Kaimin Chan

An important function of Nrf2 in combating oxidative stress:Detoxification of acetaminophen

Nrf2, a member of the “cap ‘n collar” group of transcription factors, is important for protecting cells against oxidative damage. We investigated its role in the detoxification of acetaminophen [N-acetyl-p-aminophenol (APAP)]-induced hepatotoxicity. When Nrf2 knockout (Nrf2−/−) and wild-type mice were given APAP by i.p. injection, the Nrf2−/− mice were highly susceptible to APAP treatment. With doses of APAP that were tolerated by wild-type mice, the Nrf2−/− mice died of liver failure. When hepatic glutathione was depleted after a dose of 400 mg/kg of APAP, the wild-type mice were able to compensate and regain the normal glutathione level. In contrast, the glutathione level in the Nrf2−/− mice was not compensated and remained low. This was because of the decrease in the gene expression of gcsH and gcsL as well as gss in the livers of the Nrf2−/− mice. In addition, the expression of ugt1a6 and gstpi that detoxify APAP by conjugation was also decreased. This increased susceptibility of the Nrf2−/− mice to APAP, because of an impaired capacity to replenish their glutathione stores, compounded with a decreased detoxification capability, highlights the importance of Nrf2 in the regulation of glutathione synthesis and cellular detoxification processes.

Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis

The antioxidant responsive element (ARE) mediates transcriptional regulation of phase II detoxification enzymes and antioxidant proteins such as NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferases, and glutamate-cysteine ligase. In this study, we demonstrate that NF-E2-related factor-2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes and identify Nrf2-dependent genes by oligonucleotide microarray analysis using primary cortical astrocytes from Nrf2+/+ and Nrf2−/− mice. Nrf2−/−astrocytes had decreased basal NQO1 activity and no induction bytert-butylhydroquinone compared with Nrf2+/+ astrocytes. Similarly, both basal and induced levels of human NQO1-ARE-luciferase expression in Nrf2−/− astrocytes were significantly lower than in Nrf2+/+ astrocytes. Furthermore, human NQO1-ARE-luciferase expression in Nrf2−/−astrocytes was restored by overexpression of Nrf2, whereas ARE activation in Nrf2+/+ astrocytes was completely blocked by dominant-negative Nrf2. In addition, we observed that Nrf2-dependent genes protected primary astrocytes from H2O2- or platelet-activating factor-induced apoptosis. In support of these observations, we identified Nrf2-dependent genes encoding detoxification enzymes, glutathione-related proteins, antioxidant proteins, NADPH-producing enzymes, and anti-inflammatory genes using oligonucleotide microarrays. Proteins within these functional categories are vital to the maintenance and responsiveness of a cell defense system, suggesting that an orchestrated change in gene expression via Nrf2 and the ARE gives a synergistic protective effect against oxidative stress.

Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound

ABSTRACT Keratinocyte growth factor (KGF) is a potent mitogen for epithelial cells, and it promotes survival of these cells under stress conditions. In a search for KGF-regulated genes in keratinocytes, we identified the gene encoding the transcription factor NF-E2-related factor 2 (Nrf2). Nrf2 is a key player in the cellular stress response. This might be of particular importance during wound healing, where large amounts of reactive oxygen species are produced as a defense against invading bacteria. Therefore, we studied the wound repair process in Nrf2 knockout mice. Interestingly, the expression of various key players involved in wound healing was significantly reduced in early wounds of the Nrf2 knockout animals, and the late phase of repair was characterized by prolonged inflammation. However, these differences in gene expression were not reflected by obvious histological abnormalities. The normal healing rate appears to be at least partially due to an up-regulation of the related transcription factor Nrf3, which was also identified as a target of KGF and which was coexpressed with Nrf2 in the healing skin wound. Taken together, our results reveal novel roles of the KGF-regulated transcription factors Nrf2 and possibly Nrf3 in the control of gene expression and inflammation during cutaneous wound repair.

Role of oxidative stress in rheumatoid arthritis: insights from the Nrf2-knockout mice

Objectives Increasing evidence suggests that oxidative stress may play a key role in joint destruction due to rheumatoid arthritis (RA). The aim of this study was to elucidate the role of nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that maintains the cellular defence against oxidative stress, in RA. Methods The activation status of Nrf2 was assessed in synovial tissue from patients with RA using immunohistochemistry. Antibody-induced arthritis (AIA) was induced in Nrf2-knockout and Nrf2-wild-type control mice. The severity of cartilage destruction was evaluated using a damage score. The extent of oxidative stress, the activation state of Nrf2 and the expression level of Nrf2 target genes were analysed by immunhistological staining. The expression of vascular endothelial growth factor (VEGF)-A was examined on mRNA and protein using the Luminex technique. A Xenogen imaging system was used to measure Nrf2 activity in an antioxidant response element-luciferase transgenic mouse during AIA. Results Nrf2 was activated in the joints of arthritic mice and of patients with RA. Nrf2-knockout mice had more severe cartilage injuries and more oxidative damage, and the expression of Nrf2 target genes was enhanced in Nrf2-wild-type but not in knockout mice during AIA. Both VEGF-A mRNA and protein expression was upregulated in Nrf2-knockout mice during AIA. An unexpected finding was the number of spontaneously fractured bones in Nrf2-knockout mice with AIA. Conclusion These results provide strong evidence that oxidative stress is significantly involved in cartilage degradation in experimental arthritis, and indicate that the presence of a functional Nrf2 gene is a major requirement for limiting cartilage destruction.

hMAF, a Small Human Transcription Factor That Heterodimerizes Specifically with Nrf1 and Nrf2

A 1.6-kilobase pair full-length cDNA encoding a transcription factor homologous to the Maf family of proteins was isolated by screening a K562 cDNA library with the NFE2 tandem repeat probe derived from the globin locus control region. The protein, which was designated hMAF, contains a basic DNA binding domain and an extended leucine zipper but lacks any recognizable activation domain. Expressed in vitro, the hMAF protein is able to homodimerize in solution and band-shift the NFE2 tandem repeat probe. In addition to homodimers, hMAF can also form high affinity heterodimers with two members of the NFE2/CNC-bZip family (Nrf1 and Nrf2) but not with a third family member, p45-NFE2. Although hMAF/hMAF homodimers and hMAF/Nrf1 and hMAF/Nrf2 heterodimers bind to the same NFE2 site, they exert functionally opposite effects on the activity of a linked γ-globin gene. In fact, whereas all hMAF/CNC-bZip heterodimers stimulate the activity of a γ-promoter reporter construct in K562 cells, the association into homodimers that is induced by overexpressing hMAF inhibits the activity of the same construct. Thus variations in the expression of hMAF may account for the modulation in the activity of the genes that bear NFE2 recognition sites.

OP0117 Safety and immune response of a live attenuated herpes zoster vaccine in patients with systemic lupus erythematosus: a randomised placebo-controlled trial

Objectives To evaluate the safety and immune response of a live attenuated herpes zoster (HZ) vaccine in patients with systemic lupus erythematosus (SLE) in a randomised placebo-controlled trial. Methods Adult patients who fulfilled ≥4 ACR criteria for SLE and had a SLEDAI score <6 with stable immunosuppressive treatment for 6 m were recruited. Exclusion criteria were: active infection; lymphocyte <500/mm2; reduced serum IgG/A/M level; serum creatinine >200 umol/L; a history of cancer; and high-dose immunosuppressive treatment (prednisone >15 mg/day, azathioprine >100 mg/day, MMF >500 mg/day, cyclosporin >100 mg/day, tacrolimus >3 mg/day, CYC and biologics). Participants were randomly assigned to receive HZ vaccine (Zostavax) or placebo (same volume of normal saline) given subcutaneously. Anti-VZV IgG reactivity (baseline and 6 weeks post-vaccination) was measured by an ELISA coated with inactivated varicella-zoster viral antigen (Vidas VZV IgG, bioMerieux, Marcy I’Etoile, France). Cell-mediated response to HZ was assessed by a specific VZV-stimulated IFNγ enzyme linked immunospot (ELISPOT) assay. Disease activity of SLE was assessed by the SLEDAI and PGA. Adverse events (AEs) and immune responses to HZ of the two groups were compared. Results 90 SLE patients were recruited (age 45.6±14.1 years; 93% women): 45 assigned to HZ vaccine and 45 to placebo. All participants had a history of HZ/chickenpox infection. The baseline clinical profile of the two groups of patients was similar. Only 3 patients in the vaccine and 1 patient in the placebo group had mild SLE activity (all mild thrombocytopenia). Baseline SLEDAI and PGA scores of the two groups were not significantly different (1.58±1.8 vs 1.64±1.7; p=0.86 and 0.21±0.18 vs 0.27±0.25; p=0.18, respectively). The proportion of patients receiving various immunosuppressive agents, lymphocyte count, serum creatinine, IgG/A/M levels were also similar in the two groups. The mean baseline VZV IgG index value was 3.28±1.19 and 3.45±1.07 in the vaccine and control group of patients, respectively (p=0.48). The paired VZV IgG titer at week 6 was significantly higher in the vaccine than control group, even after adjustment for baseline value (4.16±1.26 vs 3.32±1.01; p<0.001), lymphocyte count, Ig levels, SLEDAI, and other clinical variables. The% increase in VZV IgG antibody was significantly higher in the vaccinated than control patients (+59.8% vs −2.1%; p=0.01), indicating an effect of vaccine. 21 and 6 AEs were reported in the vaccinated and control patients, respectively, but none were serious. Significantly more vaccinated patients reported pain and erythema at the injection site than controls (31% vs 7%; p<0.01) (mild in all and subsided in a few days). Other AEs more commonly reported with vaccination included dizziness (2%), arthralgia (2%) and subjective fever (4%). Two vaccinated patients (4.4%) had mild flare of skin/joint disease, and one control patients (2.2%) had mild increase in proteinuria between week 0 and 6. None of the patients had clinical HZ infection post-vaccination. Conclusions In patients with stable SLE who were not receiving intensive immunosuppression, the live attenuated HZ vaccine was well tolerated and provoked an expected antibody response. Study of the cell-mediated response to HZ post-vaccination is in progress. Disclosure of Interest None declared