Background: 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.
Methods: 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.
Results: 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.
Conclusion: 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.