C57BL/6J Nrf2 (+/+; wildtype) mice genotypes were identified. Amongst these, in small intestine and liver, 1291 and 750 genes respectively were identified as Nrf2-dependent and upregulated, and 1370 and 943 genes respectively as Nrf2-dependent and downregulated. Based on their biological functions, these genes can be categorized into molecular chaperones and heat shock proteins, ubiquitination/proteolysis, apoptosis/cell cycle, electron transport, detoxification, cell growth/differentiation, signaling molecules/interacting partners, kinases and phosphatases, transport, biosynthesis/metabolism, nuclear assembly and processing, and genes related to calcium and glucose homeostasis. Phase II detoxification/antioxidant genes as well as putative interacting partners of Nrf2 such as nuclear corepressors and coactivators, were also identified as Nrf2-dependent genes. The identification of TM-regulated and Nrf2-dependent genes in the unfolded protein response to ER stress not only provides potential novel insights into the gestalt biological effects of TM on the toxicogenomics and spatial regulation of global gene expression profiles in cancer pharmacology and toxicology, but also points to the pivotal role of Nrf2 in these biological processes.
doi:10.1016/j.toxlet.2006.10.012
PMCID: PMC1847389  PMID: 17127020
Tunicamycin; endoplasmic reticulum stress; Nuclear Factor-E2-related factor 2; microarray; global gene expression profiles
15.  Keap1 Controls Postinduction Repression of the Nrf2-Mediated Antioxidant Response by Escorting Nuclear Export of Nrf2▿  
Molecular and Cellular Biology  2007;27(18):6334-6349.
The transcription factor Nrf2 regulates cellular redox homeostasis. Under basal conditions, Keap1 recruits Nrf2 into the Cul3-containing E3 ubiquitin ligase complex for ubiquitin conjugation and subsequent proteasomal degradation. Oxidative stress triggers activation of Nrf2 through inhibition of E3 ubiquitin ligase activity, resulting in increased levels of Nrf2 and transcriptional activation of Nrf2-dependent genes. In this study, we identify Keap1 as a key postinduction repressor of Nrf2 and demonstrate that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2-antioxidant response element (ARE) signaling by escorting nuclear export of Nrf2. We provide evidence that ubiquitination of Nrf2 is carried out in the cytosol. Furthermore, we show that Keap1 nuclear translocation is independent of Nrf2 and the Nrf2-Keap1 complex does not bind the ARE. Collectively, our results suggest the following mechanism of postinduction repression: upon recovery of cellular redox homeostasis, Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the NES in Keap1. Once in the cytoplasm, the Keap1-Nrf2 complex associates with the E3 ubiquitin ligase, resulting in degradation of Nrf2 and termination of the Nrf2 signaling pathway. Hence, postinduction repression of the Nrf2-mediated antioxidant response is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination and degradation machinery.
doi:10.1128/MCB.00630-07
PMCID: PMC2099624  PMID: 17636022
16.  Nuclear factor-E2-related factor 2 expression in liver is critical for induction of NAD(P)H:quinone oxidoreductase 1 during cholestasis 
Cell Stress & Chaperones  2006;11(4):356-363.
Bile duct ligation (BDL) causes hepatocellular oxidative stress and injury. The transcription factor nuclear factor-E2-related factor (Nrf2) induces expression of numerous genes including NAD(P)H:quinone oxidoreductase 1 (Nqo1) during periods of oxidative stress. Therefore, we hypothesized that BDL increases liver expression of mouse antioxidant genes in an Nrf2-dependent manner. BDL or sham surgeries were performed on male C57BL/6, Nrf2-null, and wild-type mice. Livers were collected at 1, 3, and 7 days after surgery for analysis of messenger ribonucleic acid (mRNA) levels of Nrf2-responsive genes as well as Nqo1 protein and activity. BDL increased mRNA expression of multiple Nrf2 genes in mouse liver, compared to sham-operated controls. Follow-up studies investigating protein expression, enzyme activity, and Nrf2 dependency were limited to Nqo1. Nqo1 protein expression and activity in mouse livers was increased 2- to 3-, and 4- to 5-fold at 3 and 7 days after BDL, respectively. Studies also showed that BDL increases Nqo1 mRNA, protein expression, and enzyme activity in livers from wild-type mice, but not in Nrf2-null mice. In conclusion, expression of Nrf2-dependent genes is increased during cholestasis. These studies also demonstrate that Nqo1 expression and activity in mouse liver are induced via an Nrf2-dependent mechanism.
doi:10.1379/CSC-217.1
PMCID: PMC1759988  PMID: 17278884
17.  Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-κB and AP-1 
Molecular and Cellular Biology  2005;25(14):5933-5946.
Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tert-Butylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidant-responsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-κB family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-κB binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-κB family members.
doi:10.1128/MCB.25.14.5933-5946.2005
PMCID: PMC1168815  PMID: 15988009
18.  Nrf1 Is Critical for Redox Balance and Survival of Liver Cells during Development 
Molecular and Cellular Biology  2003;23(13):4673-4686.
The Nrf1 transcription factor belongs to the CNC subfamily of basic leucine zipper proteins. Knockout of Nrf1 is lethal in mouse embryos, but nothing is known about the cell types that absolutely require its function during development. We show by chimera analysis that Nrf1 is essential for the hepatocyte lineage. Mouse embryonic stem cells lacking Nrf1 developed normally and contributed to most tissues in adult chimeras where Nrf1 is normally expressed. Nrf1-deficient cells contributed to fetal, but not adult, liver cells. Loss of Nrf1 function resulted in liver cell apoptosis in late-gestation chimeric fetuses. Fetal livers from mutant embryos exhibited increased oxidative stress and impaired expression of antioxidant genes, and primary cultures of nrf1−/− fetal hepatocytes were sensitive to tert-butyl hydroperoxide-induced cell death, suggesting that impaired antioxidant defense may be responsible for the apoptosis observed in the livers of chimeric mice. In addition, cells deficient in Nrf1 were sensitized to the cytotoxic effects of tumor necrosis factor (TNF). Our results provide in vivo evidence demonstrating an essential role of Nrf1 in the survival of hepatocytes during development. Our results also suggest that Nrf1 may promote cell survival by maintaining redox balance and protecting embryonic hepatocytes from TNF-mediated apoptosis during development.
doi:10.1128/MCB.23.13.4673-4686.2003
PMCID: PMC164851  PMID: 12808106

Results 1-18 (18)