alpha-Crystallin distribution in retinal pigment epithelium and effect of gene knockouts on sensitivity to oxidative stress.
OBJECTIVE
To investigate the susceptibility of retinal pigment epithelium (RPE) from alphaA (-/-) and alphaB (-/-) mice to oxidative stress, and the subcellular changes of alphaA and alphaB-crystallins under oxidative stress.


METHODS
The effect of hydrogen peroxide (H(2)O(2)) on apoptosis in RPE from alphaA (-/-), alphaB (-/-), and wild type (wt) mice was assessed by TUNEL and AnnexinV/Propidium Iodide assays. H(2)O(2)-induced changes in caspase-3 activity and mitochondrial permeability transition (MPT) were determined. Human RPE in early passages (2-4) were starved in 1% FBS-containing Dulbecco's modified Eagle medium (DMEM) and treated with H(2)O(2) for 24 h. Gene expression was quantitated by real time PCR. Confocal microscopy was used to examine alpha-crystallin compartmentalization. Whole cell and mitochondrial alpha-crystallin protein amounts were examined by transmission electron microscopy (TEM) and Western blot analysis.


RESULTS
RPE from alphaA (-/-), alphaB (-/-) mice exhibited increased susceptibility to apoptosis induced by H(2)O(2), increased caspase-3 activation, and increased MPT. Treatment of human RPE with H(2)O(2) resulted in a dose-dependent decrease in alphaB-crystallin mRNA expression. Confocal microscopy and subcellular fractionation of RPE showed that H(2)O(2) treatment decreased cytosolic and mitochondrial pools of alphaB-crystallin but caused no change in alphaA-crystallin content. TEM confirmed changes in expression of alphaA and alphaB-crystallins with oxidative stress.


CONCLUSIONS
Lack of alpha-crystallins renders RPE cells more susceptible to apoptosis from oxidative stress. Mitochondrial alpha-crystallins may play an important role in the protection from increased susceptibility of RPE in oxidative stress.
Yaung
Jennifer
J
Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Jin
Manlin
M
Barron
Ernesto
E
Spee
Christine
C
Wawrousek
Eric F
EF
Kannan
Ram
R
Hinton
David R
DR
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D010539:Permeability; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D014018:Tissue Distribution / Q000187:drug effects; D038202:alpha-Crystallin A Chain / Q000235:genetics / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000235:genetics / Q000378:metabolism; D038201:alpha-Crystallins / Q000172:deficiency* / Q000378:metabolism*
D016877:Oxidants; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D038201:alpha-Crystallins; D006861:Hydrogen Peroxide; D053148:Caspase 3