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...

Sodium butyrate, an erythroid differentiation inducer and a histone deacetylase inhibitor, increases G␣ i2 levels in differentiating K562 cells. Here we show that sodium butyrate induces G␣ i2 gene transcription via sequences at ؊50/؊36 and ؊92/؊85 in the G␣ i2 gene promoter. Both sequences contain core sequence motif for Sp1 binding; electrophoretic mobility shift as well as supershift assays confirmed binding to Sp1. Transcription from the G␣ i2 gene promoter was also activated by two other histone deacetylase inhibitors, trichostatin A and Helminthsporium carbonium toxin (HC toxin), which also induce erythroblastic differentiation in K562 cells. However, hydroxyurea, a potent erythroid differentiation inducer in these cells, did not activate transcription from this gene promoter, indicating that promoter activation is inducer-specific. Mutations within the Sp1 sites at ؊50/؊36 and ؊92/؊85 in the G␣ i2 gene promoter substantially decreased transcriptional activation by sodium butyrate, trichostatin A, or HC toxin. Transfection with constitutively activated ERKs indicated that this promoter can be activated through the MEK-ERK signal transduction pathway. Inhibition of the MEK-ERK pathway with U0126 or reduction in the expression of endogenous ERK with an antisense oligonucleotide to ERK significantly inhibited sodium butyrate-and HC toxin-induced transcription but had no effect on trichostatin A-induced transcription. Inhibition of the JNK and p38 MAPKs, using selective inhibitors, had no effect on sodium butyrate-induced transcription. In cells in which sodium butyrate induction of promoter activation had been inhibited by various concentrations of U0126, constitutively activated ERK2 reversed this inhibition. These results show that the MEK-ERK signal transduction pathway is important in butyrate signaling, which eventually converges in the cell nucleus.There is compelling evidence that the ␣-subunits of heterotrimeric G-proteins 1 can influence cell differentiation in different ways, depending on the cell type. For example, G␣ s has been shown to suppress dexamethasone-induced differentiation of 3T3-L1 cells, leading to suppression of adipogenesis in these cells (1). G␣ 12 and G␣ 13 have been implicated in the retinoic acid-mediated differentiation of P19 mouse embryonal carcinoma cells (2, 3). During Me 2 SO-induced neutrophilic differentiation of human myeloid HL-60 cells, the expression of G␣ 16 is decreased by 90%, whereas the expression of G␣ i2 is increased by 160% (4, 5); this suggests an association between cell differentiation and these G-protein ␣-subunits. In F9 teratocarcinoma cells, the levels of G␣ i2 decrease as the cells are induced to differentiate (6). Sodium butyrate-induced erythroblastic differentiation of K562 cells requires the presence of G␣ i2 , since pertussis toxin or an antisense oligonucleotide to a portion of the G␣ i2 gene blocks the sodium butyrate-induced effect (7). The expression of genes for some proteins has been reported to be influenced by butyrate (8 -15), but none of...

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Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein ␤ (C/ EBP␤؊/؊ mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP␤؊/؊ mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, ␤-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP␤؊/؊ mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI␣ and 27% increase in RII␣ in the particulate fraction of the livers of C/EBP␤؊/؊ mice relative to wild type mice, with no change in the catalytic subunit (C␣). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP␤؊/؊ mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP␤؊/؊ mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP␤ influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.