FIGURE 4.

AKT activation by IGF-I prevents FOXO3-mediated cell death in astrocytes during oxidative stress. A, astrocyte death 12 h after H₂O₂ (100 μm) addition was determined by measuring lactate dehydrogenase (LDH) released to the medium. Transfection of astrocytes with DN-FOXO3 protected them (***, p < 0.001 versus H₂O₂-treated cells transfected with control construct; n = 3). B, IGF-I (100 nm) increased phospho-FOXO3 (pFOXO3) (Thr-32) and FOXO3 levels in the cytosolic fraction 4 h after its addition (*, p < 0.05 versus control cells; n = 3). Co-administration of H₂O₂ (100 μm) enhanced this effect (**, p < 0.01 versus control cells; *, p < 0.05 versus IGF-I-treated cells; n = 3). β-Actin served as a loading control. C, astrocytes were co-transfected with a luciferase reporter vector containing six copies of the DAF16 family protein-binding element and either the WT FOXO3 or MFOXO3 (insensitive to IGF-I) construct. IGF-I (100 mm) significantly reduced FOXO3 activity in non-H₂O₂-treated astrocytes transfected with WT FOXO3 (**, p < 0.01 versus control cells; n = 4). Alternative expression of MFOXO3 abrogated IGF-I effects (*, p < 0.05 versus WT FOXO3-transfected cells; n = 4). H₂O₂ (100 μm) treatment increased FOXO3 transcriptional activity; however, it did not prevent IGF-I-mediated FOXO3 inactivation (***, p < 0.001 versus H₂O₂-treated cells; n = 4). Alternative expression of MFOXO3 abrogated IGF-I effects on H₂O₂-treated astrocytes (***, p < 0.001 versus WT FOXO3-transfected cells; n = 4). D, astrocyte death 12 h after H₂O₂ (100 μm) addition was determined by measuring lactate dehydrogenase released to the medium. IGF-I (100 mm) co-administration significantly prevented H₂O₂-mediated lactate dehydrogenase release (***, p < 0.001 versus H₂O₂-treated cells; n = 3). Transfection of astrocytes with MFOXO3 (insensitive to IGF-I) significantly prevented the cytoprotective effect on IGF-I in oxidative stress conditions (***, p < 0.001 versus H₂O₂-treated cells and WT FOXO3-transfected cells; n = 3). E, JIP-1-transfected astrocytes showed lower FOXO3 activity than control astrocytes in the presence of H₂O₂ (100 μm) (**, p < 0.01 versus cells transfected with control construct; n = 4). F, H₂O₂ (50–100 μm) co-administration up-regulated phospho-JNK1 (pJNK1) and -2 (pJNK2) (Thr-183 and Tyr-185) levels in IGF-I (100 nm)-treated astrocytes. Pretreatment with the PI3K/AKT inhibitor LY294002 (25 μm) prevented AKT phosphorylation (Ser-473) (pAKT)and significantly enhanced JNK phosphorylation (**, p < 0.01 and *, p < 0.05 versus H₂O₂-treated cells without LY294002; n = 3). G, astrocytes transfected with MEKK-CA showed a significantly higher FOXO3 activity than control astrocytes (*, p < 0.05 versus cells transfected with control construct; n = 4). Co-transfection with an AKT-CA construct prevented this effect (***, p < 0.001 versus cells only transfected with MEKK-CA; n = 4). Error bars represent S.E. a.u., absorbance units.