Abstract
Purpose: :
Endoplasmic reticulum (ER) stress has been implicated in cell survival and apoptosis. However, the role of ER stress in retinal pigment epithelium (RPE) damage and its implication in age-related macular degeneration (AMD), the leading cause of irreversible vision loss in the aging population, is poorly understood. The purpose of this study is to test the hypothesis that cigarette smoking, a single risk factor for AMD, induces ER stress resulting in apoptosis in RPE cells, and that inhibition of ER stress alleviates cigarette smoking-triggered RPE injury and cell death.
Methods: :
Human RPE (ARPE19) cells were exposed to hydroquinone (HQ), a potent oxidant in cigarette smoke, for 0-24 h. ER stress markers were determined by Western blot analysis, RT-PCR, real-time RT-PCR and immunocytochemistry. Apoptosis and cell death were detected by TUNEL assay and trypan blue staining. ER stress was manipulated by chemical chaperone 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA). Over-expression of X-box binding protein 1 (XBP1) was achieved by adenoviruses expressing spliced XBP1.
Results: :
Expression of GRP78, a major ER chaperone implicated in protein folding, was rapidly increased in HQ-treated ARPE-19 cells. Consistently, XBP1 splicing is enhanced as early as 1 h after HQ treatment, indicative of the activation of the ER stress sensor IRE1α. Simultaneously, phosphorylation of PERK, another prominent ER stress sensor, was increased in HQ-treated cells. These changes were accompanied by sequential phosphorylation of eIF2α, elevated nuclear level of ATF4 and pro-apoptotic transcription factor CHOP, and increased expression of cleaved caspase 3. These results indicate an activation of ER stress-associated apoptotic cascade. Inhibition of ER stress by 4-PBA or TUDCA remarkably alleviated HQ-induced apoptosis of RPE cells. In addition, over-expression of XBP1, a key regulator of unfold protein response to eliminate ER stress, inhibited eIF2α phosphorylation and abolished CHOP and ATF4 induction in HQ-treated cells.
Conclusions: :
Taken together, these results suggest that ER stress plays a causal role in RPE cell apoptosis induced by cigarette smoking in AMD. Inhibition of ER stress by chemical chaperones or by enhancing XBP1 activity may represent a novel therapeutic strategy to prevent and treat AMD .
Keywords: age-related macular degeneration • retinal pigment epithelium • apoptosis/cell death