SUMO-Modification of Human Nrf2 at K110 and K533 Regulates Its Nucleocytoplasmic Localization, Stability and Transcriptional Activity.

BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox homeostasis. Post-translational modifications (PTMs) that mediate activation of Nrf2, in the cytosol and its release from Keap1, have been extensively studied but PTMs that impact its biology after activation are beginning to emerge. In this regard, PTMs like acetylation, phosphorylation, ubiquitination and sumoylation contribute towards the Nrf2 subcellular localization, and its transactivation function. We previously demonstrated that Nrf2 traffics to the promyelocytic leukemia-nuclear bodies (PML-NB), where it is a target for modification by small ubiquitin-like modifier (SUMO) proteins (sumoylation), but the site(s) for SUMO conjugation have not been determined. In this study, we aim to identify SUMO-2 conjugation site(s) and explore the impact, sumoylation of the site(s) have on Nrf2 stability, nuclear localization and transcriptional activation of its target gene expression upon oxidative stress. METHODS: The putative SUMO-binding sites in Nrf2 for human isoform1 (NP_006155.2) and mouse homolog (NP_035032.1) were identified using a computer-based SUMO-predictive software (SUMOplot™). Site-directed mutagenesis, immunoblot analysis, and ARE-mediated reporter gene assays were used to assess the impact of sumoylation on these site(s) in vitro. Effect of mutation of these sumoylation sites of Nrf2 on expression of Heme Oxygenase1 (HO-1) was determined in HEK293T cell. RESULTS: Eight putative sumoylation sites were identified by SUMOplot™ analysis. Out of the eight predicted sites only one 532LKDE535 of human (h) and its homologous 524LKDE527 of mouse (m) Nrf2, exactly matches the SUMO-binding consensus motif. The other high probability SUMO-acceptor site identified was residue K110, in the motifs 109PKSD112 and 109PKQD112 of human and mouse Nrf2, respectively. Mutational analysis of putative sumoylation sites (human (h)/mouse (m)K110, hK533 and mK525) showed that these residues are needed for SUMO-2 conjugation, nuclear localization and ARE driven transcription of reporter genes and the endogenous HO-1 expression by Nrf2. These residues also stabilized Nrf2, as evident from shorter half-lives of the mutant protein compared to wild-type Nrf2. CONCLUSION: Our findings indicate that SUMO-2mediated sumoylation of K110 and K533 in human Nrf2 regulates in part its transcriptional activity by enhancing its stabilization and nuclear localization.

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.

Trafficking of the transcription factor Nrf2 to promyelocytic leukemia-nuclear bodies: implications for degradation of NRF2 in the nucleus.

Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1.

Dual role of C/EBPα as an activator and repressor of Gαi2 gene transcription.

The G-protein Gαi2 mediates signaling in a variety of processes. Induced expression of Gαi2 by butyrate and various transcription factors has been established, but transcriptional suppression has not previously been explored. Using HepG2 and K562 cells in culture, we show here that whereas both C/EBPα and C/EBPß induced transcription from the Gαi2 gene promoter, C/EBPα, but not C/EBPß, inhibited butyrate-induced Gαi2 expression. Because the transcriptional effect of butyrate on this gene promoter is largely mediated by the transcription factor Sp1, we investigated whether C/EBPα influenced Sp1-induced Gαi2 gene transcription. Binding of C/EBPα to a C/EBP response element in Gαi2 gene promoter inhibited Sp1-induced promoter activity. ChIP analysis showed decreased butyrate-induced recruitment of Sp1 to the Gαi2 gene promoter in response to C/EBPα treatment. Incubating cells with acetate or transfecting them with expression plasmid for either the acetyltransferase p300 or CREB-binding protein (CBP) reversed the antagonistic effect of C/EBPα on Sp1-dependent gene transcription, suggesting that the mechanistic basis for the antagonism is related to the squelching of co-activator acetyltransferase(s) by C/EBPα or the acetylation of Sp1 and/or C/EBPα. This work reveals that C/EBPα plays a dual role as an activator and as a repressor of Gαi2 gene transcription.

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 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.

Metabolic dysregulation in the SHROB rat reflects abnormal expression of transcription factors and enzymes that regulate carbohydrate metabolism.

The Koletsky (SHROB) strain of rats is spontaneously hypertensive and displays insulin resistance, hyperglucagonemia and hypertriglyceridemia but is normoglycemic under fasting conditions. The aim of this study was to unravel the pattern of expression of genes encoding key regulatory enzymes involved in carbohydrate metabolism in the liver and kidney that may be impacted in this strain. We found that SHROB animals have decreased beta-adrenergic receptor density and, consequently, blunted increases in cAMP levels in response to beta-adrenergic agonists. They also have lower levels of hepatic as well as renal phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) mRNA and protein than their lean littermates. Expression of the genes for glycogen phosphorylase and glycogen synthase was also decreased. Hepatocytes from the SHROB animals exhibited glycogen depletion of only 50% compared to 86% by hepatocytes from lean littermates when challenged with either glucagon or forskolin to stimulate adenylyl cyclase. The expression of C/EBPalpha and C/EBPbeta, two key transcription factors that are essential for the coordinated expression of genes involved in glucose homeostasis, was depressed in livers of the SHROB rats, as were levels of HNF-4alpha, PPARalpha and PGC-1alpha. We conclude that overproduction of glucose is prevented in the SHROB rats by decreased expression of the genes for glycogen phosphorylase and the gluconeogenic enzymes PEPCK and G6Pase, which may prevent progression to diabetes in this model.

Valproic acid-induced gene expression through production of reactive oxygen species.

Valproic acid (VPA) is a widely used anticonvulsive agent that has profound antiproliferative effects in many cell types, as well as inductive effects on a number of genes. The mechanism of its gene-inducing effect has been reported to involve transcription factors, Sp1 and activator protein-1. Using two well-characterized antioxidant response element (ARE)-driven gene promoters, i.e., mouse heme oxygenase-1 and human NAD(P)H:quinone oxidoreductase 1 genes as tools to monitor the transcriptional response to VPA, we show here that VPA-induced gene transcription was abrogated by antioxidants. With the human Galpha(i2) gene promoter, which was previously used to establish the involvement of Sp1 in the transcriptional action of VPA, we found that VPA-induced gene transcription was also blocked by antioxidants. Mutation of the ARE (5'-TGACtggGC-3') in this promoter abrogated the transcriptional response to VPA. With such mutants, the NADPH oxidase inhibitor, diphenyleneiodonium, had no effect on VPA-induced transcription. In gel mobility shift assays, VPA-induced binding of nuclear proteins to a DNA probe containing the relevant ARE sequence in the Galpha(i2) gene promoter was decreased in nuclear extracts from cells pretreated with antioxidants. Chromatin immunoprecipitation assays showed that the prototype redox-sensitive transcription factors, Nrf2, small Maf protein(s), and c-Fos, were recruited to this promoter in VPA-treated cells. Overall, this study reveals that the mechanism of the transcriptional response to VPA includes VPA-induced production of reactive oxygen species which induce the activation of redox-sensitive transcription factors that interact with the ARE.

Facilitating understanding of the purine nucleotide cycle and the one-carbon pool: Part I: The purine nucleotide cycle.

Some metabolic processes are readily understood because they are circumscribed in metabolic pathways that have clearly identifiable beginning points, end products, and other features. Other metabolic pathways that do not appear to be straightforward pose difficulties for students. One such metabolic process, the purine nucleotide cycle, is discussed here.

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 alpha5 and beta1 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 alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.

Several transcription factors are recruited to the glucose-6-phosphatase gene promoter in response to palmitate in rat hepatocytes and H4IIE cells.

Fatty acids and glucose are strong modulators of the expression of glucose-6-phosphatase (Glc-6-Pase), an enzyme that plays a key role in glucose homeostasis. PUFA inhibit, whereas SFA and monounsaturated fatty acids induce the expression of the Glc-6-Pase gene. Palmitate and oleate are the most abundant fatty acid species in circulation during food deprivation in mammals. Although dietary fats have been shown to modulate the expression of genes involved in both lipid and carbohydrate metabolism in liver, little is known regarding the molecular mechanism of transcriptional response of the Glc-6-Pase gene to long-chain fatty acids. Using H4IIE hepatoma cells and hepatocytes from adult rats, we investigated the mechanism of the induction of this gene by palmitate and oleate. Both of these fatty acids stimulated Glc-6-Pase gene transcription but did not affect the stability of its mRNA. In transient transfection assays, transcription from the Glc-6-Pase gene promoter was markedly enhanced by both palmitate and oleate but not by arachidonate. Chromatin immunoprecipitation analysis was used to show that palmitate induced the recruitment of an array of transcription factors viz hepatic nuclear factor(NF)-4alpha, CAAT/enhancer binding proteinbeta, PPARalpha, chicken ovalbumin upstream promoter transcription factor (COUP-TF), cAMP regulatory element binding protein, and NF-kappaB to this gene promoter. Although it is presently unclear how these various transcription factors interact at this promoter, the data are consistent with the view that multiple regulatory elements in the Glc-6-Pase gene promoter are responsible for the modulation of gene transcription by fatty acids.

Transcriptional activation of the human Galphai2 gene promoter through nuclear factor-kappaB 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 Galpha(i2) in K562 cells. Redox-sensing chemicals abrogated this transcriptional effect. A dominant negative I-kappaB double mutant (S32A/S36A) suppressed PMA-induced transcription by 54-62%, suggesting involvement of nuclear factor-kappaB (NF-kappaB). SN50, a cell-permeable peptide that inhibits nuclear import of stress-responsive transcription factors (such as NF-kappaB), inhibited PMA- and tBHQ-induced transcription. Deletion of an NF-kappaB-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 Galpha(i2) gene promoter; the cytoskeleton-associated protein, Keap1, abrogated this effect. Taken together, the present studies reveal that transcription factors that bind NF-kappaB and/or antioxidant response elements play an activating role in the transcription of the human Galpha(i2) gene.

Sp family of transcription factors is involved in valproic acid-induced expression of Galphai2.

Valproic acid-induced gene expression has been attributed to the DNA-binding activity of the transcription factor activator protein 1 (AP-1). Using K562 cells, we have studied valproic acid-induced transcription from the human Galpha(i2) gene promoter, which lacks AP-1-binding motifs. We find that valproic acid-induced expression of Galpha(i2) is inhibited by mithramycin A, a compound that interferes with Sp1 binding to GC boxes in DNA. Three Sp1-binding sequences, located at +68/+75, -50/-36, and -92/-85 in the promoter, accounted for about 60% of this transcriptional effect, as judged by transient transfection assays. Electrophoretic mobility shift assays indicated that these sites bind members of the Sp family of transcription factors. Binding to DNA was inhibited by mithramycin A and was greater in nuclear extracts from cells treated with valproic acid than in control cells. Okadaic acid, calyculin A, and fostriecin, which are potent inhibitors of protein phosphatase, suppressed the transcriptional response to valproic acid. This inhibitory effect was not observed when promoter constructs containing mutations in the referenced Sp1-binding sites were used for transfections. In nuclear extracts from cells cultured in the presence of these inhibitors, the binding of Sp1/Sp3 to DNA probes was much less than in control cells. Alkaline phosphatase treatment of nuclear extracts resulted in enhanced binding of Sp proteins to the DNA probes. These results are consistent with the idea that dephosphorylating conditions enhanced Sp binding to the DNA probes as well as Sp-mediated transcription induced by valproic acid. This study demonstrates that the gene expression-inducing effect of valproic acid occurs, in part, through the Sp family of transcription factors.

Regulation of glucose-6-phosphatase gene expression in cultured hepatocytes and H4IIE cells by short-chain fatty acids: role of hepatic nuclear factor-4alpha.

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-4alpha (alpha isoforms of hepatic nuclear factor 4). HNF-4alpha also enhanced promoter activity induced by other short-chain fatty acids. A dominant negative form of HNF-4alpha abrogated the fatty acid-induced promoter activity, a finding that accentuates a role for HNF-4alpha in the transcription process studied here. In cells transfected with HNF-4alpha, 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-4alpha. Region-751/-466 of this promoter contains seven putative HNF-4alpha-binding motifs. Binding of HNF-4alpha to this region was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, indicating that HNF-4alpha is recruited to the Glc-6-Pase gene promoter during short-chain fatty acid-induced transcription from this promoter.