SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

Abstract

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.

Keywords: FOXO3a; SIRT3; Superoxide; gene expression.

Figure 1

The daf-16 homolog, FOX3a forms a complex with SIRT3. (A) FOXO3a binds to SIRT3 in vitro. Cos-7 cells were transfected with either SIRT3 wild-type (p-myc-hSIRT3-wt) or deacetylation mutant (p-myc-hSIRT3-mt) vectors and cell lysates were immunoprecipitated (IPd) with an anti-Myc antibody followed by Western analysis with an anti-FOXO3a antibody. (B) HCT116 cell lysates were IPd with either an anti-FOXO3a or anti-SIRT3 antibody, resolved by SDS-PAGE, and immunoblotted with anti-FOXO3a antibody. (C) Mitochondrial factions from HCT116 cells were IPd with either an anti-FOXO3a or anti-SIRT3 antibody and immunoblotted with anti-FOXO3a antibody.

Figure 2

The daf-16 homolog, FOX3a is a mitochondrial protein. Mitochondrial protein fraction from heart (A) and liver (B) (DNA Technologies, ReadyWestern™) were analyzed by western analysis for the presence of the FOXO3a protein. The PVDF membrane was also probed with both and a-tubulin and a-cytochrome c antibodies that localize to the cytoplasm and mitochondria, respectively. (C) Mitochondrial protein fractions from of pcDNA, wt-SIRT3, or mt-SIRT3 cell lines were isolated and immunoreactive FOXO3a protein levels were determined.

Figure 3

SIRT3 activates FOXO3a dependent gene expression. (A) pMnSOD-Luc was transiently co-transfected with CMV based FOXO3a expression vectors encoding the wild-type (pCMV-wt-FOXO3a), dominant negative (pCMV-m-FOXO3a), or dominant positive (pCMV-N-FOXO3a) FOXO3a genes. Results for all the experiments presented in this figure were normalized to β-galactosidase activity and the results are presented as luciferase/β-galactosidase relative units. (B) SIRT3 activates FOXO3a dependent gene expression. A reporter plasmid containing three FOXO DNA-binding sites (p3x-FOXO-Luc) was transfected with either pCMV-SIRT3-wt or pCMV-SIRT3-mt and luciferase activity was determined as described above. (C) The p3x-FOXO-Luc reporter plasmid was transfected with wild type or mutant FOXO3a expression vectors (pCMV-wt-FOXO3a or pCMV-m-FOXO3a respectively) along with wild type or mutant SIRT3 expression vectors (pCMV-SIRT3-wt or pCMV-SIRT3-mt) and luciferase activity was determined and normalized to β-galactosidase as described above. All experiments are mean of at least three separate experiments and error bars for all data shown represent one standard deviation and statistical significance was established by Student's t-test. * Indicates P < 0.05 by t-test. (D) ChIP analysis of FOXO3a finding to the MnSOD and SCO2 promoters in HCT116 cells that overexpress either a wild-type or deacetylation null SIRT3 gene. Cells were fixed with 1% formaldehyde to crosslink protein-DNA interactions, sonicated, and fixed cells were immunoprecipitated with either an anti-FOXO3a antibody. DNA was eluted and purified before analysis specific primers and visualized by ethidium bromide staining.

Figure 4

Overexpression of SIRT3 induced MnSOD expression. (A) A MnSOD promoter luciferase reporter plasmid (pMnSOD-Luc) was transfected into wt-SIRT3, mt-SIRT, and vector control cell lines. Luciferase activity was normalized to β-galactosidase activity and presented as luciferase/β-galactosidase relative units. All results are the mean of at least three separate experiments. Error bars around data points represent one standard deviation about the arithmetic mean. (B) pMnSOD-Luc was co-transfected into HCT116 cells with either pCMV-SIRT3-wt or pCMV-SIRT3-mt expression vectors or luciferase assays were done as described above. * Indicates P < 0.05 by t-test.

Figure 5

SIRT3 expression alters intracellular superoxide levels. (A) Altered steady-state levels of superoxide as shown by increased oxidation of DHE in HCT116 SIRT3 overexpressing cells. Control (pcDNA vector), wild-type SIRT3 (wt-SIRT3), or a deacetylation SIRT3 mutant (mut-SIRT3) cell lines were analyzed by flow cytometry for the amount of hydrolyzed DHE per 10,000 cells represented as Mean Florescent Intensity (MFI). Each bar represents the average of three separate experiments. Error bars indicate standard error. * Indicates P < 0.05 by t-test. (B) SIRT3 overexpressing cells contain increased GSH/GSSG redox ratios. Total levels of glutathione (GSH) was determined and normalized to total protein (left panel). GSSG/GSH ratios were determined and represented as % GSSG (right panel). Error bars represent of standard deviation of three independent experiments. * Indicates P < 0.05 by t-test.