Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys 588 and Lys 591 of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export.The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a key oxidative stress response modifier that induces transcription of a variety of genes through binding to the antioxidant response element (ARE) 4 in target gene promoters (1-3). Nrf2-dependent activation of ARE-driven gene promoters is generally understood to lead to induction of cytoprotective proteins, which enable cells to combat oxidative insult (2-4). However, overexpression of Nrf2 in cancerous cells may actually be deleterious because it may enable them to sustain growth and become chemo-resistant to various prooxidant chemotherapeutic drugs (5-7).In nonstressed cells, Nrf2 activity is thought to be repressed by Keap1 (Kelch-like ECH-associated protein 1) (8 -10), a cytoskeleton-associated protein that, when complexed with Nrf2, promotes ubiquitin-mediated degradation of Nrf2. In one model for the activation of Nrf2 in stressed cells, electrophileor reactive oxygen species-induced release of Nrf2 is proposed to involve covalent modifications of Keap1 and/or Nrf2 in the cytoplasm. Such modifications include oxidation of key cysteine residues in Keap1 (11), phosphorylation of Nrf2 at Ser 40 by protein kinase C (12, 13), and switching of Cullin-3...

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494 -511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins ␣5 and ␤1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin ␣-␤ heterodimer nuclear import receptor system plays a critical role in the import process.Nuclear factor erythroid 2-related factor 2 (Nrf2), 4 in association with the cytoskeleton-associated Kelch-like protein Keap1, functions as a sensor of oxidative and electrophilic stress in cells (1-4). In non-stressed cells, Nrf2 is transcriptionally inactive because of the repressive effect of Keap1 in the cytoplasm (4 -6). Reactive oxygen species or electrophilic agents induce modifications of this complex that allow Nrf2 to translocate into the nucleus to mediate activation of a variety of genes (2, 5-12). The promoters of such genes contain antioxidant response element(s) (AREs), at which Nrf2, in association with small Maf proteins or other basic region-leucine zipper transcription factors (1, 13-18), interacts to regulate gene transcription. As determined by microarray analyses, such genes include those that code for proteins that function in DNA repair, enzymes that catalyze phase II reactions in drug metabolism, signal transduction proteins, and many others that function in protein trafficking, chaperone system/stress response, and apoptosis (19,20).Electrophile-induced Nrf2 release from the Keap1-Nrf2 complex appears to involve not only modification of specific cysteine residues in Keap1 (7-10) but also switching of Cullin 3-dependent ubiquitination from Nrf2 to Keap1, leading to the de...

We undertook a case-control study to investigate the association between chemicals in maternal drinking water consumed during pregnancy and congenital heart disease in the offspring. Two hundred and seventy affected children and 665 healthy children were enrolled in the study. Information on contaminant levels in maternal drinking water was available from records of routine water analysis of samples taken from public taps in the communities where the mothers resided during pregnancy. Mothers provided information during a telephone interview on their health, pregnancy management, and demographic characteristics. Nine inorganic metals were analysed for detection of an association with congenital heart disease. The chemical exposures of particular interest were arsenic, lead, mercury and selenium. None of the chemicals was associated materially with an increase in the frequency of congenital heart disease overall. Arsenic exposure at any detectable level was associated with a threefold increase in occurrence of coarctation of the aorta (prevalence odds ratio = 3.4, 95% confidence interval = 1.3-8.9). Detectable traces of selenium in drinking water were associated with a lower frequency of any congenital heart disease than was observed among children exposed to drinking water not containing detectable levels of selenium (prevalence odds ratio = 0.62, 95% confidence limits = 0.40-0.97). A dose-response effect was observed over four levels of selenium exposure. Non-differential errors in the measurement and classification of exposure to contaminants routinely monitored in drinking water could account for lack of positive findings. In addition, most of the contaminant levels were below the maximum levels set by the Environmental Protection Agency, so that lack of evidence of effect may have been due to the low exposure levels in this population.

Given that condom use is not directly under a woman's control, the sexual division of power may play an important role in sexual behavior among pregnant women. We assessed the influence of factors related to the theory of gender and power (e.g., relationship power, abuse history, and sexual communication) on sexual behavior (e.g., two or more partners in the year prior to pregnancy, condom use, condom-use intentions, and STI diagnosis) among 196 pregnant women recruited from five community dispensaries in rural Haiti. Results showed that gender and power factors significantly related to sexual behavior. Gender and power factors were most significant for condom use and intention to use condoms, accounting for 18 and 25% of the variance above and beyond HIV knowledge and demographic covariates, respectively. These results suggest the need to create prevention interventions that restore power imbalances, provide support for women suffering abuse, and strengthen communication skills.