Putative Antioxidant Response Elements Research Articles

AGE-DEPENDENT CARTILAGE DEGENERATION THROUGH NRF2/ARE MODULATION OF SOX9 EXPRESSION

Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9.In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05.While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE1, ARE2) were identified in the SOX9 promoter region. ARE2 mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE1 did not. A functional ARE2 site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion.Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression.

Nrf2/ARE Signaling Directly Regulates SOX9 to Potentially Alter Age-Dependent Cartilage Degeneration.

Oxidative stress is implicated in osteoarthritis, and nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway maintains redox homeostasis. We investigated whether Nrf2/ARE signaling controls SOX9. SOX9 expression in human C-28/I2 chondrocytes was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap ‘‘n’’ collar homology-associated protein 1 (Keap1). To verify whether Nrf2 transcriptionally regulates SOX9, putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays. SOX9 promoter activities without and with Nrf2-inducer methysticin were compared. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Nrf2-specific RNAi significantly decreased SOX9 expression, whereas Keap1-specific RNAi increased it. Putative ARE sites (ARE1, ARE2) were identified in the SOX9 promoter region. ARE2 mutagenesis significantly reduced SOX9 promoter activity, but ARE1 excision did not. Functional ARE2 site was essential for methysticin-mediated induction of SOX9 promoter activity. Young Nrf2-knockout mice revealed significantly lower Sox9-positive chondrocytes, and old Nrf2-knockout animals showed thinner cartilage and more cartilage degeneration. Our results suggest Nrf2 directly regulates SOX9 in articular cartilage, and Nrf2-loss can develop mild osteoarthritis at old age. Pharmacological Nrf2 induction may hold the potential to diminish age-dependent cartilage degeneration through improving SOX9 expression.

ZFARED: A Database of the Antioxidant Response Elements in Zebrafish

Background: Antioxidant Response Elements (ARE) play a key role in the expression of Nrf2 target genes by regulating the Keap1-Nrf2-ARE pathway, which offers protection against toxic agents and oxidative stress-induced diseases. Objective: To develop a database of putative AREs for all the genes in the zebrafish genome. This database will be helpful for researchers to investigate Nrf2 regulatory mechanisms in detail. Methods: To facilitate researchers functionally characterize zebrafish AREs, we have developed a database of AREs, Zebrafish Antioxidant Response Element Database (ZFARED), for all the protein-coding genes including antioxidant and mitochondrial genes in the zebrafish genome. The front end of the database was developed using HTML, JavaScript, and CSS and tested in different browsers. The back end of the database was developed using Perl scripts and Perl-CGI and Perl- DBI modules. Results: ZFARED is the first database on the AREs in zebrafish, which facilitates fast and efficient searching of AREs. AREs were identified using the in-house developed Perl algorithms and the database was developed using HTML, JavaScript, and Perl-CGI scripts. From this database, researchers can access the AREs based on chromosome number (1 to 25 and M for mitochondria), strand (positive or negative), ARE pattern and keywords. Users can also specify the size of the upstream/promoter regions (5 to 30 kb) from transcription start site to access the AREs located in those specific regions. Conclusion: ZFARED will be useful in the investigation of the Keap1-Nrf2-ARE pathway and its gene regulation. ZFARED is freely available at http://zfared.buc.edu.in/.

17-β estradiol exerts anti-inflammatory effects through activation of Nrf2 in mouse embryonic fibroblasts.

Several reports indicate crosstalk between the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and estrogen, which has a protective effect in colorectal cancer (CRC). The aim of this study was to investigate the role of Nrf2 signaling in the anti-inflammatory effect of estrogen using Nrf2 knockout (Nrf2 KO) mouse embryonic fibroblasts (MEFs), a powerful system to test the function of target genes due to their easy accessibility, and rapid growth rates. After inducing inflammation by tumor necrosis factor alpha (TNF-α), the effects of 17β-estradiol (E2) on the expression of proinflammatory mediators [i.e., NF-κB and inducible nitric oxide synthase (iNOS)] and estrogen receptors were evaluated by Western blot. In wild type (WT) MEFs, E2 treatment ameliorated TNF-α-induced nuclear translocation of NF-κB and expression of its target protein iNOS. Estrogen receptor beta (ERβ) expression was decreased by TNF-α-induced inflammation and restored by E2 treatment. When treated to WT MEFs, E2 induced nuclear translocation of Nrf2. The inhibitory effect of E2 on TNF-α-induced enhancement of iNOS was markedly dampened in Nrf2 KO MEFs. Notably, ERβ expression was significantly diminished in Nrf2 KO MEFs compared to that in WT cells. Promoter Database (EPD) revealed two putative anti-oxidant response elements (AREs) within the mouse ERβ promoter. Furthermore, in WT MEFs, E2 treatment repressed TNF-α-induced expression of iNOS protein and recovered by 4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo(1,5-a)pyrimidin-3-yl)phenol (PHTPP), a selective ERβ antagonist, treatment, but not in Nrf2 KO MEFs. In conclusion, Nrf2 plays a pivotal role in the anti-inflammatory of estrogen by direct regulating the expression of ERβ.

Genome-wide identification and analysis of Nrf2 binding sites – Antioxidant response elements in zebrafish

In the post-genomic era, deciphering the Nrf2 binding sites - antioxidant response elements (AREs) is an essential task that underlies and governs the Keap1-Nrf2-ARE pathway – a cell survival response pathway to environmental stresses in the vertebrate model system. AREs regulate the transcription of a repertoire of phase II detoxifying and/or oxidative-stress responsive genes, offering protection against toxic chemicals, carcinogens, and xenobiotics. In order to identify and analyze AREs in zebrafish, a pattern search algorithm was developed to identify AREs and computational tools available online were utilized to analyze the identified AREs in zebrafish. This study identified the AREs within 30 kb upstream from the transcription start site of antioxidant genes and mitochondrial genes. We report for the first time the AREs of all the known protein coding genes in the zebrafish genome. Western blotting, RT2 profiler array PCR, and qRT-PCR were performed to test whether AREs influence the Nrf2 target genes expression in the zebrafish larvae using sulforaphane. This study reveals unique AREs that have not been previously reported in the cytoprotective genes. Nine TGAG/CNNNTC and six TGAG/CNNNGC AREs were observed significantly. Our findings suggest that AREs drive the dynamic transcriptional events of Nrf2 target genes in the zebrafish larvae on exposure to sulforaphane. The identified abundant putative AREs will define the Keap1-Nrf2-ARE network and elucidate the precise regulation of Nrf2-ARE pathway in not only diseases but also in embryonic development, inflammation, and aerobic respiration. Our results help to understand the dynamic complexity of the Nrf2-ARE system in zebrafish.

Hepatic Lipid Accumulation and Nrf2 Expression following Perinatal and Peripubertal Exposure to Bisphenol A in a Mouse Model of Nonalcoholic Liver Disease.

Background:Exposure to chemicals during critical windows of development may re-program liver for increased risk of nonalcoholic fatty liver disease (NAFLD). Bisphenol A (BPA), a plastics component, has been described to impart adverse effects during gestational and lactational exposure. Our work has pointed to nuclear factor E2-related factor 2 (Nrf2) being a modulator of hepatic lipid accumulation in models of NAFLD.Objectives:To determine if chemical exposure can prime liver for steatosis via modulation of NRF2 and epigenetic mechanisms.Methods:Utilizing BPA as a model exposure, pregnant CD-1 mice were administered BPA via osmotic minipumps from gestational day 8 through postnatal day (PND)16. The offspring were weaned on PND21 and exposed to same dose of BPA via their drinking water through PND35. Tissues were collected from pups at week 5 (W5), and their littermates at week 39 (W39).Results:BPA increased hepatic lipid content concomitant with increased Nrf2 and pro-lipogenic enzyme expression at W5 and W39 in female offspring. BPA exposure increased Nrf2 binding to a putative antioxidant response element consensus sequence in the sterol regulatory-element binding protein-1c (Srebp-1c) promoter. Known Nrf2 activators increased SREBP-1C promoter reporter activity in HepG2 cells. Methylated DNA immunoprecipitation-PCR and pyrosequencing revealed that developmental BPA exposure induced hypomethylation of the Nrf2 and Srebp-1c promoters in livers of W5 mice, which was more prominent in W39 mice than in others.Conclusion:Exposure to a xenobiotic during early development induced persistent fat accumulation via hypomethylation of lipogenic genes. Moreover, increased Nrf2 recruitment to the Srebp-1c promoter in livers of BPA-exposed mice was observed. Overall, the underlying mechanisms described a broader impact beyond BPA exposure and can be applied to understand other models of NAFLD. https://doi.org/10.1289/EHP664

Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes

ABSTRACTAutophagy is a highly coordinated process that is controlled at several levels including transcriptional regulation. Here, we identify the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) as a regulator of autophagy gene expression and its relevance in a mouse model of Alzheimer disease (AD) that reproduces impaired APP (amyloid β precursor protein) and human (Hs)MAPT/TAU processing, clearance and aggregation. We screened the chromatin immunoprecipitation database ENCODE for 2 proteins, MAFK and BACH1, that bind the NFE2L2-regulated enhancer antioxidant response element (ARE). Using a script generated from the JASPAR's consensus ARE sequence, we identified 27 putative AREs in 16 autophagy-related genes. Twelve of these sequences were validated as NFE2L2 regulated AREs in 9 autophagy genes by additional ChIP assays and quantitative RT-PCR on human and mouse cells after NFE2L2 activation with sulforaphane. Mouse embryo fibroblasts of nfe2l2-knockout mice exhibited reduced expression of autophagy genes, which was rescued by an NFE2L2 expressing lentivirus, and impaired autophagy flux when exposed to hydrogen peroxide. NFE2L2-deficient mice co-expressing HsAPPV717I and HsMAPTP301L, exhibited more intracellular aggregates of these proteins and reduced neuronal levels of SQSTM1/p62, CALCOCO2/NDP52, ULK1, ATG5 and GABARAPL1. Also, colocalization of HsAPPV717I and HsMAPTP301L with the NFE2L2-regulated autophagy marker SQSTM1/p62 was reduced in the absence of NFE2L2. In AD patients, neurons expressing high levels of APP or MAPT also expressed SQSTM1/p62 and nuclear NFE2L2, suggesting their attempt to degrade intraneuronal aggregates through autophagy. This study shows that NFE2L2 modulates autophagy gene expression and suggests a new strategy to combat proteinopathies.

Abstract B33: Regulation and function of Nrf2-associated long noncoding RNA

Abstract We hypothesized that the long non coding RNA (lncRNA), Loc344887, is induced by Nrf2 activation and acts as a transcriptional co-activator of the Nrf2 target gene NQO1. Nrf2 is a transcription factor that is important in oxidative stress responses. Upon oxidative stress, Nrf2 is released from its binding partner Keap1. Nrf2 translocates to the nucleus, partners with small Maf proteins, binds to antioxidant response elements (ARE) and induces detoxifying and metabolizing genes such as NQO1, HMOX1, and GSTs. We propose that Loc344887 is acting as a co-activator of Nrf2, either directly in the Nrf2-Maf complex, or indirectly by some other downstream mechanism, in order to regulate NQO1 expression. Loc344887 has significantly reduced expression in human colon tumors when compared to normal colon tissue (TCGA database). Human HCT116 colon cancer cells and CCD841 non-cancer colonic epithelial cells replicate this expression difference between tumor and non-transformed. We used these cell lines to study the regulation and function of Loc344887. HCT116 and CCD841 cells were treated with 15 μM sulforaphane (SFN), a dietary isothiocyanate known to have chemopreventative properties due to Nrf2 activation. Loc344887 increased 40-fold in HCT116 cells, but was not induced in CCD841 cells. Loc344887 was also induced by oltipraz and tert-butylhydroquinone, known Nrf2 activators. Keap1 knockdown upregulated Loc344887, and inducibility of Loc344887 by SFN was lost when Nrf2 was knocked down using RNAi. The gene promoter of Loc344887 contains putative AREs, and chromatin immunoprecipitation assays identified putative Nrf2 and MafK binding regions. Because Loc344887 is regulated by Nrf2, we hypothesized that Loc344887 may be involved in NRF2 gene activation. Knockdown of Loc344887 caused a loss of NQO1 induction by both SFN and Keap1 knockdown; however, this effect was not seen for HMOX1. Time-course assays showed that Loc344887 is induced as early as 1 h post-SFN treatment and peaks at ~8 h, similar to HMOX1. NQO1, however, was induced much slower, peaking after 24 h. This suggests that other factor(s), such as Loc344887, needs to be present for full induction to occur. This study shows that Loc344887 is an Nrf2 target gene and is required for complete NQO1 induction, but not for HMOX1 activation. Thus, there is a differential response for Nrf2 target genes that depends, in part, on lincRNAs as co-regulators. Further studies are required to access the mechanism of NQO1 regulation. We hypothesize that once Loc344887 is induced, it acts as a scaffold for MafK, Nrf2 and/or other transcription factors on the NQO1 promoter. Loc344887 localizes primarily to the nucleus, and preliminary RNA immunoprecipitation experiments show that Loc344887 binds directly to MafK. Further studies will also test whether Loc344887 is required for regulation of other Nrf2 target genes, or if other lncRNAs are involved in Nrf2 target gene induction. Studies supported in part by Chancellor's Research Initiative funding from Texas A&M University, and by NIH grants CA090890, CA122959, P30 ES00210, P30 ES023512. Citation Format: Gavin Johnson, Laura Beaver, David E. Williams, Emily Ho, Roderick H. Dashwood. Regulation and function of Nrf2-associated long noncoding RNA. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B33.

Abstract 122: Activation of Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) Enhances Cyclooxygenase 2 Expression via Promoter Antioxidant Response Element in Preglomerular Vascular Smooth Muscle Cells (PGVSMCs)

Nrf2 is a key cytoprotective transcription factor driving many antioxidant and anti-inflammatory genes expression via binding to antioxidant response elements (AREs) in promoters of targeting genes. However, it was reported that Nrf2 also increased expression of pro-inflammatory and pro-oxidative genes IL-6 and NOX4 through the same mechanism. We detected 2 putative ARE consensus sequences within 1500 nucleotides (nts) located in the genomic region upstream of the transcription start site in rat cyclooxygenase 2 (COX2) gene. Therefore, we hypothesized that activation of Nrf2-dependent AREs upregulates COX2 expression in rat PGVSMCs. Incubation of the cells for 24 hours with Nrf2 activator, tert-butylhydroquinone (tBHQ), did not significantly alter Nrf2 expression but dose-dependently increased COX2 gene and protein in a dose response manner (5-20μM). Twenty μM of tBHQ treatment enhanced expression of COX2 gene and protein by 10.7±2.2 and 9.2±3.4 fold (both p<0.01, N=3), COX2 activity (from COX2-based product of fluorescent resorufin) by 229.9±28.8% (p<0.005, N=6) but reactive oxygen species (ROS) generated in the cells (from oxidized dihydroethidium-based fluorescent intensity) were reduced by 35.1±10.2% (p<0.01, N=4). tBHQ also significantly promoted Nrf2 nuclear translocation by 86±7.4% (p<0.05, N=3) and potentiated Nrf2 activity by 387.5±19.2% (p<0.005, N=4) determined with cell fraction immunoblotting and Nrf2-ARE binding-based enzyme-linked immunosorbent assay. Nrf2 expression was dramatically knocked down with markedly decreased COX2 expression and activity in the cells transfected with Nrf2 specific siRNA and treated with tBHQ compared to those transfected with the scramble control siRNA and treated with tBHQ. Chromatin immunoprecipitation (CHIP)-based PCR analysis revealed an enhanced recruitment of Nrf2 to the endogenous COX2 promoter spanning the ARE (-417 to -426 nts) after 24 hour-tBHQ treatment but knockdown of Nrf2 gene with siRNA significantly diminished the tBHQ-mediated role, suggesting that there is a functional ARE located in COX2 promoter. In conclusion, these results show a novel role of Nrf2 in inducing COX2 expression through binding to promoter ARE in the absence of increased ROS in rat PGVSMCs.

Activation of nuclear factor erythroid 2-related factor 2 coordinates dimethylarginine dimethylaminohydrolase/PPAR-γ/endothelial nitric oxide synthase pathways that enhance nitric oxide generation in human glomerular endothelial cells.

Dimethylarginine dimethylaminohydrolase (DDAH) degrades asymmetric dimethylarginine, which inhibits nitric oxide (NO) synthase (NOS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that binds to antioxidant response elements and transcribes many antioxidant genes. Because the promoters of the human DDAH-1 and DDAH-2, endothelial NOS (eNOS) and PPAR-γ genes contain 2 to 3 putative antioxidant response elements, we hypothesized that they were regulated by Nrf2/antioxidant response element. Incubation of human renal glomerular endothelial cells with the Nrf2 activator tert-butylhydroquinone (20 μmol·L(-1)) significantly (P<0.05) increased NO and activities of NOS and DDAH and decreased asymmetric dimethylarginine. It upregulated genes for hemoxygenase-1, eNOS, DDAH-1, DDAH-2, and PPAR-γ and partitioned Nrf2 into the nucleus. Knockdown of Nrf2 abolished these effects. Nrf2 bound to one antioxidant response element on DDAH-1 and DDAH-2 and PPAR-γ promoters but not to the eNOS promoter. An increased eNOS and phosphorylated eNOS (P-eNOSser-1177) expression with tert-butylhydroquinone was prevented by knockdown of PPAR-γ. Expression of Nrf2 was reduced by knockdown of PPAR-γ, whereas PPAR-γ was reduced by knockdown of Nrf2, thereby demonstrating 2-way positive interactions. Thus, Nrf2 transcribes HO-1 and other genes to reduce reactive oxygen species, and DDAH-1 and DDAH-2 to reduce asymmetric dimethylarginine and PPAR-γ to increase eNOS and its phosphorylation and activity thereby coordinating 3 pathways that enhance endothelial NO generation.

Identification of a functional antioxidant response element within the eighth intron of the human ABCC3 gene.

The ATP-binding cassette (ABC) family of transporters, including ABCC3, is a large family of efflux pumps that plays a pivotal role in the elimination of xenobiotics from the body. ABCC3 has been reported to be induced during hepatic stress conditions and through the progression of some forms of cancer. Several lines of evidence have implicated the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in this induction. However, although rodent models have been investigated, a functional antioxidant response element (ARE) in the human ABCC3 gene has not been identified. The purpose of this study was to identify and characterize the ARE(s) responsible for mediating the Nrf2-dependent induction of the human ABCC3 gene. A high-throughput chromatin immunoprecipitation-sequencing analysis performed in A549 cells revealed a specific interaction between Nrf2 and the eighth intron of the human ABCC3 gene rather than the more prototypical flanking region of the gene. Subsequent in silico analysis of the intron identified two putative ARE elements that contained the core consensus ARE sequence commonly found in several Nrf2-responsive genes. Functional characterization of these two AREs using luciferase-reporter constructs with ARE mutant constructs revealed that one of these putative AREs is functionally active. Finally, DNA pull-down assays confirmed specific binding of these intronic AREs by Nrf2 in vitro. Our findings identify a functional Nrf2 response element within the eighth intron of the ABCC3 gene, which may provide mechanistic insight into the induction of ABCC3 during antioxidant response stimuli.

CO Induces Nrf2-Dependent Heme Oxygenase-1 Transcription by Cooperating with Sp1 and c-Jun in Rat Brain Astrocytes.

Upregulation of heme oxygenase 1 (HO-1) by carbon monoxide (CO) delivered by CO-releasing molecules (CORMs) may be utilized as a therapeutic intervention for neurodegenerative diseases. This study was to delineate the two putative anti-oxidant response elements (AREs) in modulating HO-1 gene by participating with its promoter elements in rat brain astrocytes (RBA-1). CORM-2-induced HO-1 expression was mediated through superoxide, p38 mitogen-activated protein kinase(MAPK), extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), protein tyrosine kinase 2 (Pyk2), platelet-derived growth factor receptor (PDGFR), and phosphatidylinositol 3'-kinase (PI3K/Akt), revealed by the pharmacological inhibitors or knockdown of these signaling molecules. CORM-2-enhanced HO-1 promoter activity was inhibited by co-transfection with small interfering RNA (siRNA) of c-Jun, specificity protein 1 (Sp1), or nuclear factor-erythroid 2-related factor 2 (Nrf2). Immunoprecipitation assay showed that CORM-2 increased the association of nuclear Nrf2 with Sp1 and c-Jun. Furthermore, chromatin immunoprecipitation (ChIP) assay confirmed that Nrf2, Sp1, and c-Jun are associated with the proximal ARE binding site on HO-1 promoter, suggesting that Nrf2/Sp1/c-Jun cooperations are key transcription factors modulating HO-1 expression. Mechanistically, CORM-2-induced ARE promoter activity was reduced by the inhibitors of reactive oxygen species (ROS), p38 MAPK, Pyk2, MAPK/ERK kinases 1 and 2 (MEK1/2), PDGFR, and PI3K/Akt or the siRNAs of c-Jun, SP1, and Nrf2. These findings suggested that CORM-2 increases formation of c-Jun, Sp1, and Nrf2 complex and binding with ARE1 binding site, which is mediated through both ROS/p38 MAPK and Pyk2-dependent PDGFR/PI3K/Akt/Erk1/2 pathways, resulting in HO-1 expression in RBA-1 cells.

Nrf2 Pathway Regulates Multidrug-Resistance-Associated Protein 1 in Small Cell Lung Cancer

Although multidrug-resistance-associated protein-1 (MRP1) is a major contributor to multi-drug resistance (MDR), the regulatory mechanism of Mrp1 still remains unclear. Nrf2 is a transcription factor that regulates cellular defense response through antioxidant response elements (AREs) in normal tissues. Recently, Nrf2 has emerged as an important contributor to chemo-resistance in tumor tissues. In the present study, the role of Nrf2-ARE pathway on regulation of Mrp1 was investigated. Compared with H69 lung cancer cells, H69AR cells with MDR showed significantly higher Nrf2-ARE pathway activity and expression of Mrp1 as well. When Nrf2 was knocked down in H69AR cells, MRP1's expression decreased accordingly. Moreover, those H69AR cells with reduced Nrf2 level restored sensitivity to chemo-drugs. To explore how Nrf2-ARE pathway regulates Mrp1, the promoter of Mrp1 gene was searched, and two putative AREs—ARE1 and ARE2—were found. Using reporter gene and ChIP assay, both ARE1 and ARE2 showed response to and interaction with Nrf2. In 40 cases of cancer tissues, the expression of Nrf2 and MRP1 was measured by immunohistochemistry (IHC). As the quantitive data of IHC indicated, both Nrf2 and MRP1 showed significantly higher expression in tumor tissue than adjacent non-tumor tissue. And more important, the correlation analysis of the two genes proved that their expression was correlative. Taken together, theses data suggested that Nrf2-ARE pathway is required for the regulatory expression of Mrp1 and implicated Nrf2 as a new therapeutic target for MDR.

Involvement of Nrf2 activation in the upregulation of S100A9 by exposure to inorganic arsenite

This study examined whether S100A8 and S100A9, which comprise a complex called calprotectin, are upregulated by exposure to sodium arsenite [As(III)] in nine lines of human-derived cells. HaCaT skin keratinocyte cells, U937 leukemic monocyte cells, and UROtsa urothelial cells showed increased mRNA levels of S100A8 and S100A9 after a 2-week exposure to As(III). To understand the mechanisms regulating S100A9 upregulation in response to As(III), we tested S100A9 promoter-dependent luciferase activity in HaCaT cells transfected with S100A9 promoter (-1000/+429)-fused luciferase cDNA. The results indicated that exposure to As(III) stimulated S100A9 promoter-dependent luciferase activity. In addition, the transcription NF-E2-related factor2 (Nrf2) was strongly activated in HaCaT cells exposed to As(III). Since two putative antioxidant response elements were found in the S100A9 promoter (ARE1, 5'-ACAGGCAGGG-3' from -897 to -887; ARE2, 5'-ATCTTCCGGAG-3' from -78 to -67), we constructed deletion mutants of each ARE on S100A9 promoter-fused luciferase cDNA. The results indicated that ARE2 is the responsible element for the activation of S100A9 transcription in response to As(III). This report is the first to demonstrate that As(III) enhanced S100A8 and S100A9 expression in human-derived cells and As(III)-induced S100A9 expression is dependent on Nrf2 activation.

Abstract 91: Activation of The Antioxidant Response Element (ARE) in Human Renal Glomerular Endothelial Cells (HRGECs) Reduces Asymmetric Dimethylarginine (ADMA) And Increases Nitric Oxide by Transcriptional Activation of Dimethylarginine Dimethylaminohydrolase (DDAH) and Endothelial Nitric Oxide Synthase (eNOS)

The E2-related factor 2 (Nrf2) binds to AREs and transcriptionally activates several genes that protect against oxidative stress. DDAH phosphorylates and activates eNOS and degrades ADMA which is an endogenous inhibitor of eNOS. We detected 2-3 putative AREs in the promoter regions of the human genes for DDAH-1 and -2, peroxisome proliferator-activated receptor-γ (PPAR-γ) and eNOS. PPAR-γ can increase eNOS expression and eNOS phosphorylation. Therefore, we hypothesized that AREs regulate the DDAH/eNOS/PPAR-γ/ADMA/ NO pathway. Tert-butylhydroquinone (tBHQ) activates Nrf2 by translocation to the nucleus. Incubation of HRGECs for 24 hours with tBHQ enhanced cell proliferation dose-dependently (5-20μM). tBHQ (20μM; n=4) increased medium nitrite by 69 ± 3.7% (P&lt;0.01), intracellular NO (from DAF-FM fluorescence ) by 62 ± 17.1% (P&lt;0.05), and NOS and DDAH activities( from conversion of [ 3 H]-arginine or [ 14 C]-ADMA to [ 3 H]- or [ 14 C] -citrulline) by 58 ± 3.6% and 47 ± 8.5% (P&lt;0.05) and decreased ADMA (by capillary zone electrophoresis) from 1.2±0.03 vs. 2.3±0.16μM/L, (P&lt;0.001, n=6). tBHQ increased the mRNAs and proteins for eNOS by 79 ± 9% and 43 ± 17%, for DDAH-1 by 129 ± 10% and 62 ± 21%, and for DDAH-2 by 119 ± 25% and 48 ± 18% (all P&lt;0.05) and increased the protein expression of PPAR-γ and eNOS phosphorylation and increased tBHQ nuclear translocation markedly. Knockdown of the cell Nrf2 by transfection with a specific siRNA abolished all these effects and attenuated cell proliferation. Chromatin immunoprecipitation (CHIP)-based PCR assays demonstrated that tBHQ enhanced the binding of Nrf2 to one ARE region in the promoters for DDAH-1 and -2 but there was no binding to the eNOS promoter. In conclusion, activation of a specific ARE in the promoter region of the DDAH-1 and -2 genes causes their transcriptional activation. The ensuing enhancement of DDAH activity enhances ADMA metabolism. Nrf2 also increases eNOS expression, NOS activity and NO generation without binding to the ARE for eNOS, likely by upregulation of PPAR-γ. Thus, Nrf2 improves endothelial cell function by coordinating a diverse set of pathways that enhance NO generation.

Nrf2-ARE pathway regulates induction of Sestrin-2 expression

The Sestrin2 (Sesn2) gene encodes a conserved antioxidant protein that is induced on oxidative stress and protects cells against reactive oxygen species. NF-E2-related factor-2 (Nrf2) is an essential transcription factor that regulates expression of a variety of antioxidant genes via binding to the antioxidant-response element (ARE), but the role of Nrf2 in Sesn2 gene expression has not been elucidated yet. The present study investigated whether the Nrf2-ARE pathway regulates Sesn2 gene expression and the identification of the molecular mechanism. The Nrf2 activators, tert-butylhydroquinone (t-BHQ) and sulforaphane (SFN), up-regulated Sesn2 expression in a dose- and time-dependent manner in hepatocytes. Also, t-BHQ increased Sesn2 mRNA and luciferase gene activity, whereas the levels of Sesn1 and Sesn3 mRNA were not affected by t-BHQ treatment. The specific role of Nrf2 in Sesn2 induction was verified by using Nrf2 overexpression plasmid and Nrf2 knockout or knockdown cells. In silico analysis of the 5′ upstream region of Sesn2 gene identified a putative ARE sequence. Deletion of the putative ARE demonstrated that the ARE from −550 to −539bp in the human Sesn2 promoter was critical for the Nrf2-mediated response. Moreover, SFN injection increased Sesn2 mRNA and protein levels in the livers of mice. Knockdown experiments with Sesn2 siRNA showed that Sesn2 is required for the Nrf2-mediated cytoprotective activity against hydrogen peroxide. Our results suggest that the Nrf2-ARE pathway is critical for Sesn2 gene expression and might protect against oxidative stress.

The prostaglandin F synthase activity of the human aldose reductase AKR1B1 brings new lenses to look at pathologic conditions

Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1β in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1β is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1β particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies.

Human CYP2A6 is regulated by nuclear factor-erythroid 2 related factor 2

Human CYP2A6 is responsible for the metabolism of nicotine and coumarin as well as the metabolic activation of tobacco-related nitrosamines. Earlier studies revealed that CYP2A6 activity was increased by dietary cadmium or cruciferous vegetables, but the underlying mechanisms remain to be clarified. In the present study, we investigated the possibility that Nrf2 might be involved in the regulation of CYP2A6. Real-time RT-PCR analysis revealed that the CYP2A6 mRNA level in human hepatocytes was significantly ( P < 0.01, 1.4-fold) induced by 10 μM sulforaphane (SFN), a typical activator of Nrf2. A computer-based search identified three putative antioxidant response elements (AREs) in the 5′-flanking region of the CYP2A6 gene at positions −1212, −2444, and −3441, termed ARE1, ARE2, and ARE3, respectively. Electrophoretic mobility shift assays demonstrated that Nrf2 bound only to ARE1. Luciferase assays using HepG2 cells revealed that the overexpression of Nrf2 significantly increased the reporter activities of the constructs containing a 30-bp fragment that included ARE1. However, the activity of the construct containing the intact 5′-flanking region (−1 to −1395) including ARE1 was not increased by the overexpression of Nrf2. In contrast, when the reporter construct was injected into mice via the tail vein, the reporter activity in the liver was significantly ( P < 0.05, 1.9-fold) increased by SFN (1 mg/head) administration. In conclusion, we found that human CYP2A6 is regulated via Nrf2, suggesting that CYP2A6 is induced under oxidative stress.

Genetic Variation and Antioxidant Response Gene Expression in the Bronchial Airway Epithelium of Smokers at Risk for Lung Cancer

Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. As a first step in applying functional genomic analysis to population studies, we have examined the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer (SC, n = 20), smokers without lung cancer (SNC, n = 24), and never smokers (NS, n = 8). Functional enrichment analysis showed that the NRF2-mediated, antioxidant response element (ARE)-regulated genes, were significantly lower in SC, when compared with expression levels in SNC. Importantly, we found that the expression of MAFG (a binding partner of NRF2) was correlated with the expression of ARE genes, suggesting MAFG levels may limit target gene induction. Bioinformatically we identified single nucleotide polymorphisms (SNPs) in putative ARE genes and to test the impact of genetic variation, we genotyped these putative regulatory SNPs and other tag SNPs in selected NRF2 pathway genes. Sequencing MAFG locus, we identified 30 novel SNPs and two were associated with either gene expression or lung cancer status among smokers. This work demonstrates an analysis approach that integrates bioinformatics pathway and transcription factor binding site analysis with genotype, gene expression and disease status to identify SNPs that may be associated with individual differences in gene expression and/or cancer status in smokers. These polymorphisms might ultimately contribute to lung cancer risk via their effect on the airway gene expression response to tobacco-smoke exposure.

Oxidized Phospholipids Regulate Expression of ATF4 and VEGF in Endothelial Cells via NRF2-Dependent Mechanism: Novel Point of Convergence Between Electrophilic and Unfolded Protein Stress Pathways

The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response. Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation. Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.