Abstract
Abstract: :
Purpose:N–retinylidene–N–retinylethanolamine (A2E), a hydrophobic fluorophore from lipofuscin, accumulates in relatively high concentrations in retinal pigment epithelial cells in Stargardt’s disease and other retinal degenerations. In fact, A2E triggers damage and cell death in the RPE. The present study aimed to define the intracellular signaling of A2E and its relationship to NF–ΚB induction and apoptosis. Methods:ARPE–19 cells grown in 6–well plates were transfected by Fugene 6 with NF–ΚB promoter sequence linked to a luciferase reporter gene and with IΚB–EGFP. Promoterless ß–galactosidase was co–transfected in ARPE–19 cells to determine the transfection efficiency. Four hours after transfection, cells were washed and further incubated 8 to 10 hr before adding inducers. Two different set of cells were used. One set of cells was serum–starved 5 hr before the addition of A2E and TNF–α/H2O2, and further incubated for 20–24 hr before harvesting. The other set of cells was not serum–starved or further incubated. Luciferase activity was measured. The IΚB–EGFP probe was measured by fluorescence confocal microscopy and Hoechst–positive cells were identified. Results:A2E and TNF–α/H2O2 enhanced the expression of NF–ΚB in ARPE cells. Low concentrations of A2E (25 µM ) had no effect on RPE; however, concentrations up to 200 µM induced NF–ΚB and apoptosis shown by Hoechst staining. Transfection with IΚB–EGFP resulted in up–regulation of NF–ΚB after 20 hours. Hoechst staining of A2E–induced ARPE–19 cells indicated apoptotic cell death by the same time. Conclusions:NF–ΚB and IΚB are activated by A2E and oxidative stress. This indicates that the IΚB–NF–ΚB complex has been dissociated. Subsequently NF–ΚB translocates to the nucleus and induces pro–inflammatory gene expression. Moreover, accumulation of A2E in ARPE–19 cells participates in apoptotic cell death through NF–ΚB. Low concentrations of A2E are not sufficient to trigger these changes. Pro–inflammatory signaling at the level of NF–ΚB triggered by A2E is a potential drug target to attenuate RPE cell apoptosis. (R01EY05121; Neurobiotech. Prog. of LA)
Keywords: apoptosis/cell death • transcription factors • retinal degenerations: cell biology