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1.  Heightened Endoplasmic Reticulum Stress in the Lungs of Patients with Chronic Obstructive Pulmonary Disease 
Rationale: Nuclear factor erythroid 2–related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD.
Objectives: To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD.
Methods: We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD.
Measurements and Main Results: Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death.
Conclusions: Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.
PMCID: PMC2796732  PMID: 19797762
Nrf2; proteasome system; endoplasmic reticulum stress; unfolded protein response; chronic obstructive pulmonary disease lungs
2.  Dysfunctional KEAP1–NRF2 Interaction in Non-Small-Cell Lung Cancer 
PLoS Medicine  2006;3(10):e420.
Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.
Methods and Findings
Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.
This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.
Biallelic inactivation ofKEAP1, a frequent genetic alteration in NSCLC, is associated with activation of the NRF2 pathway which leads to expression of genes that contribute to resistance against chemotherapeutic drugs.
Editors' Summary
Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.
Why Was This Study Done?
To improve the outlook for patients with lung cancer, researchers need to discover exactly how cancer cells become resistant to chemotherapy drugs. Detoxification enzymes, efflux pumps, and antioxidants normally protect cells from environmental toxins and from oxidants produced by the chemical processes of life. Their production is regulated by nuclear factor erythroid-2 related factor 2 (NRF2). The activity of this transcription factor (a protein that controls the expression of other proteins) is controlled by the protein Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 holds NRF2 in the cytoplasm of the cell (the cytoplasm surrounds the cell's nucleus, where the genetic material is stored) when no oxidants are present and targets it for destruction. When oxidants are present, KEAP1 no longer interacts with NRF2, which moves into the nucleus and induces the expression of the proteins that protect the cell against oxidants and toxins. In this study, the researchers investigated whether changes in KEAP1 might underlie the drug resistance seen in lung cancer.
What Did the Researchers Do and Find?
The researchers looked carefully at the gene encoding KEAP1 in tissue taken from lung tumors and in several lung cancer cell lines—tumor cells that have been grown in a laboratory. They found mutations in parts of KEAP1 known to be important for its function in half the cell lines and a fifth of the tumor samples. They also found that about half of the samples had lost part of one copy of the KEAP1 gene—cells usually have two copies of each gene. Five of the six tumors with KEAP1 mutations had also lost one copy of KEAP1—geneticists call this biallelic inactivation. This means that these tumors should have no functional KEAP1. When the researchers checked this by staining the tumors for NRF2, they found that the tumor cells had more NRF2 than normal cells and that it accumulated in the nucleus. In addition, the tumor cells made more detoxification enzymes, efflux proteins, and antioxidants than normal cells. Finally, the researchers showed that lung cancer cells with KEAP1 mutations were more resistant to chemotherapy drugs than normal lung cells were.
What Do These Findings Mean?
These results indicate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC and suggest that the loss of KEAP1 activity is one way that lung tumors can increase their NRF2 activity and develop resistance to chemotherapeutic drugs. More lung cancer samples need to be examined to confirm this result, and similar studies need to be done in other cancers to see whether loss of KEAP1 activity is a common mechanism by which tumors become resistant to chemotherapy. If such studies confirm that high NRF2 activity (either through mutation or by some other route) is often associated with a poor tumor response to chemotherapy, then the development of NRF2 inhibitors might help to improve treatment outcomes in patients with chemotherapy-resistant tumors.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute information on lung cancer and on cancer treatment
MedlinePlus entries on small cell lung cancer and NSCLC Cancer Research UK information on lung cancer
Wikipedia entries on lung cancer and chemotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
PMCID: PMC1584412  PMID: 17020408
3.  Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress 
Research highlights
► Nrf2 anti-oxidant function is impaired when HDAC activity is inhibited. ► HDAC inhibition decreases Nrf2 protein stability. ► HDAC2 is involved in reduced Nrf2 stability and both correlate in COPD samples. ► HDAC inhibition increases Nrf2 acetylation.
Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H2O2) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H2O2-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r = 0.92, p < 0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.
PMCID: PMC3061319  PMID: 21320471
ARE, anti oxidant response element; COPD, chronic obstructive pulmonary disease; DJ-1, Parkinson’s disease (PD)-associated protein; HDAC2, histone deacetylase-2; HO-1, heme oxygenase-1; H2O2, hydrogen peroxide; Keap1, Kelch-like ECH associated protein 1; MDM, monocyte-derived macrophage; Nrf2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; TSA, trichostatin A; Oxidative stress; Nrf2; Histone deacetylase 2; Protein stability; Acetylation; COPD
4.  Deletion of Keap1 in the Lung Attenuates Acute Cigarette Smoke–Induced Oxidative Stress and Inflammation 
Exposure to cigarette smoke (CS) is the primary factor associated with the development of chronic obstructive pulmonary disease (COPD). CS increases the level of oxidants in the lungs, resulting in a depletion of antioxidants, which promotes oxidative stress and the destruction of alveolar tissue. In response to CS, pulmonary epithelial cells counteract increased levels of oxidants by activating Nrf2-dependent pathways to augment the expression of detoxification and antioxidant enzymes, thereby protecting the lung from injury. We hypothesize that increasing the pathways activated by Nrf2 will afford protection against CS-induced lung damage. To this end we have developed a novel mouse model in which the cytosolic inhibitor of Nrf2, Keap1, is genetically deleted in Clara cells, which predominate in the upper airways in mice. Deletion of Keap1 in Clara cells resulted in increased expression of Nrf2-dependent genes, such as Nqo1 and Gclm, as determined by microarray analysis and quantitative PCR. Deletion of Keap1 in airway epithelium decreased Keap1 protein levels and significantly increased the total level of glutathione in the lungs. Increased Nrf2 activation protected Clara cells against oxidative stress ex vivo and attenuated oxidative stress and CS-induced inflammation in vivo. Expression of KEAP1 was also decreased in human epithelial cells through siRNA transfection, which increased the expression of Nrf2-dependent genes and attenuated oxidative stress. In conclusion, activating Nrf2 pathways in tissue-specific Keap1 knockout mice represents an important genetic approach against oxidant-induced lung damage.
PMCID: PMC2874439  PMID: 19520915
cigarette smoke; Nrf2; Keap1; inflammation; oxidative stress
5.  Cross-Regulations among NRFs and KEAP1 and Effects of their Silencing on Arsenic-Induced Antioxidant Response and Cytotoxicity in Human Keratinocytes 
Environmental Health Perspectives  2012;120(4):583-589.
Background: Nuclear factor E2-related factors (NRFs), including NRF2 and NRF1, play critical roles in mediating the cellular adaptive response to oxidative stress. Human exposure to inorganic arsenic, a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer.
Objective: We investigated the cross-regulations among NRF2, NRF1, and KEAP1, a cullin-3–adapter protein that allows NRF2 to be ubiquinated and degraded by the proteasome complex, in arsenic-induced antioxidant responses.
Results: In human keratinocyte HaCaT cells, selective knockdown (KD) of NRF2 by lentiviral short hairpin RNAs (shRNAs) significantly reduced the expression of many antioxidant enzymes and sensitized the cells to acute cytotoxicity of inorganic arsenite (iAs3+). In contrast, silencing KEAP1 led to a dramatic resistance to iAs3+-induced apoptosis. Pretreatment of HaCaT cells with NRF2 activators, such as tert-butylhydroquinone, protects the cells against acute iAs3+ toxicity in an NRF2-dependent fashion. Consistent with the negative regulatory role of KEAP1 in NRF2 activation, KEAP1-KD cells exhibited enhanced transcriptional activity of NRF2 under nonstressed conditions. However, deficiency in KEAP1 did not facilitate induction of NRF2-target genes by iAs3+. In addition, NRF2 silencing reduced the expression of KEAP1 at transcription and protein levels but increased the protein expression of NRF1 under the iAs3+-exposed condition. In contrast, silencing KEAP1 augmented protein accumulation of NRF2 under basal and iAs3+-exposed conditions, whereas the iAs3+-induced protein accumulation of NRF1 was attenuated in KEAP1-KD cells.
Conclusions: Our studies suggest that NRF2, KEAP1, and NRF1 are coordinately involved in the regulation of the cellular adaptive response to iAs3+-induced oxidative stress.
PMCID: PMC3339469  PMID: 22476201
antioxidant response; arsenic; cytotoxicity; KEAP1; keratinocyte; NRF1; NRF2
6.  Activation of Transcription Factor Nrf2 Signalling by the Sphingosine Kinase Inhibitor SKI-II Is Mediated by the Formation of Keap1 Dimers 
PLoS ONE  2014;9(2):e88168.
Anti-oxidant capacity is crucial defence against environmental or endogenous oxidative stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that plays a key defensive role against oxidative and cytotoxic stress and cellular senescence. However, Nrf2 signalling is impaired in several aging-related diseases, such as chronic pulmonary obstructive disease (COPD), cancer, and neurodegenerative diseases. Thus, novel therapeutics that enhance Nrf2 signalling are an attractive approach to treat these diseases.
Methodology/Principal Findings
Nrf2 was stabilized by SKI-II (2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole), which is a known sphingosine kinase inhibitor, in human bronchial epithelial cell line, BEAS2B, and in primary human bronchial epithelial cells, leading to enhancement of anti-oxidant proteins, such as HO-1, NQO1 and GCLM. The activation of Nrf2 was achieved by the generation of inactive dimerized form of Keap1, a negative regulator of Nrf2 expression, which was independent of sphingosine kinase inhibition. Using mice that were exposed to cigarette smoke, SKI-II induced Nrf2 expression together with HO-1 in their lungs. In addition, SKI-II reduced cigarette smoke mediated oxidative stress, macrophages and neutrophil infiltration and markers of inflammation in mice.
SKI-II appears to be a novel activator of Nrf2 signalling via the inactivation of Keap1.
PMCID: PMC3914928  PMID: 24505412
7.  Glutathione Peroxidase 2, the Major Cigarette Smoke–Inducible Isoform of GPX in Lungs, Is Regulated by Nrf2 
Disruption of NF-E2–related factor (Nrf2), a redox-sensitive basic leucine zipper transcription factor, causes early-onset and more severe emphysema due to chronic cigarette smoke. Nrf2 determines the susceptibility of lungs to cigarette smoke–induced emphysema in mice through the transcriptional induction of numerous antioxidant genes. The lungs of Nrf2−/− mice have higher oxidative stress as evident from the increased levels of lipid peroxidation (4-hydroxy-2-nonenal) and oxidative DNA damage (7,8-dihydro-8-Oxo-2′deoxyguanosine) in response to cigarette smoke. Glutathione peroxidases (GPX) are the primary antioxidant enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Among the five GPX isoforms, expression of GPX2 was significantly induced at both mRNA and protein levels in the lungs of Nrf2+/+ mice, in response to cigarette smoke. Activation of Nrf2 by specific knock down of the cytosolic inhibitor of Nrf2, Keap1, by small inhibitory RNA (siRNA) upregulated the expression of GPx2, whereas Nrf2 siRNA down-regulated the expression of GPX2 in lung epithelial cells. An ARE sequence located in the 5′ promoter–flanking region of exon 1 that is highly conserved between mouse, rat, and human was identified. Mutation of this ARE core sequence completely abolished the activity of promoter–reporter gene construct. The binding of Nrf2 to the GPX2 antioxidant response element was confirmed by chromatin immunoprecipation, electrophoretic mobility shift assays, and site-directed mutagenesis. This study shows that GPX2 is the major oxidative stress–inducible cellular GPX isoform in the lungs, and that its basal as well as inducible expression is dependent on Nrf2.
PMCID: PMC2643293  PMID: 16794261
antioxidant response element; cigarette smoke; emphysema; GPX2; Nrf2
8.  Nrf2 Dependent Sulfiredoxin-1 Expression Protects Against Cigarette Smoke-induced Oxidative Stress in Lungs 
Free radical biology & medicine  2008;46(3):376-386.
Oxidative stress results in protein oxidation and is involved in the pathogenesis of lung diseases such as chronic obstructive pulmonary disorder (COPD). Sulfiredoxin-1 (Srx1) catalyzes reduction of cysteine sulfinic acid to sulfenic acid in oxidized proteins and protects them from inactivation. This study examined the mechanism of transcriptional regulation of Srx1 and its possible protective role during oxidative stress associated with COPD. Nrf2, a transcription factor known to influence susceptibility to pulmonary diseases, upregulates Srx1 expression during oxidative stress caused by cigarette smoke exposure in the lungs of mice. Disruption of Nrf2 signaling by genetic knockout in mice or RNAi in cells downregulated the expression of Srx1. In silico analysis of the 5′-promoter flanking region of Srx1 identified multiple antioxidant response elements that are highly conserved. Reporter and chromatin-immunoprecipation assays demonstrated that ARE1 at −228 is critical for the Nrf2-mediated response. Attenuation of Srx1 expression with RNAi potentiated the toxicity of hydrogen peroxide (H2O2), whereas overexpression of Srx1 protected against H2O2 mediated cell death in vitro. Immunoblot analysis revealed dramatic decreases in Srx1 expression in lungs from patients with COPD relative to non-emphysematous lungs together with a decline in Nrf2 protein. Thus, Srx1, a key Nrf2-regulated gene, contributes to protection against oxidative injury in the lung.
PMCID: PMC2828157  PMID: 19027064
Srx1; Nrf2; oxidative stress; antioxidant response element; chronic obstructive pulmonary disease; emphysema
9.  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.
PMCID: PMC2099624  PMID: 17636022
10.  Regulation of Nrf2 – An update 
Free radical biology & medicine  2013;66:10.1016/j.freeradbiomed.2013.02.008.
Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes which encode detoxifying enzymes, drug transporters (MRPs), anti-apoptotic proteins and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase mediated degradation of Nrf2. Chemicals including antioxidants, tocopherols including α-tocopherol (vitamin E), phytochemicals and radiations antagonize the Nrf2:INrf2 interaction and leads to the stabilization and activation of Nrf2. The signaling events involve pre-induction, induction and post-induction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinase(s) in a pre-induction response which phosphorylates specific residues on Nrf2 negative-regulators, INrf2, Fyn and Bach1, leading to their nuclear export, ubiquitination and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2cysteine151 followed by PKC phosphorylation of Nrf2serine40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus resulting in a coordinated activation of gene expression. This is followed by a post-induction response that controls the ‘switching off’ of Nrf2-activated gene expression. GSK3β under the control of AKT and PI3K, phosphorylates Fyn leading to Fyn nuclear localization. Fyn phosphorylates Nrf2Y568 resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provides protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.
PMCID: PMC3773280  PMID: 23434765
Nrf2; INrf2(Keap1); Antioxidants; Vitamins; Phytochemicals; ROS; Signaling; Regulation; Chemoprotection; Oncogenesis
11.  Trolox contributes to Nrf2-mediated protection of human and murine primary alveolar type II cells from injury by cigarette smoke 
Cell Death & Disease  2013;4(4):e573-.
Cigarette smoke (CS) is a main risk factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by CS causes DNA and lung damage. Oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD. We studied the effect of oxidative stress generated by CS both in vivo and in vitro on murine primary alveolar type II (ATII) cells isolated from nuclear erythroid 2-related factor-2 (Nrf2)−/− mice. We determined human primary ATII cell injury by CS in vitro and analyzed ATII cells isolated from smoker and non-smoker lung donors ex vivo. We also studied whether trolox (water-soluble derivative of vitamin E) could protect murine and human ATII cells against CS-induced DNA damage and/or decrease injury. We analyzed oxidative stress by 4-hydroxynonenal expression, reactive oxygen species (ROS) generation by Amplex Red Hydrogen Peroxide Assay, Nrf2, heme oxygenase 1, p53 and P53-binding protein 1 (53BP1) expression by immonoblotting, Nrf2 nuclear translocation, Nrf2 and p53 DNA-binding activities, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cytokine production by ELISA. We found that ATII cells isolated from Nrf2−/− mice are more susceptible to CS-induced oxidative DNA damage mediated by p53/53BP1 both in vivo and in vitro compared with wild-type mice. Therefore, Nrf2 activation is a key factor to protect ATII cells against injury by CS. Moreover, trolox abolished human ATII cell injury and decreased DNA damage induced by CS in vitro. Furthermore, we found higher inflammation and p53 mRNA expression by RT-PCR in ATII cells isolated from smoker lung donors in comparison with non-smokers ex vivo. Our results indicate that the Nrf2 and p53 cross talk in ATII cells affect the susceptibility of these cells to injury by CS. Trolox can protect against oxidative stress, genotoxicity and inflammation induced by CS through ROS scavenging mechanism, and serve as a potential antioxidant prevention strategy against oxidative injury of ATII cells in CS-related lung diseases.
PMCID: PMC3668634  PMID: 23559007
alveolar type II cells; cigarette smoke; trolox; Nrf2; lung
12.  Genetic or Pharmacologic Amplification of Nrf2 Signaling Inhibits Acute Inflammatory Liver Injury in Mice 
Oxidative stress-mediated destruction of normal parenchymal cells during hepatic inflammatory responses contributes to the pathogenesis of immune-mediated hepatitis and is implicated in the progression of acute inflammatory liver injury to chronic inflammatory liver disease. The transcription factor NF-E2-related factor 2 (Nrf2) regulates the expression of a battery of antioxidative enzymes and Nrf2 signaling can be activated by small-molecule drugs that disrupt Keap1-mediated repression of Nrf2 signaling. Therefore, genetic and pharmacologic approaches were used to activate Nrf2 signaling to assess protection against inflammatory liver injury. Profound increases in ind of cell death were observed in both Nrf2 wild-type (Nrf2-WT) mice and Nrf2-disrupted (Nrf2-KO) mice 24-hr following intravenous injection of concanavalin A (12.5 mg/kg, ConA), a model for T cell-mediated acute inflammatory liver injury. However, hepatocyte-specific conditional Keap1 null (Alb-Cre:Keap1flox/−, cKeap1-KO) mice with constitutively enhanced expression of Nrf2-regulated antioxidative genes as well as Nrf2-WT mice but not Nrf2-KO mice pretreated with three daily doses of a triterpenoid that potently activates Nrf2 (30 µmole/kg, CDDO-Im) were highly resistant to ConA-mediated inflammatory liver injury. CDDO-Im pretreatment of both Nrf2-WT and Nrf2-KO mice resulted in equivalent suppression of serum pro-inflammatory soluble proteins suggesting that the hepatoprotection afforded by CDDO-Im pretreatment of Nrf2-WT mice but not Nrf2-KO mice was not due to suppression of systemic pro-inflammatory signaling, but instead was due to activation of Nrf2 signaling in the liver. Enhanced hepatic expression of Nrf2-regulated antioxidative genes inhibited inflammation-mediated oxidative stress, thereby preventing hepatocyte necrosis. Attenuation of hepatocyte death in cKeap1-KO mice and CDDO-Im pretreated Nrf2-WT mice resulted in decreased late-phase pro-inflammatory gene expression in the liver thereby diminishing the sustained influx of inflammatory cells initially stimulated by the ConA challenge. Taken together, these results clearly illustrate that targeted cytoprotection of hepatocytes through Nrf2 signaling during inflammation prevents the amplification of inflammatory responses in the liver.
PMCID: PMC2435415  PMID: 18417483
Liver inflammation; Nrf2; Keap1; antioxidative enzymes; cytoprotection; triterpenoid
13.  Distinct Cysteine Residues in Keap1 Are Required for Keap1-Dependent Ubiquitination of Nrf2 and for Stabilization of Nrf2 by Chemopreventive Agents and Oxidative Stress 
Molecular and Cellular Biology  2003;23(22):8137-8151.
A common feature of diverse chemopreventive agents is the ability to activate expression of a genetic program that protects cells from reactive chemical species that, if left unchecked, would cause mutagenic DNA damage. The bZIP transcription factor Nrf2 has emerged as a key regulator of this cancer-preventive genetic program. Nrf2 is normally sequestered in the cytoplasm by a protein known as Keap1. Chemopreventive agents allow Nrf2 to escape from Keap1-mediated repression, although the molecular mechanism(s) responsible for activation of Nrf2 is not understood. In this report, we demonstrate that Keap1 does not passively sequester Nrf2 in the cytoplasm but actively targets Nrf2 for ubiquitination and degradation by the proteosome under basal culture conditions. We have identified two critical cysteine residues in Keap1, C273 and C288, that are required for Keap1-dependent ubiquitination of Nrf2. Both sulforaphane, a chemopreventive isothiocyanate, and oxidative stress enable Nrf2 to escape Keap1-dependent degradation, leading to stabilization of Nrf2, increased nuclear localization of Nrf2, and activation of Nrf2-dependent cancer-protective genes. We have identified a third cysteine residue in Keap1, C151, that is uniquely required for inhibition of Keap1-dependent degradation of Nrf2 by sulforaphane and oxidative stress. This cysteine residue is also required for a novel posttranslational modification to Keap1 that is induced by oxidative stress. We propose that Keap1 is a component of a novel E3 ubiquitin ligase complex that is specifically targeted for inhibition by both chemopreventive agents and oxidative stress.
PMCID: PMC262403  PMID: 14585973
14.  Gene expression profiling following NRF2 and KEAP1 siRNA knockdown in human lung fibroblasts identifies CCL11/Eotaxin-1 as a novel NRF2 regulated gene 
Respiratory Research  2012;13(1):92.
Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system.
Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools.
An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts.
These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.
PMCID: PMC3546844  PMID: 23061798
Asthma; NRF2; KEAP1; Oxidative stress; Eotaxin regulation; Microarray profiling
15.  KPNA6 (Importin α7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿  
Molecular and Cellular Biology  2011;31(9):1800-1811.
The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.
PMCID: PMC3133232  PMID: 21383067
16.  N-Acetylcysteine Protects Murine Alveolar Type II Cells from Cigarette Smoke Injury in a Nuclear Erythroid 2–Related Factor–2–Independent Manner 
Emphysema is caused by the cigarette smoke (CS)–induced destruction of alveolar wall septa, and CS is the main risk factor for chronic obstructive pulmonary disease (COPD). To study the mechanisms of response to this insult, we focused on oxidant-induced lung injury and the potential role of nuclear erythroid 2–related factor–2 (Nrf2), which is a key regulator of the antioxidant defense system. We studied the protective role of N-acetylcysteine (NAC) against the injury of alveolar type II (ATII) cells induced by CS in vivo and in vitro. ATII cells were isolated and purified using magnetic MicroBeads (Miltenyi Biotec, Auburn, CA) from Nrf2−/− mice and wild-type mice. We analyzed pulmonary injury, inflammation, glutathione (GSH) concentrations, the expression of glutathione cysteine ligase catalytic subunit mRNA, glutathione cysteine ligase modifier subunit mRNA, and glutathione reductase mRNA, and Nrf2, heme oxygenase–1, and nicotinamide adenine dinucleotide phosphate–reduced:quinone oxireductase levels by Western blotting, TUNEL assay, and immunocytofluorescence for 4-hydroxynonenal as a marker of oxidative stress. We found that CS induced greater injury in ATII cells obtained from Nrf2−/− mice than from wild-type mice. Furthermore, NAC attenuated the injuries by CS in ATII cells obtained from wild-type mice both in vivo and in vitro. Moreover, NAC decreased the injury of ATII cells obtained from Nrf2−/− mice. Our results suggest that Nrf2–GSH signaling is important for the protective activity of NAC. In addition, in ATII cells deficient in Nrf2, this compound can provide partial protection through its reactive oxygen species–scavenging activities. Targeting the antioxidant system regulated by Nrf2 may provide an effective strategy against lung injury in COPD.
PMCID: PMC3707381  PMID: 23492188
murine alveolar type II cells; lung; Nrf2; NAC; cigarette smoke
17.  Regulatory Nexus of Synthesis and Degradation Deciphers Cellular Nrf2 Expression Levels 
Molecular and Cellular Biology  2013;33(12):2402-2412.
Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine.
PMCID: PMC3700104  PMID: 23572560
18.  Nrf2 deficiency impairs the barrier function of mouse esophageal epithelium 
Gut  2013;63(5):711-719.
As a major cellular defense mechanism, the Nrf2/Keap1 pathway regulates expression of genes involved in detoxification and stress response. Our previous study revealed activation of the Nrf2/Keap1 pathway at the maturation phase during mouse esophageal development, suggesting a potential function in epithelial defense. Here we hypothesize that Nrf2 is involved in the barrier function of esophageal epithelium, and plays a protective role against gastroesophageal reflux disease (GERD).
Human esophageal biopsy samples, mouse surgical models and Nrf2-/- mice were used to assess the role of the Nrf2/Keap1 pathway in esophageal mucosal barrier function. Trans-epithelial electrical resistance (TEER) was measured with mini-Ussing chambers. Hematoxylin and eosin (HE) staining and transmission electron microscopy were used to examine cell morphology, while gene microarray, immunohistochemistry, Western blotting and ChIP analysis were used to assess the expression of pathway genes.
Nrf2 was expressed in normal esophageal epithelium and activated in GERD of both humans and mice. Nrf2 deficiency and gastroesophageal reflux in mice, either alone or in combination, reduced TEER and increased intercellular space diameter in esophageal epithelium. Nrf2 target genes and gene sets associated with oxidoreductase activity, mitochondrial biogenesis and energy production were down-regulated in the esophageal epithelium of Nrf2-/- mice. Consistent with the antioxidative function of Nrf2, a DNA oxidative damage marker (8OHdG) dramatically increased in esophageal epithelial cells of Nrf2-/- mice compared with those of wild-type mice. Interestingly, ATP biogenesis, Cox IV (a mitochondrial protein) and Claudin-4 (Cldn4) expression were down-regulated in the esophageal epithelium of Nrf2-/- mice, suggesting that energy-dependent tight junction integrity was subject to Nrf2 regulation. ChIP analysis confirmed the binding of Nrf2 to Cldn4 promoter.
Nrf2 deficiency impairs esophageal barrier function through disrupting energy-dependent tight junction. Elucidating the role of this pathway in GERD has potential implications for the pathogenesis and therapy of the disease.
PMCID: PMC3883925  PMID: 23676441
Nrf2; esophagus; TEER; GERD
19.  Genetic and Pharmacologic Evidence Links Oxidative Stress to Ventilator-induced Lung Injury in Mice 
Rationale: Mechanical ventilation (MV) is an indispensable therapy for critically ill patients with acute lung injury and the adult respiratory distress syndrome. However, the mechanisms by which conventional MV induces lung injury remain unclear.
Objectives: We hypothesized that disruption of the gene encoding Nrf2, a transcription factor that regulates the induction of several antioxidant enzymes, enhances susceptibility to ventilator-induced lung injury (VILI) and that antioxidant supplementation attenuates this effect.
Methods: To test our hypothesis and to examine the relevance of oxidative stress in VILI, we assessed lung injury and inflammatory responses in Nrf2-deficient (Nrf2−/−) mice and wild-type (Nrf2+/+) mice after an acute (2-h) injurious model of MV with or without administration of antioxidant.
Measurements and Main Results: Nrf2−/− mice displayed greater levels of lung alveolar and vascular permeability and inflammatory responses to MV as compared with Nrf2+/+ mice. Nrf2 deficiency enhances the levels of several proinflammatory cytokines implicated in the pathogenesis of VILI. We found diminished levels of critical antioxidant enzymes and redox imbalance by MV in the lungs of Nrf2−/− mice; however, antioxidant supplementation to Nrf2−/− mice remarkably attenuated VILI. When subjected to a clinically relevant prolong period of MV, Nrf2−/− mice displayed greater levels of VILI than Nrf2+/+ mice. Expression profiling revealed lack of induction of several VILI genes, stress response and solute carrier proteins, and phosphatases in Nrf2−/− mice.
Conclusions: Our data demonstrate for the first time a critical role for Nrf2 in VILI, which confers protection against cellular responses induced by MV by modulating oxidative stress.
PMCID: PMC2176106  PMID: 17901416
acute lung injury; antioxidant enzymes; mechanical ventilation; Nrf2; inflammation
Chronic obstructive pulmonary disease (COPD) is associated with high incidence of morbidity and mortality. Oxidative stress is intimately associated with the progression and exacerbation of COPD and therefore targeting oxidative stress with antioxidants or boosting the endogenous levels of antioxidants is likely to have beneficial outcome in the treatment of COPD. Among the various antioxidants tried so far, thiol antioxidants and mucolytic agents, such as glutathione, N-acetyl-L-cysteine, N-acystelyn, erdosteine, fudosteine, and carbocysteine; Nrf2 activators, and dietary polyphenols (curcumin, resveratrol, green tea, and catechins/quercetin) have been reported to increase intracellular thiol status alongwith induction of GSH biosynthesis. Such an elevation in the thiol status in turn leads to detoxification of free radicals and oxidants as well as inhibition of ongoing inflammatory responses. In addition, specific spin traps, such as a-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (ECSOD mimetic), porphyrins (AEOL 10150 and AEOL 10113), and a SOD mimetic M40419 have also been reported to inhibit cigarette smoke-induced inflammatory responses in vivo in the lung. Since a variety of oxidants, free radicals and aldehydes are implicated in the pathogenesis of COPD; it is possible that therapeutic administration of multiple antioxidants and mucolytics will be effective in management of COPD. However, a successful outcome will critically depend upon the choice of antioxidant therapy for a particular clinical phenotype of COPD, whose pathophysiology should be first properly understood. This article will review the various approaches adopted to enhance lung antioxidant levels, antioxidant therapeutic advances and recent past clinical trials of antioxidant compounds in COPD.
PMCID: PMC2744584  PMID: 19124382
COPD; Smokers; Oxidants; Thiol; Glutathione; Antioxidants; Lungs
21.  Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy 
Molecular Cancer  2011;10:37.
Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer.
Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001).
Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.
PMCID: PMC3098205  PMID: 21489257
22.  Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response 
PLoS ONE  2009;4(8):e6588.
The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.
PMCID: PMC2719090  PMID: 19668370
23.  Network Inference Algorithms Elucidate Nrf2 Regulation of Mouse Lung Oxidative Stress 
PLoS Computational Biology  2008;4(8):e1000166.
A variety of cardiovascular, neurological, and neoplastic conditions have been associated with oxidative stress, i.e., conditions under which levels of reactive oxygen species (ROS) are elevated over significant periods. Nuclear factor erythroid 2-related factor (Nrf2) regulates the transcription of several gene products involved in the protective response to oxidative stress. The transcriptional regulatory and signaling relationships linking gene products involved in the response to oxidative stress are, currently, only partially resolved. Microarray data constitute RNA abundance measures representing gene expression patterns. In some cases, these patterns can identify the molecular interactions of gene products. They can be, in effect, proxies for protein–protein and protein–DNA interactions. Traditional techniques used for clustering coregulated genes on high-throughput gene arrays are rarely capable of distinguishing between direct transcriptional regulatory interactions and indirect ones. In this study, newly developed information-theoretic algorithms that employ the concept of mutual information were used: the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), and Context Likelihood of Relatedness (CLR). These algorithms captured dependencies in the gene expression profiles of the mouse lung, allowing the regulatory effect of Nrf2 in response to oxidative stress to be determined more precisely. In addition, a characterization of promoter sequences of Nrf2 regulatory targets was conducted using a Support Vector Machine classification algorithm to corroborate ARACNE and CLR predictions. Inferred networks were analyzed, compared, and integrated using the Collective Analysis of Biological Interaction Networks (CABIN) plug-in of Cytoscape. Using the two network inference algorithms and one machine learning algorithm, a number of both previously known and novel targets of Nrf2 transcriptional activation were identified. Genes predicted as novel Nrf2 targets include Atf1, Srxn1, Prnp, Sod2, Als2, Nfkbib, and Ppp1r15b. Furthermore, microarray and quantitative RT-PCR experiments following cigarette-smoke-induced oxidative stress in Nrf2+/+ and Nrf2−/− mouse lung affirmed many of the predictions made. Several new potential feed-forward regulatory loops involving Nrf2, Nqo1, Srxn1, Prdx1, Als2, Atf1, Sod1, and Park7 were predicted. This work shows the promise of network inference algorithms operating on high-throughput gene expression data in identifying transcriptional regulatory and other signaling relationships implicated in mammalian disease.
Author Summary
A variety of conditions including certain cancers and heart diseases, diabetes mellitus, and rheumatoid arthritis have been associated with the generation of high levels of highly reactive molecular species under conditions known as “oxidative stress.” A number of protein molecules have been identified as participants in an elaborate response to oxidative stress. Sustained elevated generation of reactive species can overwhelm this response and lead to disease conditions. In these studies, we make use of data generated from over 250 studies (microarrays) in which messenger RNA levels of the gene precursors of mouse lung proteins have been examined collectively. We have made use of computational approaches to help identify the key regulatory relationships among the proteins that respond to oxidative stress. Nrf2, a protein known as a master regulator of oxidative stress response, was a principal focus of our studies. Among the novel regulatory targets of Nrf2 we identified is Als2, a protein involved in amyotrophic lateral sclerosis (Lou Gehrig's disease). We also identify important candidate three-party regulatory relationships, one of which involves the recently discovered Srxn1, an antioxidant protein that reverses S-glutathionylation, a common posttranslational modification associated with diseases such as Parkinson's disease, diabetes, hyperlipidemia, Friedreich's ataxia, renal cell carcinoma, and HIV/AIDS. These studies demonstrate the utility of network inference algorithms and affirm that Nrf2 has a direct regulatory role over the expression of other genes responding to oxidative stress.
PMCID: PMC2516606  PMID: 18769717
24.  TGFβ1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts 
Chronic alcohol ingestion induces the expression of transforming growth factor beta-1(TGFβ1), inhibits nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated activation of the antioxidant response element (ARE), depletes alveolar glutathione pools, and potentiates acute lung injury. In this study, we examined the mechanistic relationship between TGFβ1 and Nrf2-ARE signaling in the experimental alcoholic lung.
Wild-type mice were treated ± alcohol in drinking water for 8 weeks and their lungs were assessed for Nrf2 expression. In parallel, mouse lung fibroblasts were cultured ± alcohol and treated ± sulforaphane (SFP; an activator of Nrf2), ±TGFβ1, ±TGFβ1 neutralizing antibody, and/or ±activin receptor-like kinase 5 inhibitors (to block TGβ1 receptor signaling) and then analyzed for the expression of Nrf2, Kelch-like ECH-associated protein 1 (Keap1) and TGFβ1, Nrf2-ARE activity, and the expression of the Nrf2-ARE-dependent antioxidants glutathione s-transferase theta 2 (GSTT2) and glutamate-cysteine ligase catalytic subunit (GCLC). Finally, silencing RNA (siRNA) of Nrf2 was then performed prior to alcohol exposure and subsequent analysis of TGFβ1 expression.
Alcohol treatment in vivo or in vitro decreased Nrf2 expression in murine whole lung and lung fibroblasts, respectively. In parallel, alcohol exposure in vitro decreased Keap1 gene and protein expression in lung fibroblasts. Furthermore, alcohol exposure increased TGFβ1 expression but decreased Nrf2-ARE activity and expression of the ARE-dependent genes for GSTT2 and GCLC. These effects of alcohol were prevented by treatment with SFP; in contrast, Nrf2 SiRNA expression exacerbated alcohol-induced TGFβ1 expression. Finally, TGFβ1 treatment directly suppressed Nrf2-ARE activity whereas blocking TGFβ1 signaling attenuated alcohol-induced suppression of Nrf2-ARE activity.
Alcohol-induced oxidative stress is mediated by TGFβ1, which suppresses Nrf2-ARE-dependent expression of antioxidant defenses and creates a vicious cycle that feeds back to further increase TGFβ1 expression. These effects of alcohol can be mitigated by activation of Nrf2, suggesting a potential therapy in individuals at risk for lung injury due to alcohol abuse.
PMCID: PMC4244649  PMID: 25421510
Nrf2; Antioxidant Response Element; TGFβ1; Glutathione; Lung Fibroblasts; Alcohol
25.  Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex 
Molecular and Cellular Biology  2004;24(24):10941-10953.
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.
PMCID: PMC533977  PMID: 15572695

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