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. 2009 May 22;284(21):14476-84.
doi: 10.1074/jbc.M807397200. Epub 2009 Mar 26.

Mutual dependence of Foxo3a and PGC-1alpha in the induction of oxidative stress genes

Yolanda Olmos  1 , Inmaculada Valle, Sara Borniquel, Alberto Tierrez, Estrella Soria, Santiago Lamas, Maria Monsalve
Affiliations

Mutual dependence of Foxo3a and PGC-1alpha in the induction of oxidative stress genes

Yolanda Olmos et al. J Biol Chem. .

Abstract

Oxidative stress is a hallmark of metabolism-related diseases and a risk factor for atherosclerosis. FoxO factors have been shown to play a key role in vascular endothelial development and homeostasis. Foxo3a can protect quiescent cells from oxidative stress through the regulation of detoxification genes such as sod2 and catalase. Here we show that Foxo3a is a direct transcriptional regulator of a group of oxidative stress protection genes in vascular endothelial cells. Importantly, Foxo3a activity requires the transcriptional co-activator PGC-1alpha, because it is severely curtailed in PGC-1alpha-deficient endothelial cells. Foxo3a and PGC-1alpha appear to interact directly, as shown by co-immunoprecipitation and in vitro interaction assays, and are recruited to the same promoter regions. The notion that Foxo3a and PGC-1alpha interact directly to regulate oxidative stress protection genes in the vascular endothelium is supported by the observation that PGC-1alpha transcriptional activity at the sod2 (manganese superoxide dismutase) promoter requires a functional FoxO site. We also demonstrate that Foxo3a is a direct transcriptional regulator of PGC-1alpha, suggesting that an auto-regulatory cycle regulates Foxo3a/PGC-1alpha control of the oxidative stress response.

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Figures

FIGURE 1.
FIGURE 1.
Foxo3a regulates oxidative stress protection in HUVEC. A, expression of genes involved in oxidative stress protection was determined by qRT-PCR in HUVEC infected with TM-Foxo3a. Expression is shown as the fold induction above the level in cells infected with control virus (GFP). 18 S RNA was used as a loading control. B, Western blot analysis of oxidative stress protection proteins in HUVEC infected with TM-Foxo3a or control (GFP) adenovirus. Right panel, Western blot quantification. C, ChIP analysis of Foxo3a localization in the promoter regions of oxidative stress protection genes in HUVEC infected with TM-Foxo3a adenovirus. β-Actin was used as a negative control. A and C, control samples were assigned the value of 1. D, ROS levels (left panel) and ΔΨm (right panel) were determined by fluorescence-activated cell sorting of DhR6G or TMREM loaded HUVEC infected with TM-Foxo3a or control (GFP) adenovirus. A, B, and D, data are the means ± S.D. *, p < 0.05.
FIGURE 2.
FIGURE 2.
Requirement of Foxo3a to maintain oxidative stress protection systems. A, HUVEC were transfected with control or Foxo3a specific small interference RNAs and mRNA expression levels of target genes were determined by qRT-PCR. B, mRNA expression of oxidative stress protection genes in Foxo3a+/+ and Foxo3a-/- MEF. 18 S RNA was used as loading control, and MMP13 was used as negative control. Control samples were assigned the value of 100%. The data are the means ± S.D. *, p < 0.05.
FIGURE 3.
FIGURE 3.
PGC-1α induced expression of oxidative stress protection genes is reduced in the absence of Foxo3a. Up-regulation of oxidative stress protection systems by PGC-1α was determined in Foxo3a+/+ and Foxo3a-/- MEF that were infected with PGC-1α (Ad-P) or a control adenovirus (Ad). Upper panel, mRNA expression levels were determined by qRT-PCR. 18 S RNA was used as loading control. Control samples were assigned the value of 1. The data are the means ± S.D. *, p < 0.05. Lower panel, Western blot analysis.
FIGURE 4.
FIGURE 4.
Foxo3a-dependent expression of oxidative stress protection genes is reduced in the absence of PGC-1α. Induction of oxidative stress protection genes by Foxo3a was determined in PGC-1α+/+ and PGC-1α-/- MEF (A and B) and MAEC (C and D) that were infected with TM-Foxo3a or a control adenovirus. A and C, mRNA expression levels were determined by qRT-PCR. 18 S RNA was used as loading control. B and D, Western blot analysis. Right panels, Western blot quantification. E, PGC-1α+/+ and PGC-1α-/- MAEC or MEF were infected with TM-Foxo3a or a control adenovirus, and ROS levels were determined by fluorescence-activated cell sorting of CM-H2DCFDA loaded cells. Control samples were assigned the value of 1. The data are the means ± S.D. *, p < 0.05.
FIGURE 5.
FIGURE 5.
Foxo3a interacts with PGC-1α. A, HUVEC were infected with PGC-1α (Ad-P) or control adenovirus (Ad). PGC-1α was immunoprecipitated (IP), and Foxo3a was detected in the immunoprecipitated material by Western blotting (WB) but not in a control immunoprecipitation. Input, Western blot of whole cell lysate. B, HUVEC were infected with a PGC-1α adenovirus (Ad-P). Foxo3a was immunoprecipitated and PGC-1α was detected in the immunoprecipitated material by Western blot. C, mammalian two-hybrid assay. HUVEC were co-transfected with the Gal4 luciferase reporter plasmid 5×UAS-Luc, the indicated amounts of G4-DBD-PGC-1α (encoding PGC-1α fused to the DNA-binding domain of Gal4 to provide a DNA tethering domain), and a TM-Foxo3a expression vector, or the corresponding control vectors. Luciferase activity was determined 24 h post-transfection. D, GST pulldown assay mapping the interaction between Foxo3a and PGC-1α. GST-fusions with the indicated PGC-1α fragments (fragments A–D) were immobilized on glutathione beads and incubated with 35S-labeled Foxo3a. After extensive washing, the samples were separated by SDS/PAGE, and bound 35S-labeled Foxo3a protein was detected by autoradiography.
FIGURE 6.
FIGURE 6.
PGC-1α is a co-activator of Foxo3a. A, BAEC were transfected with the FoxO activity luciferase reporter construct 3×IRE-luc (left panel) or with the 2 kb sod2 promoter-luciferase reporter vector (right panel) and with the indicated amounts of Foxo3a and PGC-1α expression vectors or the corresponding controls. B, BAEC were transfected with the wild type 2-kb sod2 promoter (wt) or version containing a point mutation in the functional FoxO site (mut) and with the indicated amounts of expression vectors encoding Foxo3a (left panel) or PGC-1α (right panel) or the corresponding controls. Luciferase activity was determined 24 h post-transfection. Control samples were assigned the value of 1. The data are the means ± S.D. *, p < 0.05.
FIGURE 7.
FIGURE 7.
Proposed model of Foxo3a regulation of oxidative stress protection genes. Foxo3a induces PGC-1α expression and interacts with PGC-1α to regulate the expression of oxidative stress protection genes.
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