Purchase this article with an account.
Ram Kannan, Guorui Dou, Parameswaran G. Sreekumar, Stephen J. Ryan, David R. Hinton; Signaling Mechanisms Involved in ER Stress-induced Caspase 4 Activation and Mitochondrial Dysfunction in Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):885.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Endoplasmic reticulum (ER) stress has been linked to several pathological conditions including age-related macular degeneration. However, the mechanism by which ER stress induces RPE cell death is not well understood. The aim of the present study was to investigate the role of caspase 4 in ER stress-mediated RPE cell death and its associated signaling pathways.
Expression levels of cleaved caspase 3, caspase 4 along with GRP78 and CHOP were examined in primary cultured human RPE cells treated with varying doses of tunicamycin (TM, 0.1-10 ug/ml) for 1-24h. Caspase 4 expression was determined by confocal immunofluorescent microscopy using ER tracker and by western blot analysis. Generation of reactive oxygen species (ROS) was quantified by confocal microscopy and flow cytometry using MitoSox. The effect of tunicamycin (TM) on caspase 3 activation, cytochrome c release and mitochondrial permeability transition (MPT) was also studied. Further, the effect of unfolded protein response (UPR) inhibitors salubrinal (eIF2alpha activator), 4-(2-aminoethyl) benzene sulfonyl fluoride (AEBSF, inhibitor of ATF6 proteolysis) on TM-induced ER stress was also studied.
ER stress with TM increased with time as indicated by increased expression of GRP78 and CHOP in RPE. A dose dependent increase in cleaved caspase 4 expression with TM treatment was found. This was accompanied by an increase in ROS production, cytochrome c release and caspase 3 activation. Both salubrinal and AEBSF reduced ER stress induced apoptosis significantly (p<0.05 vs untreated controls). 2h pretreatment with salubrinal significantly (p<0.05) activated phosphorylation of eIF2alpha while AEBSF pretreatment inhibited proteolysis and inhibition of ATF6.
ER stress induced by TM results in activation of caspase 4 and leads to cell death by participation of mitochondrial events (ROS, caspase 3). Our data also suggest that the PERK-eIF2alpha pathway may be involved in this process.
This PDF is available to Subscribers Only