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Oxidant-mediated Akt Activation in Human RPE Cells

From the Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina.

PURPOSE. To determine whether a model oxidant, hydrogen peroxide (H₂O₂), influences Akt activation and, if so, whether Akt activation promotes retinal pigment epithelial (RPE) cell survival.

METHODS. Cultured human RPE cells were pretreated with medium alone, with LY294002 (LY), an inhibitor of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt, or with Akt/protein kinase B signaling inhibitor (API)-2, a specific Akt inhibitor, and then were stimulated with H₂O₂ at different doses for various times. Akt phosphorylation was evaluated by Western blot using antibody against phosphorylated Akt (Ser473). The effect of Akt blockade on RPE cell viability was assessed by tetrazolium salt (WST-1) assay and a lactate dehydrognease (LDH) release assay. Caspase-mediated cytokeratin cleavage, an early apoptosis marker, was assessed by M30 antibody staining. Caspase-independent apoptosis was determined by nuclear translocation of apoptosis-inducing factor (AIF). RPE cell morphology was evaluated by electron microscopy. The effect of H₂O₂ on downstream Akt targets was examined by Western blot using antibody against phosphorylated forkhead in rhabdomyosarcoma (FKHR) and phosphorylated glycogen synthase kinase (GSK)-3ß.

RESULTS. H₂O₂ induced Akt phosphorylation in a dose-dependent manner and also induced the phosphorylation of downstream effectors FKHR and GSK-3ß. LY markedly inhibited H₂O₂-mediated Akt phosphorylation and significantly enhanced caspase-associated and caspase-independent RPE cell death.

CONCLUSIONS. A model oxidant, H₂O₂, induces PI3K and thereby activates Akt. Akt activation enhances RPE cell survival and thus may protect RPE cells from oxidant-induced cell death under normal circumstances and in disease states such as age-related macular degeneration (AMD).

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