Phosphatidylinositol 3 kinase pathway and 4-hydroxy-2-nonenal-induced oxidative injury in the RPE.

PURPOSE 4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation product in the retina and the retinal pigment epithelium. The purpose of the present study was to investigate how NF-E2-related factor-2 (Nrf2) and phosphatidylinositol 3 (PI3K) pathways affect the responses of cultured human retinal pigment epithelial (RPE) cells to 4-HNE. METHODS Cultured ARPE-19 cells were treated with different concentrations of 4-HNE and a PI3K inhibitor, LY294002. Intracellular glutathione (GSH) was measured by high-performance liquid chromatography (HPLC). The transcriptional activity of Nrf2 was measured by dual luciferase assay after transient transfection with reporter plasmids. The mRNA level of glutamate cysteine ligase (GCL) was quantified by real-time RT-PCR. Formation of HNE adduct on heat shock cognate protein 70 (Hsc70) was measured by immunoprecipitation and Western blot analyses. RESULTS Treatment with 4-HNE increased Nrf2 activity and GSH synthesis in a dose-dependent manner in cultured RPE cells. The modulatory subunit of GCL was upregulated by 4-HNE. Antioxidant responses were largely abolished by pretreatment with LY294002. The modification of Hsc70 by 4-HNE was increased when PI3K was inhibited. CONCLUSIONS The Nrf2-dependent antioxidant response protects against 4-HNE toxicity, and this protective mechanism is dependent on the functions of the PI3K pathway.