Acetylation-deacetylation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) regulates its transcriptional activity and nucleocytoplasmic localization

Abstract

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.

FIGURE 1.

Effect of CBP on Nrf2-dependent gene transcription. A, Nrf2-dependent transcription from the Gα_i2 gene promoter is enhanced by CBP and inhibited by E1A. K562 cells (1 × 10⁵ cells in 1 ml of medium/well) seeded in 24-well plates for 24 h were co-transfected with 0.2 μg each of empty vector (pCI-Neo) or heterologous Nrf2 (pCI-Nrf2) as inducer and 0.2 μg of pGα_i2(−1214/+115)-luc with or without expression plasmid for CBP (0.2 μg) or E1A (0.2 μg). The total amount of DNA in each well was 0.8 μg; the empty vector (pCMV5) was used to make up the total to this amount, as needed. The cells were harvested 24 h after transfection to measure promoter activity (28, 31, 32). The data are the means ± S.E. of duplicate assays from four separate cell cultures. *, significantly different (p < 0.02) from control (vector only); **, significantly different (p < 0.02) from the Nrf2 effect; ***, significantly different (p < 0.02) from Nrf2 + CBP. B, Nrf2-induced transcription from HO-1-ARE-luc and hNQO1-ARE-luc reporters is enhanced by CBP and inhibited by E1A. The HO-1-ARE-luc contains three copies of ARE (5′-CGGACCTTGACTCAGCAGAAAA-3′) cloned upstream of the mouse HO-1 minimal promoter (−32 to +72 bp) (29). The hNQO1-ARE-luc (human NAD(P)H:quinone oxidoreductase 1-ARE-luc) contains a single copy of ARE, derived from the human NQO1 promoter, placed upstream of a minimal promoter containing a TATA box fused to the luciferase gene (30). All of the plasmids were transfected at 0.2 μg each, and the total amount of DNA was adjusted to 0.8 μg as in A. The data are the means ± S.E. of duplicate assays from four separate cell cultures. *, significantly different (p < 0.05) from control (vector only); **, significantly different (p < 0.02) from Nrf2-induced activity; ***, significantly different (p < 0.01) from Nrf2 + CBP. C, whole cell content of Nrf2 in samples used for the luciferase assays shown in B. Western blotting analysis (10% SDS-PAGE) was performed with 15 μg of protein. Nrf2 was detected with anti-Nrf2 antibody (sc-13032; Santa Cruz Biotechnology, Inc.). A nonspecific band in the same blot was used as loading control. The Western blot shown is representative of three such blots from three different cell cultures.

FIGURE 2.

Nrf2 is acetylated in cells during Nrf2-induced gene transcription. K562 cells (8 × 10⁵ in 8 ml of medium) were seeded in T25 flasks. After 24 h, the cells were transfected with expression plasmid for CBP (1.6 μg) with or without E1A (1.6 μg) (A) or SIRT1 (1.6 μg) with or without dominant negative SIRT1 (SIRT1-H355A) (1.6 μg) or treated with NAM (10 mm) (B), along with 0.2 μg of pCI-Nrf2 for 24 h. Cell cultures in A were treated with trichostatin A (66 nm) and NAM (10 mm) for 6 h before harvesting to inhibit deacetylase(s) (33–35). Acetylation was measured in whole cell lysates by a co-immunoprecipitation assay. The whole cell lysates (200 μg of protein) were first precipitated with anti-Nrf2 antibody (1 μg, sc-13032; Santa Cruz Biotechnology, Inc.), as described under “Experimental Procedures,” followed by Western blotting (WB) of the IP samples, using anti-acetyl-lysine antibody (antibody 9441; Cell Signaling Technology). The blots shown in A and B are representative of three different experiments. C, whole cell content of Nrf2 in CBP- and SIRT1-treated cells. Western blotting analysis (8% SDS-PAGE) was performed with 10 μg of protein. Nrf2 was detected with anti-Nrf2 antibody (sc-13032; Santa Cruz Biotechnology, Inc.). A nonspecific band in the same blot was used as loading control. The Western blot shown is representative of three such blots. D, SIRT1 inhibits Nrf2-induced transcription from prototypic ARE-driven reporter gene. K562 cells were transfected with 0.2 μg of the ARE-driven reporter minimal promoter HO-1-ARE-luc along with pCI-Nrf2 (0.2 μg) and plasmid harboring the cDNA for wild-type SIRT1 (0.2 μg) with or without dominant negative SIRT1 (SIRT1-H355A) (0.2 μg). The total amount of DNA in each well was 0.8 μg, made up by the addition of empty vector. The cells were harvested 24 h after transfection, and promoter activity was measured as in Fig. 1. The values plotted are the means ± S.E. of duplicate assays from four different cell cultures. *, significantly different (p < 0.05) from Nrf2 treatment.

FIGURE 3.

Effect of small molecule modulators of SIRT1 on Nrf2-dependent gene transcription. In A, K562 cells were transfected with 0.2 μg of HO-1-ARE-luc reporter construct, along with 0.3 μg of pCI-Nrf2 or empty vector (pCI-Neo). After 24 h, various concentrations of EX-527 were added, and the cells were harvested for promoter analysis (28, 31, 32) 2 h later (left panel). In B, cells were treated with NAM (10 mm) at the time of transfection and harvested along with cells treated with EX-527. The data are the means ± S.E. of duplicate assays from three different cell cultures. In C, K562 cells transfected with 0.3 μg of Gα_i2 gene promoter construct (pGα_i2(−1214/+115)-luc) or 0.2 μg each of either HO-1-ARE-luc or hNQO1-ARE-luc reporter were treated with resveratrol 24 h after transfection, followed by tBHQ (20 μm) 30 min later. The cells were harvested for promoter analysis 60 min after the addition of tBHQ. In D, cells transfected with 0.2 μg each of empty vector pC1-Neo or heterologous Nrf2 (pCI-Nrf2) as inducer, along with 0.3 μg of pGα_i2(−1214/+115)-luc, or 0.2 μg each of either HO-1-ARE-luc or hNQO1-ARE-luc reporter were treated with resveratrol 24 h after transfection. The cells were harvested for promoter activity 90 min after the addition of resveratrol. In C and D, the effects of resveratrol on promoter activities are presented as percentage plots, taking “no resveratrol treatment” as 100%. The values plotted are the means ± S.E. of duplicate assays from three or four experiments; error bars are not indicated if the number of experiments was less than three. Con, control.

FIGURE 4.

EMSA of Nrf2-DNA binding activity of nuclear extracts. A, K562 cells were transfected with pCI-Nrf2 or pCI-Neo (control). After 24 h the cells were incubated for 1 h with or without tBHQ (20 μm) in the absence or presence of resveratrol (50 μm). B, cells were co-transfected with expression plasmid for CBP with or without expression plasmid for E1A; the cells were harvested after 24 h. For both A and B, nuclear extracts were prepared and used for EMSA as described previously (20, 28, 32), using ³²P-labeled double-stranded DNA probe 5′-GCCCGCCCCGGCCCAGTCACAGGCTTGGTTC-3′, which contains the ARE (underlined) motif that maps at −84/−76 in the Gα_i2 gene promoter. The reactions were carried out with 2 μg of nuclear extract protein for each lane. When antibodies were used, the nuclear extract was incubated with the labeled probe for 30 min at 25 °C prior to the addition of each antibody and then incubated for an additional 30 min, followed by electrophoresis. Antibodies against Nrf2 and small Maf (Maf F/G/K (C-18) from Santa Cruz Biotechnology, Inc.) were used at 2 or 4 μg for lanes 6 and 7 and at 1 or 2 μg for lanes 8 and 9, respectively. Anti-Sp1 antibody was used at 2 μg. Nrf2-DNA binding complex is indicated by the arrow. Unlabeled probe (lane 11) was used at 20-fold excess. Ab, antibody; NE, nuclear extract; pCI-Neo, empty vector; pCI-Nrf2, expression plasmid for Nrf2.

FIGURE 5.

Mutation (Lys → Ala or Lys → Arg) of Lys⁵⁹¹ and Lys⁵⁸⁸ residues in the Neh3 domain of Nrf2 impairs Nrf2-dependent transactivation of ARE-driven reporter gene. A, schematic of putative acetylation sites outside the Neh1 domain of Nrf2, identified with the acetylation site prediction algorithm PAIL (60). Lysine to alanine substitution mutations were created at five of these sites (×) by using the QuikChange^TM site-directed mutagenesis kit from Stratagene (La Jolla, CA), and the mutants were used for the transfection experiments summarized in B. In separate experiments, lysine to arginine (Lys → Arg) or lysine to glutamine (Lys → Gln) substitution mutations were created at Lys⁵⁸⁸ and Lys⁵⁹¹ and used for the transfection experiments summarized in C. Transactivation activities of WT and mutant Nrf2 were assessed in transient transfection assays using HO-1-ARE-luc as the reporter construct (42). The values shown are the means ± S.E. of triplicate assays from four (B) or five (C) different experiments. *, significantly different (p < 0.05) from the WT.

FIGURE 6.

Nucleocytoplasmic localization of Nrf2 in cells, as assessed by fluorescence imaging. HepG2 cells were grown on coverslips in six-well plates to 50–80% confluence, then transfected with plasmid harboring cDNA for EGFP linked to the coding sequence for Nrf2 (EGFP-Nrf2, also designated GFP-Nrf2) (20), and then examined by wide field microscopy. A, effect of resveratrol on tBHQ-induced GFP-Nrf2 localization. Resveratrol (50 μm) and/or tBHQ (20 μm) were/was added to some cells 24 h after transfection. In cells treated with both resveratrol and tBHQ, resveratrol was added 30 min before the addition of tBHQ. All of the cells were harvested 1 h after the addition of tBHQ and processed for fluorescence imaging (20). Propidium iodide (red) was used to counterstain the nuclei. The data presented are representative of three experiments. B, E1A induces cytoplasmic localization of GFP-Nrf2 in cells treated with CBP. HepG2 cells grown as described in A were transfected with 1.5 μg of GFP-Nrf2 (panel a) along with an expression plasmid for the acetyltransferase CBP (0.25 μg) in the absence (panel b) or presence of an expression plasmid for E1A (0.25 μg) (panel c) or E1A plus LMB (panel d). The cells were harvested 24 h after transfection and processed for fluorescence imaging (20). LMB (10 ng/ml) was added 30 min before harvest. The data presented are representative results from three or four separate cultures. C, CBP increases nuclear fluorescence of GFP-Nrf2 compared with basal. Quantification of green fluorescence intensity in B was performed as described previously (20), using Nikon Elements Advanced Research Software (Melville, NY). ResV, resveratrol.

FIGURE 7.

Nucleocytoplasmic distribution of Nrf2 in cells, as assessed by Western blotting. K562 cells (4 × 10⁶/8 ml in T25 flask) were transfected with 4 μg each of expression plasmid for Nrf2 (pCI-Nrf2) or the empty vector (pCI-Neo) along with expression plasmid for SIRT1 (4 μg) with or without dominant negative SIRT1 (SIRT1-H355A) (4 μg) or treated with tBHQ (20 μm) for 1 h in the absence or presence of small molecule modulators (1 μm EX-527 or 50 μm resveratrol) of SIRT1. When used, EX-527 or resveratrol was added 30 min before the addition of tBHQ. Western blotting analysis (8% SDS-PAGE) was performed with 10 μg of protein. Cytoplasmic and nuclear fractions were prepared as described previously (32). Nrf2 was detected with anti-Nrf2 antibody (sc-13032; Santa Cruz Biotechnology, Inc.). A, total cell content of Nrf2 is not changed by small molecule modulators of SIRT1. B, assessment of relative purity of cytoplasmic and nuclear fractions, using antibodies against protein markers for these fractions. Markers for nuclear (p300/CβP-associated factor) and cytoplasmic (β-tubulin) fractions were used to assess the degree of potential cross-contamination between the two fractions. C and D, relative content of Nrf2 in nuclear (upper panels) and cytoplasmic fractions (lower panels) from cells treated with small molecule modulators of SIRT1 (C) or expression plasmid for SIRT1 (D). Western blotting analysis (8% SDS-PAGE) was performed with 10 μg of protein for each fraction. Quantification of the Western blots was done by densitometric scanning using UN-SCAN-IT software (Silk Scientific, Inc, Orem, UT). The results were calculated relative to the corresponding loading controls. The ratios obtained are plotted as histograms (three or four different experiments), setting the ratios obtained for the empty vector treatment as 100%. D, SIRT1-induced changes in the nucleocytoplasmic distribution of Nrf2. C, cytoplasmic fraction; W, whole cell lysate; N, nuclear fraction; Neo, empty vector (pCI-Neo); Nrf2, pCI-Nrf2; ResV, resveratrol; dnSIRT1, dominant negative SIRT1.

FIGURE 8.

Mutation (Lys → Arg) of Lys⁵⁹¹ and Lys⁵⁸⁸ residues in the Neh3 domain of Nrf2 alters nucleocytoplasmic localization of GFP-Nrf2. HepG2 cells grown on coverslips in six-well plates to 50–80% confluence, as in the legends to Fig. 6, were transfected with wild-type pEGFP-Nrf2 or Lys → Arg or Lys → Gln mutants described in the legends to Fig. 5. The cells were processed for fluorescence imaging analysis as described under “Experimental Procedures.” To stain the nuclei, the cells were incubated for 2 min at room temperature in 3 μg/ml propidium iodide and then rinsed with PBS. The coverslips were then mounted onto the slides using Aqua/Polymount (Polysciences, Inc. Warrington, PA), kept overnight at 4 °C, and visualized under a Nikon TE2000-U C1 confocal laser scanning microscope at excitation/emission wavelengths of 488/505–550 nm (for green fluorescence) and 543/560–615 nm (for red fluorescence). Using Adobe Photoshop, images of the propidium iodide (PI) and GFP fluorescence patterns were merged to visualize nuclear localization.