Effect of 4-Hydroxynonenal on EGFR Mediated Signaling Pathway in RPE Cells

Abstract

Purpose: : 4-Hydroxynonenal (4-HNE), an stable end-product of oxidative stress induced lipid peroxidation (LPO) is an important second messenger molecule involved in the regulation of various cellular processes including proliferation, transformation and apoptosis. Previous studies have shown that 4-HNE activates EGFR but the mechanism(s) of activation, its effect on downstream signaling components, and its biological significance are not understood. Since LPO has been implicated in the mechanisms of retinopathy we have studied the effects of 4-HNE on the downstream components of EGFR signaling pathway in RPE cells in order to elucidate the physiological significance of 4-HNE mediated activation of EGFR.

Methods: : SV40-Transformed human fetal male RPE 28 cells cultured in DMEM containing 10% FBS and antibiotics in a humidified incubator at 37^oC in 5% CO₂ atmosphere were treated with 4-HNE (0-70 µM) for 12 h. The extent of total cell death was measured by MTT and apoptosis was determined by TUNNEL assays. Expression of EGFR and its downstream targets pERK/ERK and pAKT/AKT were compared in 4-HNE treated RPE cells by immunoblotting and immunofluorescence studies. These effects of 4-HNE were compared in presence and absence of EGFR, ERK and AKT inhibitors.

Results: : Up to 5 µM final concentration in medium, 4-HNE induced time and concentration dependent sustained activation of EGFR in RPE cells. 4-HNE also activated EGFR downstream targets pERK/ERK and pAKT/AKT. Combined treatments of 4-HNE (1µM and 5µM,) with EGFR inhibitor AG1478 (0.1 µM -10µM), MEK inhibitor UO126 (0.5 µM -50µM), and PI3K inhibitor LY 294002 (0.5 µM -50µM), in separate experiments, caused enhanced cell death in RPE cells strongly indicating that the activation of EGFR mediated by 4-HNE is a protective mechanism against oxidative stress.

Conclusions: : These results suggest that activation of EGFR and its downstream pathway by 4-HNE is an adaptive response in RPE cells for protection from oxidative stress.

Keywords: growth factors/growth factor receptors • oxidation/oxidative or free radical damage • retinal pigment epithelium