Hydrogen Peroxide– and Growth Factor–Mediated Akt Activation in Human RPE Cells

Abstract

Abstract: : Purpose: Retinal pigment epithelial (RPE) cell death and survival are crucially important in the normal eye, and in diseases such as age–related macular degeneration (AMD). It has been proposed that oxidative stress contributes to RPE cell death and that growth factors promote RPE cell survival. The mechanisms by which oxidative stress influences RPE cell death and growth factors enhance RPE cell survival are not fully understood. Akt also called protein kinase B, is a critical mediator of cell death and survival signals. In the present study, we determined whether hydrogen peroxide and growth factors influence Akt activation, and if so, whether Akt blockade affects RPE cell survival. Methods: Human RPE cells were harvested from donor eyes and propagated in culture. RPE cells were pretreated with or without LY 294002, a inhibitor of phosphatidylinositol 3–kinase (PI3–K) and it's downstream effector Akt, for 30 min or 60 min and then treated with or without hydrogen peroxide (H₂O₂) or growth factors [platelet derived growth factor (PDGF), insulin–like growth factor (IGF)–1, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)–α, or interleukin (IL)–1ß] at different dosages for varying times. Phosphorylated Akt (Serine 473) and total Akt were evaluated by Western blot. H₂O₂–induced cell death was evaluated by a colorimetric assay based on the cleavage of the tetrazolium salt WST–1, trypan blue exclusion assay, and visualization of cell morphology by inverted microscopy. Results: H₂O₂ caused RPE cell death in a dose–dependent, and time–dependent manner. Stimulation of RPE cells with H₂O₂ for 15 min and 30 min produced a marked increase in Akt phosphorylation in a concentration–dependent manner. Following 30 min stimulation, PDGF, IGF–1, and EGF caused rapid phosphorylation of Akt in a dose–dependent manner. TNF–α slightly phosphorylated Akt, while VEGF and IL–1ß did not affect Akt phosphorylation. Both H₂O₂ and growth factor–mediated Akt phosphorylation were blocked by LY294002. Total Akt was not greatly affected by these treatments. LY294002 enhanced H₂O₂–induced RPE cell death. Conclusions: H₂O₂ and growth factors induce PI3–K that activates Akt. The enhanced RPE cell survival conferred by Akt activation may help to protect RPE cells from oxidant–induced cell death normally, and in diseases such as AMD.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • apoptosis/cell death