Abstract 10582: GSK-3β Translocates to Mitochondria by Oxidant Stress in a Kinase Activity-Dependent Manner and Contributes to Lethal ROS Production
Background: Activity of glycogen synthase kinase-3β (GSK-3β) is a determinant of the threshold for mitochondrial permeability transition (MPT) and thus a determinant of cell resistance to reperfusion injury. However, it remains unclear where and how GSK-3β modulates MPT. Here we examined the hypothesis that GSK-3β translocates from the cytosol to mitochondria in a kinase activity-dependent manner, leading to augmented production of reactive oxygen species (ROS), a trigger of MPT.
Methods and Results: H9c2 cells were transfected with EGFP-tagged GSK-3β (WT), constitutive active GSK-3β (S9A) or kinase inactive GSK-3β (K85R). Forty-eight hours after the transfection, the mitochondria were stained with Mito-Tracker red and then observed by time-lapse microscopy (Eclipse Ti-E, Nikon). Mitochondrial translocation of WT and S9A, but not that of K85R, was observed after exposure of cells to H2O2 (100 μ M), and the translocation of GSK-3β was followed by development of apoptotic phenotypes (blebbing and cell shrinkage). The prevalence of apoptosis was significantly higher in S9A-transfected cells and lower in K85R-transfected cells than in WT-transfected cells (48±9% and 22±4% vs 31±5%). Production of ROS determined by dihydrodichlorofluorescein diacetate staining after 2-hr hypoxia/30-min reoxygenation or treatment with antimycin A (100 μ M), an inhibitor of complex III, was significantly suppressed by LiCl (30 mM), a GSK-3β inhibitor, by 38% and 27%, respectively. Under the condition of normoxia without oxidant stress, activation of PI3K-Akt-GSK-3β signaling by erythropoietin (10 units/ml) did not change the level of total mitochondrial GSK-3β, but it increased Ser9-phospho-GSK-3β in mitochondria by 35% and inhibited H2O2-induced apoptosis (45±7% vs 27±5%).
Conclusion: In response to oxidant stress, GSK-3β translocates to the mitochondria in a kinase activity-dependent manner and enhances production of cytotoxic ROS from mitochondria. PI3K-Akt-GSK-3β signaling, leading to inactivation of GSK-3β by phosphorylation at Ser9 in the cytosol and mitochondria, appears to have dual protective mechanisms against MPT: inhibition of mitochondrial recruitment of cytosolic GSK-3β and inhibition of ROS production by mitochondrial GSK-3β.
- © 2011 by American Heart Association, Inc.