Ifeanyi J. Arinze

Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization

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 Lys588 and Lys591 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.

Multiple Nuclear Localization Signals Function in the Nuclear Import of the Transcription Factor Nrf2

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 α5 and β1 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 α-β heterodimer nuclear import receptor system plays a critical role in the import process.

Regulation of Glucose-6-phosphatase Gene Expression in Cultured Hepatocytes and H4IIE Cells by Short-chain Fatty Acids ROLE OF HEPATIC NUCLEAR FACTOR-4α

Mechanisms underlying dietary nutrient regulation of glucose-6-phosphatase (Glc-6-Pase) gene expression are not well understood. Here we investigated the effects of short-chain fatty acids on the expression of this gene in primary cultures of rat hepatocytes and H4IIE hepatoma cells. Propionate, butyrate, valerate, and caproate induced severalfold increases in the expression of Glc-6-Pase mRNA. In reporter gene assays, propionate, valerate, caproate, and also octanoate increased Glc-6-Pase promoter activity by 6-16-fold. Butyrate, by itself, had little or no effect on promoter activity, but it induced a robust increase (45-fold) in promoter activity in cells co-transfected with a plasmid expressing the transcription factor HNF-4α (α isoforms of hepatic nuclear factor 4). HNF-4α also enhanced promoter activity induced by other short-chain fatty acids. A dominant negative form of HNF-4α abrogated the fatty acid-induced promoter activity, a finding that accentuates a role for HNF-4α in the transcription process studied here. In cells transfected with HNF-4α, short-chain fatty acids and trichostatin A, an inhibitor of histone deacetylase, synergistically enhanced promoter activity, suggesting that hyperacetylation of histones is an important component of the transactivation of the Glc-6-Pase gene promoter by HNF-4α. Region-751/-466 of this promoter contains seven putative HNF-4α-binding motifs. Binding of HNF-4α to this region was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, indicating that HNF-4α is recruited to the Glc-6-Pase gene promoter during short-chain fatty acid-induced transcription from this promoter.

Transcriptional Activation of the Human Gαi2 Gene Promoter through Nuclear Factor-κB and Antioxidant Response Elements

Very little is known regarding molecular mechanism(s) underlying transcriptional regulation of any G-protein gene despite the importance of G-protein expression in modulating cellular processes. Here we show that phorbol myristate acetate (PMA) and tert-butylhydroquinone (tBHQ), which induce oxidative stress in cells, up-regulate transcription of Gαi2 in K562 cells. Redox-sensing chemicals abrogated this transcriptional effect. A dominant negative I-κB double mutant (S32A/S36A) suppressed PMA-induced transcription by 54–62%, suggesting involvement of nuclear factor-κB (NF-κB). SN50, a cell-permeable peptide that inhibits nuclear import of stress-responsive transcription factors (such as NF-κB), inhibited PMA- and tBHQ-induced transcription. Deletion of an NF-κB-binding motif that maps at +10/+19 in the promoter resulted in 55–60% suppression of PMA-induced transcription, and 81% suppression of tBHQ-induced transcription. Mutation of an antioxidant response element (ARE) that maps at –84/–76 in the promoter resulted in 51 and 86% decrease in PMA- and tBHQ-induced transcription, respectively. In electrophoretic mobility shift assays, this element formed complexes with the transcription factors NF-E2p45 and Nrf2 that are prototypic for binding to the ARE, as well as with c-Fos, which can also interact with the ARE. Chromatin immunoprecipitation analysis demonstrated recruitment of these transcription factors to the promoter. Exogenously transfected Nrf2 transactivated the Gαi2 gene promoter; the cytoskeleton-associated protein, Keap1, abrogated this effect. Taken together, the present studies reveal that transcription factors that bind NF-κB and/or antioxidant response elements play an activating role in the transcription of the human Gαi2 gene.

On the development of phosphoenolpyruvate carboxykinase and gluconeogenesis in guinea pig liver

Summary The activity of cytosol phosphoenolpyruvate carboxykinase is negligible in near term fetal guinea pig liver but the mitochondrial form of the enzyme is present in amounts comparable to that found in the adult. If Cesarean delivered newborns are examined 24 hours after delivery, the activity of the mitochondrial enzyme remains unchanged whereas the cytosol activity almost equals that in the mitochondria. Livers taken from near term fetuses and perfused immediately after Cesarean section synthesize glucose from lactate, pyruvate, and glycerol, and from lactate-pyruvate mixtures.

Mitochondrial redox state and the regulation of gluconeogenesis in the isolated, perfused cat liver

Previous studies from this laboratory [l-4] have advanced the hypothesis that the oxidation-reduction state is a major factor regulating the rate of PEP? formation by isolated guinea pig, rabbit and human liver mitochondria. The difference in the regulation of gluconeogenesis in the perfused guinea pig and rabbit liver as compared to rat liver [5] underscores the importance of mitochondrial PEPCK, which makes up SO-60% of the total hepatic activity of this enzyme in many mammalian species [6,7]. In this paper we have tested the influence of redox alterations in the mitochondria on the overall rate of glucogenesis in the perfused liver of the cat, a non-ruminant carnivorous animal in which the intracellular distribution of hepatic PEPCK [7] is similar to that of the human. The results show that, as in guinea pig liver, shifting the mitochondrial oxidation-reduction potential towards increased reduction inhibits gluconeogenesis. The gluconeogenic flux via mitochondrial PEPCK accounts for at least half of the overall rate of glucose production from lactate.

Arkadia (RING Finger Protein 111) Mediates Sumoylation-Dependent Stabilization of Nrf2 Through K48-Linked Ubiquitination

Background/Aims: The transcription factor Nrf2 is a master regulator of the antioxidant defense system, protecting cells from oxidative damage. We previously reported that the SUMO-targeted E3 ubiquitin ligase (STUbL), RING finger protein 4 (RNF4) accelerated the degradation rate of Nrf2 in promyelocytic leukemia-nuclear body (PML-NB)-enriched fractions and decreased Nrf2-mediated gene transcription. The mechanisms that regulate Nrf2 nuclear levels are poorly understood. In this study, we aim to explore the role of the second mammalian STUbL, Arkadia/RNF111 on Nrf2. Methods: Arkadia mediated ubiquitination was detected using co-immunoprecipitation assays in which whole cell lysates were immunoprecipated with anti-Nrf2 antibody and Western blotted with anti-hemagglutinin (HA) antibody or anti-Lys-48 ubiquitin-specific antibody. The half-life of Nrf2 was detected in whole cell lysates and promyelocytic leukemia-nuclear body enriched fractions by cycloheximide-chase. Reporter gene assays were performed using the antioxidant response element (ARE)-containing promoter Heme oxygenase-1 (HO-1). Results: We show that Arkadia/RNF111 is able to ubiquitinate Nrf2 resulting in the stabilization of Nrf2. This stabilization was mediated through Lys-48 ubiquitin chains, contrary to traditionally degradative role of Lys-48 ubiquitination, suggesting that Lys-48 ubiquitination of Nrf2 protects Nrf2 from degradation thereby allowing Nrf2-dependent gene transcription. Conclusion: Collectively, these findings highlight a novel mechanism to positively regulate nuclear Nrf2 levels in response to oxidative stress through Arkadia-mediated K48-linked ubiquitination of Nrf2.

PML-Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2-Nrf2 in the Nucleus

Background/Aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Methods: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. Results: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Conclusion: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells.