April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Signaling Mechanisms Involved in ER Stress-induced Caspase 4 Activation and Mitochondrial Dysfunction in Human RPE Cells
Ram Kannan; Guorui Dou; Parameswaran G. Sreekumar; Stephen J. Ryan; David R. Hinton
Author Affiliations & Notes
  • Ram Kannan
    Arnold and Mabel Beckman Macular Researcn Center, Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • Guorui Dou
    Arnold and Mabel Beckman Macular Researcn Center, Doheny Eye Institute, Los Angeles, California
  • Parameswaran G. Sreekumar
    Arnold and Mabel Beckman Macular Researcn Center, Doheny Eye Institute, Los Angeles, California
  • Stephen J. Ryan
    Arnold and Mabel Beckman Macular Researcn Center, Doheny Eye Institute, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • David R. Hinton
    Arnold and Mabel Beckman Macular Researcn Center, Doheny Eye Institute, Los Angeles, California
    Pathology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • Footnotes
    Commercial Relationships  Ram Kannan, None; Guorui Dou, None; Parameswaran G. Sreekumar, None; Stephen J. Ryan, None; David R. Hinton, None
  • Footnotes
    Support  NIH grants EY01545, EY03040 and grants from RPB and Arnold and Mabel Beckman Foundation.
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 885. doi:
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      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.

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: retinal pigment epithelium • mitochondria • apoptosis/cell death 
© 2011, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2024 Association for Research in Vision and Ophthalmology.
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