Photo-oxidative Stress Down-modulates the Activity of Nuclear Factor-κB via Involvement of Caspase-1, Leading to Apoptosis of Photoreceptor Cells

doi:10.1074/jbc.274.6.3734

Photo-oxidative Stress Down-modulates the Activity of Nuclear Factor-κB via Involvement of Caspase-1, Leading to Apoptosis of Photoreceptor Cells*

From the Department of Anatomy and Cell Biology, North Texas Eye Research Institute at University of North Texas Health Science Center, Fort Worth, Texas 76107, the ^§Department of Pediatrics, Louisiana State University Medical Center, Shreveport, Louisiana 71130, the ^‡Department of Molecular Oncology, Cytokine Research Laboratory, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and the ^¶Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois 60612

Abstract

The mechanisms of photoreceptor cell death via apoptosis, in retinal dystrophies, are largely not understood. In the present report we show that visible light exposure of mouse cultured 661W photoreceptor cells at 4.5 milliwatt/cm² caused a significant increase in oxidative damage of 661W cells, leading to apoptosis of these cells. These cells show constitutive expression of nuclear factor-κB (NF-κB), and light exposure of photoreceptor cells results in lowering of NF-κB levels in both the nuclear and cytosolic fractions in a time-dependent manner. Immunoblot analysis of IκBα and p50, and p65 (RelA) subunits of NF-κB, suggested that photo-oxidative stress results in their depletion. Immunocytochemical studies using antibody to RelA subunit of NF-κB further revealed the presence of this subunit constitutively both in the nucleus and cytoplasm of the 661W cells. Upon exposure to photo-oxidative stress, a depletion of the cytoplasmic and nuclear RelA subunit was observed. The depletion of NF-κB appears to be mediated through involvement of caspase-1. Furthermore, transfection of these cells with a dominant negative mutant IκBα greatly enhanced the kinetics of down modulation of NF-κB, resulting in a faster photo-oxidative stress-induced apoptosis. Taken together, these studies show that the presence of NF-κB RelA subunit in the nucleus is essential for protection of photoreceptor cells against apoptosis mediated by an oxidative pathway.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ Recipient of a grant from Foundation Fighting Blindness and Knights Templer Eye Foundation, Inc.
↵** To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. Tel.: 817-735-2094; E-mail:nagarwal{at}hsc.unt.edu.
↵2 M. J. Crawford, R. R. Krishnamoorthy, H. J. Sheedlo, D. T. Organisciak, R. S. Roque, N. Agarwal, and M. R. Al-Ubaidi, submitted for publication.
Abbreviations:

NF-κB

nuclear factor-κB

EMSA

electrophoretic mobility shift assay

TUNEL

terminal deoxynucleotidyl transferase mediated fluoresceinated dUTP nick end labeling

GAPDH

glyceraldehyde phosphate dehydrogenase

GSH

glutathione-reduced

NAC

N-acetylcysteine

ALLN

N-acetylleucylleucylnorleucinal

IκBα

inhibitory subunit of NF-κB

IκB αΔN

super-repressor of IκB α

FITC

fluorescein isothiocyanate

TNF-α

tumor necrosis factor-α

ROI

reactive oxygen intermediates

MDCK

Madin-Darby canine kidney.
- Received October 8, 1998.
- Revision received November 13, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.