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. 2013 Nov 1;19(13):1507-21.
doi: 10.1089/ars.2012.4713. Epub 2013 Apr 15.

SirT1 regulation of antioxidant genes is dependent on the formation of a FoxO3a/PGC-1α complex

Yolanda Olmos  1 , Francisco J Sánchez-Gómez, Brigitte Wild, Nieves García-Quintans, Sofía Cabezudo, Santiago Lamas, María Monsalve
Affiliations

SirT1 regulation of antioxidant genes is dependent on the formation of a FoxO3a/PGC-1α complex

Yolanda Olmos et al. Antioxid Redox Signal. .

Abstract

SirT1 is a class III histone deacetylase that has been implicated in metabolic and reactive oxygen species control. In the vasculature it has been shown to decrease endothelial superoxide production, prevent endothelial dysfunction and atherosclerosis. However, the mechanisms that mediate SirT1 antioxidant functions remain to be characterized. The transcription factor FoxO3a and the transcriptional coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α) have been shown to induce the expression of antioxidant genes and to be deacetylated by SirT1.

Aims: Here we investigated SirT1 regulation of antioxidant genes and the roles played by FoxO3a and PGC-1α in this regulation.

Results: We found that SirT1 regulates the expression of several antioxidant genes in bovine aortic endothelial cells, including Mn superoxide dismutase (MnSOD), catalase, peroxiredoxins 3 and 5 (Prx3, Prx5), thioredoxin 2 (Trx2), thioredoxin reductase 2 (TR2), and uncoupling protein 2 (UCP-2) and can be localized in the regulatory regions of these genes. We also found that knockdown of either FoxO3a or PGC-1α prevented the induction of antioxidant genes by SirT1 over-expression. Furthermore, SirT1 increased the formation of a FoxO3a/PGC-1α complex as determined by co-immunoprecipitation (IP) assays, concomitantly reducing H2O2-dependent FoxO3a and PGC-1α acetylation. Data showing that FoxO3a knockdown increases PGC-1α acetylation levels and vice versa, suggest that SirT1 activity on FoxO3a and PGC-1α may be dependent of the formation of a FoxO3a/PGC-1α complex.

Innovation: A unifying mechanism for SirT1 activities is suggested.

Conclusion: We show that SirT1 regulation of antioxidant genes in vascular endothelial cells depends on the formation of a FoxO3a/PGC-1α complex.

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Figures

FIG. 1.
FIG. 1.
SirT1 regulates the expression of oxidative stress protection genes. (A–D) Analysis of oxidative stress protection genes in bovine aortic endothelial cells (BAEC) that were infected o/n with recombinant adenovirus as indicated and harvested 24 h postinfection. (A, C) qRT-PCR mRNA expression analysis. (B, D) Protein analysis by Western Blot. (E) SirT1 dependent changes in ROS levels were monitored in control and SirT1-overexpressing BAEC, treated with 50 μM H2O2 for 4 h or 20 μM DMNQ for 2 h, as indicated. Cells were labeled with CM-H2DCFDA and analyzed by flow cytometry, (left and middle panels), or labeled with MitoSOX Red and after fixation, analyzed by confocal microscopy. (F-G) Analysis of oxidative stress protection genes in serum deprived SirT1+/+ and SirT1−/− MEFs. BAEC (F) qRT-PCR. (G) Western Blot. Data are from ≥3 independent experiments. Data are means+SD (*) p≤0.05 versus control. The blots presented correspond to a representative experiment.
FIG. 2.
FIG. 2.
SirT1 is recruited to promoter regions of ROS detoxification genes upon serum starvation. (A–E) Chromatin immunoprecipitation assays of promoter regions corresponding to the indicated ROS detoxification genes. Cross-linked chromatin Promoter occupancy was analyzed by qPCR using specific primers. β-actin CDS was used to control for non specific enrichment. (A) SirT1 binding sites. (B) H4K16 acetylation levels at SirT1 sites. (C) Pol II recruitment. The position of the analyzed fragments is indicated, using as reference (+1), the position of the ATG. (D, E) Analysis of prx3 and ucp-2 genes. Left panels, analysis of Pol II at the transcriptional start region and along the gene. Central panel, analysis of “elongating” Pol II levels (Pol II phosphorylated in the Ser2 positions of the CTD repeats of the Pol II largest subunit) at the transcriptional start region and downstream of the ATG. Right panels, SirT1 recruitment along the genes, upstream and downstream of the ATG. The position of the analyzed fragments is indicated, using as reference (+1), the position of the ATG. (F) mRNA and (G) protein levels of SirT1 target genes in confluent BAEC cells in 10% fetal bovine serum (FBS) or serum deprived (0.5% FBS) o/n were analyzed by qRT-PCR and western blot, respectively. Data are from ≥3 independent experiments. Data are means+SD (*) p≤0.05 versus control. The blots presented correspond to a representative experiment.
FIG. 3.
FIG. 3.
SirT1 requires PGC-1α to induce the expression of oxidative stress protection genes. (A, B) BAEC were coinfected o/n with Ad-SirT1 and Ad-shPGC-1α, or the respective control adenoviruses. 12 h postinfection, cells were deprived of serum o/n and harvested. Expression of the oxidative stress protection genes was analyzed by qRT-PCR (A) and Western Blot (B). (C, D). BAEC whole cell extracts were subjected to immunoprecipitation (IP) with a specific PGC-1α antibody or normal IgG, as a control, and analyzed by western blot as indicated. Input indicates a 1/10 of whole cell extract not subjected to immunoprecipitation. (C) BAEC were infected o/n with Ad-PGC-1α and 8 h postinfection were treated with 10 mM nicotinamide for 15 h. (D) BAEC were coinfected o/n with Ad-SirT1 and Ad-PGC-1α, or the respective control adenoviruses. 24 h postinfection cells were deprived of serum o/n, and then treated with 50 μM H2O2 or vehicle for 4 h. (E) Confluent BAEC were grown in galactose media and in the absence of serum for 48 h. Whole cell extracts were subjected to immunoprecipitation (IP) with specific PGC-1α or SirT1 antibody or normal IgG, as a control, and analyzed by western blot as indicated. Data are from ≥3 independent experiments. Data are means+SD (*) p≤0.05 versus control. The blots presented correspond to a representative experiment.
FIG. 4.
FIG. 4.
FoxO3a is required for SirT1-induction of antioxidant genes. (A, B) BAEC were first infected o/n with Ad-SirT1 or a control adenovirus, 36 h later cells were infected o/n with Ad-shFoxO3a or a control adenovirus and finally serum deprived for 8 h. Expression levels of ROS detoxification genes were determined by qRT-PCR (A) and Western Blot (B). (*) p≤0.05 versus control. (C, D) BAEC whole cell extracts were subjected to immunoprecipitation (IP) with a specific FoxO3a (C) or anti-HA (D) antibody or normal rabbit IgG, as a control, and analyzed by western blot as indicated. Input indicates a 1/10 of whole cell extract not subjected to immunoprecipitation. (C) BAEC were treated with 10 mM nicotinamide for 15 h (D) BAEC were coinfected o/n with Ad-SirT1 and Ad-FoxO3a-WT. 24 h postinfection the cells were serum deprived o/n, and then treated for 4 h with 50 μM H2O2 or vehicle (E) Confluent BAEC were grown in galactose media in the absence of serum for 48 h. Whole cell extracts were subjected to immunoprecipitation (IP) with a specific FoxO3a or SirT1 antibody or normal IgG, as a control, and analyzed by western blot as indicated. Data are from ≥3 independent experiments. Data are means+SD (*) p≤0.05 versus control. The blots presented correspond to a representative experiment.
FIG. 5.
FIG. 5.
SirT1 regulates the formation of a PGC-1α/FoxO3a complex. (A) BAEC were coinfected o/n with Ad-PGC-1α and the indicated adenoviruses. 12 h postinfection, cells were deprived of serum o/n, and then treated for 4 h with 50 μM H2O2. Whole cell extracts were subjected to immunoprecipitation (IP) with a specific PGC-1α antibody or normal IgG, as a control, and analyzed by Western Blot as indicated. Input indicates a 1/10 of whole cell extract not subjected to immunoprecipitation. (B) SIRT1+/+ and SIRT1−/− MEFs were infected o/n with Ad-PGC1α and 12 h postinfection cells were deprived of serum o/n. Cell extracts were immunoprecipitated with an specific anti-PGC1α antibody (PGC-1α) or rabbit IgG as control (IgG) and analyzed by western blot. (C) BAEC were coinfected o/n with Ad-FoxO3a-WT and Ad-SirT1 or the corresponding controls, 12 h postinfection cells were deprived of serum o/n, and then treated for 4 h with 50 μM H2O2. Protein levels were analyzed by Western Blot (D) BAEC were infected o/n with Ad-SirT1, Ad-shSirT1 or the corresponding control adenovirus, 12 h postinfection, cells were deprived of serum o/n, and then fixed and analyzed by immunofluorescence with FoxO3a specific antibodies. The blots presented correspond to a representative experiment from ≥3 independent experiments.
FIG. 6.
FIG. 6.
FoxO3a and PGC-1α influence each other's level of acetylation. (A, B) BAEC were infected o/n with Ad-shPGC-1α (A) Ad-shFoxO3a (B) or the corresponding control adenoviruses, 12 h postinfection, cells were deprived of serum o/n and harvested. Whole cell extracts were subjected to immunoprecipitation (IP) with FoxO3a (A) or PGC-1α (B) specific antibodies, or normal IgG, as a control. Acetylation levels were analyzed by western blot. (C, D) BAEC were infected o/n with Ad-PGC-1α or the control Ad-S and 48 h postinfection the cells were harvested. (C) FoxO3a phosphorylation was analyzed in WCE by western blot. (D) Immunofluorescence analysis of FoxO3a. Left panel confocal microscopy images. Right panel, FoxO3a cytoplasm/nuclear ratio. Data are from ≥3 independent experiments. Data are means+SD (*) p≤0.05 versus control.
FIG. 7.
FIG. 7.
PGC-1α activity is reduced in the absence of SirT1. SIRT1+/+ and SIRT1−/− MEFs were infected o/n with Ad-PGC1α or a control adenovirus (Ad-Shuttle), 12 h postinfection cells were deprived of serum o/n and harvested. (A) Cell extracts were immunoprecipitated with a specific anti-PGC1α antibody (PGC-1α) or IgG as control (IgG) and PGC-1α acetylation was analyzed by western blot. (B) qRT-PCR and Western Blot analysis of oxidative stress protection proteins. Control samples were assigned the value of 1. Data are from ≥3 independent experiments. Data are means+SD (*) p≤0.05 versus control. (C) Schematic diagram of the proposed model. In stress conditions, if SirT1 is not active, both PGC-1α and FoxO3a are acetylated and targeted for degradation. In the presence of SirT1, deacetylation of PGC-1α and FoxO3a facilitates the formation of an active transcriptional complex that induces antioxidant genes and facilitates survival in stress conditions.
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