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


CONCLUSIONS
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.
He
Shikun
S
Department of Pathology, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave., HMR 209, Los Angeles, CA 90033, USA.
Yaung
Jennifer
J
Kim
Yeong Hoon
YH
Barron
Ernesto
E
Ryan
Stephen J
SJ
Hinton
David R
DR
D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D005333:Fetus / Q000166:cytology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
C497249:DDIT3 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051742:Transcription Factor CHOP; D020122:tert-Butylhydroperoxide