Endoplasmic reticulum stress induced by oxidative stress in retinal pigment epithelial cells

• DOI: 10.1007/s00417-008-0770-2
• Publication Date: 2008-02-15T03:06:10Z
• Authors: Shikun He; Jennifer Yaung; Yeong Hoon Kim; Ernesto Barron; Stephen J. Ryan; David R. Hinton
• Keywords: Aged, 80 and over; 0303 health sciences; Reverse Transcriptase Polymerase Chain Reaction; Blotting, Western; Endoplasmic Reticulum; Up-Regulation; Oxidative Stress; 03 medical and health sciences; Fetus; tert-Butylhydroperoxide; Humans; RNA, Messenger; Pigment Epithelium of Eye; Reactive Oxygen Species; Endoplasmic Reticulum Chaperone BiP; Cells, Cultured; Heat-Shock Proteins; Transcription Factor CHOP; Aged; Molecular Chaperones

Induction of glucose-regulated protein (GRP)-78 in the endoplasmic reticulum (ER) is a protective mechanism cells use to adapt to ER stress. We evaluated the expression of GRP-78 and its regulation by an oxidant tert-butyl hydroperoxide (tBH) in human retinal pigment epithelium (RPE) cells.We used a carboxy-H2-DCFDA staining method to detect tBH-induced accumulation of reactive oxygen species (ROS) in RPE cells, and analyzed the expression of GRP-78 in normal human fetal and adult retinas and in cultured human RPE cells by immunohistochemistry. The effects of tBH (10-100 microM) on GRP-78 and on growth arrest and DNA damage inducible genes 153 (GADD153) protein and mRNA expression were studied using Western blot and real-time polymerase chain reaction.Sections of fetal retinas were negative for GRP-78. Adult retinas showed moderate cytoplasmic GRP-78 staining in the RPE and choroid. tBH-induced ROS accumulation in RPE cells showed partial colocalization with the ER. GRP-78 and GADD153 mRNA and protein expression in cultured RPE cells were significantly upregulated by treatment with tBH.tBH increases oxidative stress, increases accumulation of ROS in the ER, and upregulates expression of GRP-78 and GADD153. This supports the connection between oxidative stress and ER stress, and suggests that GRP-78 may serve a protective role in the RPE response to oxidative stress.