FOXO3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression

Abstract

Forkhead transcription factors of the O class (FOXOs) are important targets of the phosphatidylinositol 3-kinase/Akt pathway, and are key regulators of the cell cycle, apoptosis and response to oxidative stress. FOXOs have been shown to have tumour suppressor function and are important for stem cell maintenance. We have performed a detailed analysis of the transcriptional programme induced in response to Forkhead-box protein O3a (FOXO3a) activation. We observed that FOXO3a activation results in the repression of a large number of nuclear-encoded genes with mitochondrial function. Repression of these genes was mediated by FOXO3a-dependent inhibition of c-Myc. FOXO3a activation also caused a reduction in mitochondrial DNA copy number, expression of mitochondrial proteins, respiratory complexes and mitochondrial respiratory activity. FOXO3a has been previously implicated in the detoxification of reactive oxygen species (ROS) through induction of manganese-containing superoxide dismutase (SOD2). We observed that reduction in ROS levels following FOXO3a activation was independent of SOD2, but required c-Myc inhibition. Hypoxia increases ROS production from the mitochondria, which is required for stabilisation of the hypoxia-inducible factor-1α (HIF-1α). FOXO3a activation blocked the hypoxia-dependent increase in ROS and prevented HIF-1α stabilisation. Our data suggest that FOXO factors regulate mitochondrial activity through inhibition of c-Myc function and alter the hypoxia response.

Figures

Figure 1

FOXO3a downregulates mitochondrial genes independent of regulatory transcription factors. DLD-1 colon cancer cells expressing inducible FOXO3a.A3.ER (DL23 cells) were treated with 100 nM 4-OHT to induce activation of FOXO3a. The expression profile from these cells following FOXO3a activation was analysed using Affymetrix exon microarrays. (a) GSEA was used to determine gene sets significantly regulated by FOXO3a. Selected mitochondrial gene sets are shown. NES, normalised enrichment score; q-values, FDR-adjusted P-value. (b) Enrichment plot of the top mitochondrial gene set (MITODB_2002) found to be significantly associated with downregulation by FOXO3a. (c) Heatmap showing the expression profiles of genes significantly regulated by FOXO3a in DL23 cells that are also present in the HUMAN_MITODB_6_2002 gene set. Values show fold change over the median across all samples. (d) DL23 cells were treated with 4-OHT or solvent for 4 or 24 h. Expression of PGC1β, PRC, TFAM. TFB1M and TFB2M was determined by qRT-PCR. (e) DL23 cells were transfected with the indicated siRNAs or mock treated for 72 h and stimulated with 4-OHT for the final 24 h (NT, non-targeting control). Expression of the indicated genes after 24 h of solvent (white bars) or 4-OHT treatment (black bars) was determined by qRT-PCR. Data shown are representative of three independent experiments. (f) Silencing of PGC1β, PRC, NRF1, GABPA and ERRα was confirmed by qRT-PCR. Data shown are representative of three independent experiments. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=4)

Figure 2

Induction of the Mad/Mxd family contributes to the downregulation of mitochondrial regulators by FOXO3a. (a) Charts showing the functional classification of genes present in the MITODB gene set (upper), MITODB genes that are downregulated by FOXO3a (middle) and previously identified c-Myc-regulated mitochondrial genes that are downregulated by FOXO3a activation (lower). (b) Heatmap showing the expression profile of genes found to be significantly regulated by FOXO3a in DL23 cells, which have been previously identified to be c-Myc-regulated mitochondrial genes. (c) Silencing of Mad/Mxd proteins partially rescues repression of mitochondrial genes. DL23 cells were transfected with 100 nM of control (siCtrl, white bars), siRNAs targeting Mxi1 (grey bars) or pools of siRNAs targeting all Mad/Mxd genes (Mad1, Mxi1, Mad3 and Mad4; black bars) for 72 h and treated with 4-OHT for the final 24 h. Expression levels of the indicated mitochondrial genes were determined by qRT-PCR. Values represent mRNA expression levels of 4-OHT-treated cells relative to solvent-treated controls. (d) Silencing of Mad1, Mxi1 and Mad4 was confirmed by qRT-PCR. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=4)

Figure 3

Downregulation of mitochondrial genes by FOXO3a is mediated by inhibition of c-Myc. (a) Parental DLD-1 cells were transfected with 100 nM of either non-targeting (siNT), control (siCtrl) or c-Myc (siMyc) siRNA for 48 h. Expression levels of the indicated mitochondrial genes was determined by qRT-PCR. (b) Silencing of c-Myc was confirmed by immunoblotting lysates from cells transfected in parallel. β-Actin is shown as a loading control. (c) DL23 cells were infected with pWZL-Blast-c-Myc and selected for 96 h. Cells were then treated with 4-OHT or solvent for 24 h. Expression of c-Myc was determined by immunoblotting. β-Actin is shown as a loading control. (d) Expression levels of Mxi1 following FOXO3a activation in DL23 parental cells or DL23 cells expressing c-Myc (DL23+c-Myc) was determined by qRT-PCR. (e) Expression of the indicated mitochondrial genes in DL23 (white and grey bars) or DL23+c-Myc (hatched and black bars) was determined by qRT-PCR. Statistical analysis of the effect of 4-OHT treatment in DL23 or DL23+c-Myc cells was performed by comparing the fold change relative to ethanol-treated control in each cell line. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=3)

Figure 4

FOXO3a induces GSK3-dependent phosphorylation and proteasomal degradation of c-Myc. (a) DL23 cells were induced with 4-OHT or solvent for 24 h. In all, 25 μM MG132 was added for the final 1, 4 or 5 h, as indicated. c-Myc protein levels were determined by immunoblotting. β-Actin is shown as a loading control. (b) DL23 cells were treated with 4-OHT or solvent for 15 min before addition of 2 μg/ml cycloheximide (CHX). Cells were lysed after 15, 30, 45, 60 or 90 min of CHX treatment. c-Myc protein levels were determined by immunoblotting. β-Actin is shown as a loading control. (c) Quantitation of c-Myc protein levels in the presence of CHX in DL23 cells treated with 4-OHT or solvent from three independent experiments. Data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=3). (d) DL23 cells were treated with 4-OHT or solvent for 24 h. In all, 25 μM MG132 was added for the final 1, 3 or 5 h, as indicated. Levels of phosphorylated and total c-Myc were determined by immunoblotting. β-Tubulin is shown as a loading control. (e) DL23 cells were treated with 4-OHT or solvent for 24 h in the presence of DMSO or 5 μM of the GSK3 inhibitor SB216763. In all, 25 μM MG132 was added for the final 1, 2.5 or 5 h, as indicated. Levels of GSK3α/β, β-catenin and phosphorylated and total c-Myc were determined by immunoblotting. β-Actin is shown as a loading control

Figure 5

Regulation of mitochondrial function by FOXO3a. (a) RPE.FOXO3a.A3.ER (RPE-F) cells were treated with solvent (white bars) or 100 nM 4-OHT (black bars) for 24 h. Expression of the indicated mitochondrial genes was determined by qRT-PCR. (b) RPE-F cells were treated with 4-OHT of solvent for 24 or 48 h. Expression levels of COX1 and Tomm20 were determined by immunoblotting. β-Actin is shown as a loading control. (c) Ratio of mtDNA to nuclear DNA (mtDNA/nDNA) was determined by qPCR using DNA from RPE-F cells treated with solvent (white bars) or 4-OHT (black bars) for 48 h. (d) RPE-F cells were transfected with 100 nM of either control (white bars) or a pool of siRNAs targeting TFAM (black bars) for 72 h. Ratio of mtDNA to nDNA was determined by qPCR. Silencing of TFAM was confirmed by qRT-PCR. (e) RPE-F cells were treated with ethanol or 4-OHT for 48 h, fixed in formaldehyde, stained using 100 nM MTG and visualised using confocal microscopy. Scale bar, upper panels 20 μm, lower panels 5 μm. (f) Quantification of area occupied by MTG stain. The area occupied by MTG in each cell was measured from confocal images using Metamorph software in 20 cells from two independent experiments. (g) Oxygen consumption was measured in RPE-F cells treated with solvent (white bars) or 4-OHT (black bars) for 48 h. In all, 50 mM DCA or solvent (DMSO) was added for the final 24 h before measurement. (h) OCRs were measured in RPE-F or RPE-F+c-Myc cells following 48 h of solvent (white bars) or 4-OHT (black bars). In all, 2 μM FCCP was added to determine mitochondrial capacity. (i) RPE-F or RPE-F+c-Myc cells were treated with solvent for 24 h (white bars) or 4-OHT (black bars). Expression of PDK4 was determined by qRT-PCR. (j) RPE-F or RPE-F+c-Myc cells were treated with solvent or 4-OHT for 48 h. Total cell lysates were used to detect OXPHOS complexes by immunoblotting. β-Actin is shown as a loading control. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=3). The symbol ‘**' indicates statistical significance, as determined by Student's t-test (P=1 × 10−8, n=20). NS, nonsignificant

Figure 6

FOXO3a-dependent reduction in ROS is independent of SOD2 and is rescued by re-expression of c-Myc. (a) RPE-F cells were transfected with 100 nM of either controls (siCtrl) or siRNAs targeting SOD2 (siSOD2) for 72 h and treated with 4-OHT or solvent for the final 48 h. ROS levels were determined by measuring fluorescence of CM-H2DCFDA by flow cytometry. Expression levels of SOD2 were determined by qRT-PCR. (b) RPE-F cells were treated with 4-OHT or solvent for 48 h before treatment with 15 mM of the SOD2 inhibitor DETC for 90 min. ROS levels were determined by measuring fluorescence of CM-H2DCFDA by flow cytometry. (c) RPE-F cells were induced with 4-OHT or solvent for 24 h. Expression of CAT was determined by qRT-PCR. Data shown are representative of three independent experiments. (d) RPE-F or RPE-F+c-Myc cells were treated with 4-OHT or solvent for 48 h. ROS levels were determined by measuring fluorescence of CM-H2DCFDA by flow cytometry. (e) Expression levels of SOD2 mRNA in RPE-F and RPE-F+c-Myc cells treated with 4-OHT or solvent for 48 h were determined by qRT-PCR. Data shown are representative of three independent experiments. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=3). NS, nonsignificant

Figure 7

Endogenous FOXO1 and FOXO3a are involved in the downregulation of mitochondrial genes in hypoxic cells. (a) Parental RPE-hTERT cells were cultured in normoxic (20% O2) or hypoxic (<0.5% O2) conditions for 24 h. Expression of mitochondrial target genes was determined by qRT-PCR. (b) RPE-hTERT cells were transfected with siRNAs targeting FOXO1 or FOXO3a. At 72 h post transfection, cells were placed in normoxic or hypoxic conditions for 24 h. Expression of FOXO1 and FOXO3a was determined by qRT-PCR. (c) Expression of mitochondrial target genes in the same RNA samples as those in b was determined by qRT-PCR. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=3)

Figure 8

FOXO3a activation prevents ROS induction in hypoxia independent of SOD2 induction and blocks HIF-1α stabilisation. (a) RPE-F cells were pretreated with solvent (white bars) or 4-OHT (black bars) for 6 h and placed in normoxic (20% O2) or hypoxic (<0.5% O2) conditions for a further 48 h. ROS levels were determined by measuring fluorescence of CM-H2DCFDA by flow cytometry. (b) RPE-F cells were transfected with 100 nM of either control (siCtrl) or siRNAs targeting SOD2 (siSOD2) before being treated with 4-OHT or solvent in either normoxic or hypoxic conditions for 48 h. ROS levels were determined by measuring fluorescence of CM-H2DCFDA by flow cytometry. (c) RPE-F or RPE-F+Myc cells were treated with 4-OHT or solvent for 24 h and then placed in normoxic or hypoxic conditions for a further 24 h. Expression of HIF-1α was determined by immunoblotting. β-Actin is shown as a loading control. (d) DL23 or DL23+c-Myc cells were treated and analysed as in c. (e) RPE-F and RPE-F+c-Myc and DL23 and DL23-c-Myc cells were treated with 4-OHT or solvent for 24 h. HIF-1α mRNA levels were determined by qRT-PCR. Data shown are representative of three independent experiments. (f) DL23 cells were treated with 4-OHT or solvent for 24 h and placed in hypoxic conditions for a further 24 h. In all, 25 μM MG132 was added for the final 4 h. Expression of HIF-1α was determined by immunoblotting. β-Actin is shown as a loading control. (g) DL23 cells were treated with 4-OHT or solvent for 24 h and then placed in normoxic or hypoxic conditions for a further 24 h. Boluses of TBP were applied at the indicated concentrations every 20 min for the final 2 h. Expression of HIF-1α was determined by immunoblotting. β-Tubulin is shown as a loading control. (h) Diagram of FOXO action on mitochondrial activity. Induction of PDK4 by FOXO3a reduces TCA cycle activity and respiration. Inhibition of c-Myc function by FOXO3a lowers mitochondrial gene expression, reduces the formation of ROS and blocks stabilisation of HIF-1α. (ETC, electron transport chain). All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by Student's t-test (P<0.05, n=3). NS, nonsignificant