Oxidative stress-specific interaction between FANCD2 and FOXO3a

Abstract

The molecular pathway by which Fanconi anemia (FA) proteins function in oxidative stress response has not been defined. Here we report the functional interaction of the FA protein Fanconi anemia complementation group D2 (FANCD2) and the forkhead transcription factor forkhead box O 3a (FOXO3a). FOXO3a colocalized with FANCD2 foci in response to oxidative stress. The FANCD2-FOXO3a complex was not detected in cells deficient for the FA core complex component FANCA but could be restored in corrected cells. Consistent with this, a nonmonoubiquitinated FANCD2 mutant failed to bind FOXO3a. Although both mitomycin C and ionizing radiation induced FANCD2 monoubiquitination, neither could induce the association of FANCD2 and FOXO3a. Overexpression of FOXO3a reduced abnormal accumulation of reactive oxygen species, enhanced cellular resistance to oxidative stress, and increased antioxidant gene expression in corrected but not mutant FA-D2 cells. The novel oxidative stress response pathway identified in this study, in which FANCD2 and FOXO3a converge, probably contributes to cellular antioxidant defense.

Figure 1

Oxidative stress–induced formation of the FANCD2-FOXO3a complex. (A) FOXO3a colocalizes with FANCD2 after H₂O₂ treatment in normal (JY) lymphoblasts. Cells were treated with H₂O₂ (0.5mM) for 6 hours and stained with antibodies against FOXO3a and FANCD2. Colocalization of nuclear FOXO3a with FANCD2 is shown as merged images. (B) Whole-cell lysates of normal lymphoblasts (JY) and HeLa cells treated with 0.5mM H₂O₂ for 6 hours were subjected to immunoprecipitation (IP) with an anti-FANCD2 antibody or an isotype IgG (negative control) followed by immunoblotting (IB) analysis with antibodies against FOXO3a, FOXO1, FOXO4, and FANCD2. (C) HeLa cells were transduced with vector or Flag-FOXO3a and treated with 0.5mM H₂O₂ for 6 hours. Cell lysates were prepared, immunoprecipitated using anti-Flag agarose, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunoblotted. Blots were probed with antibodies against FANCD2 or Flag. (D) Normal human lymphoblasts (JY) were treated with H₂O₂ (0.5mM for 6 hours), IR (5 Gy), or MMC (0.5μM for 18 hours), and whole-cell lysates were subjected to IP with an anti-FANCD2 antibody or an isotype IgG (negative control) followed by IB analysis with antibodies against FOXO3a and FANCD2. (E) HSC72 (human FA-A lymphoblast) cells were transduced with retrovirus carrying empty vector (MIEG3) or FANCA, and treated with H₂O₂ at 0.5mM for 6 hours. Cell extracts were subjected to IP with an anti-FANCD2 antibody or an isotype IgG (negative control) followed by IB analysis with antibodies against FOXO3a and FANCD2. (F) PD20 (human FA-D2 lymphoblast) cells transduced with retrovirus carrying empty vector wt-FANCD2 or K561R-FANCD2 were treated with H₂O₂ at 0.5mM for 6 hours. Cell extracts were prepared, immunoprecipitated using anti-Flag antibody conjugated to agarose, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunoblotted with antibodies against FANCD2 and FOXO3a.

Figure 2

Coexpression of FANCD2 and FOXO3a increases cellular resistance to oxidative stress. (A) PD20 cells were infected with retroviruses carrying vector, wt-FANCD2, K561R-FANCD2, FOXO3a, or both wt-FANCD2 and FOXO3a. The transduced cells were treated with H₂O₂ at the indicated doses for 16 hours. Cell survival was determined by luminescence and is shown as the percentage of the untreated cells. Mean values and SD from 3 independent determinations are shown. Statistical significance between vector and wt-FANCD2 + FOXO3a or wt-FANCD2 samples: **P < .01; *P < .05. (B) Transduced cells described in panel A were labeled with fluorescein isothiocyanate–conjugated CM-H₂DCFDA, and levels of ROS were determined by flow cytometric analysis. (C) Expression of FOXO3a-targeted genes encoding antioxidant proteins. Transduced cells described in panel A were treated with H₂O₂ at 100μM for 12 hours. Total RNA was prepared, and gene expression was analyzed by real-time PCR. Data were presented as fold change in mRNA expression relative to glyceraldehyde-3-phosphate dehydrogenase in vector-transduced cells, which was given an arbitrary level of 1.0 for each gene. Results are mean ± SD of 3 independent experiments.