Nitric Oxide Leads to Cytoskeletal Reorganization and Apoptosis in Retinal Pigment Epithelium

Abstract

Purpose: The molecular mechanism of oxidative stress-induced apoptosis in the RPE remains elusive. Our proteomics approach demonstrated that early biosignatures, including prohibitin, PP2A, vimentin, and nitric oxide synthase might be involved in the initiation of apoptosis as a positive or negative signal. Our quantitative analysis investigates how nitric oxide reorganizes cytoskeletal proteins in the RPE under oxidative stress and how the reorganization affects apoptotic signaling in the RPE.

Methods: Quantitative analysis using Chemiluminescence detection was performed to measure nitric oxide released from ARPE-19 cells exposed to bright fluorescent light (7000 lux), oxidative stress (200 µM tert-butyl hydroperoxide), reoxygenation (2 hrs hypoxic condition by N2 then 21% O2) and lipopolysaccharide (5-10 µg/mL). Early biomarkers, including inducible nitric oxide synthase (iNOS), tyrosine nitration, vimentin, and PP2A were analyzed by SDS-PAGE and Western blotting. Phosphorylations and nitrations were analyzed by mass spectrometry and phospho/Y-NO2 Western blotting, respectively. Immunocytochemistry and scanning electron microscopy were performed to visualize protein localization and cell morphology.

Results: Light, oxidative stress and reoxygenation play a positive regulatory role in the production of nitric oxide in the RPE. LPS, oxidative stress and light treatment significantly upregulated iNOS expression in RPE cells in concentration or time-dependent manner. Western blotting showed increased nitration in various proteins under oxidative stress. Immunostaining demonstrated vimentin filament disorganization in the RPE under light induced stress conditions. Scanning electron microscopy revealed cell surface changes during oxidative stress-induced RPE apoptosis. Analysis of AMD donor eyes, diabetic, and aged model demonstrated that tyrosine nitration increased compared to age-matching control.

Conclusions: Oxidative stress-induced disassembly of vimentin intermediate filaments may cause vimentin phosphorylation in the RPE. The study implies dual roles of NO that 1-10 nmole/(min x cm2) may protect RPE cell by PP2A and vimentin phosphorylation, whereas >100 nmole/(min x cm2) generated under oxidative stress may induce apoptosis, leading to AMD. Modulation of nitric oxide might be assigned as a therapeutic intervention to maintain the balanced phosphorylation status for cytoskeletal proteins and apoptosis.

Keywords: 701 retinal pigment epithelium • 617 nitric oxide