α-Crystallin distribution in retinal pigment epithelium and effect of gene knockouts on sensitivity to oxidative stress

PURPOSE: To investigate the susceptibility of retinal pigment epithelium (RPE) from αA (-/-) and αB (-/-) mice to oxidative stress, and the subcellular changes of αA and αB-crystallins under oxidative stress. METHODS: The effect of hydrogen peroxide (H(2)O(2)) on apoptosis in RPE from αA (-/-), αB (-/-), 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 α-crystallin compartmentalization. Whole cell and mitochondrial α-crystallin protein amounts were examined by transmission electron microscopy (TEM) and Western blot analysis. RESULTS: RPE from αA (-/-), αB (-/-) 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 αB-crystallin mRNA expression. Confocal microscopy and subcellular fractionation of RPE showed that H(2)O(2) treatment decreased cytosolic and mitochondrial pools of αB-crystallin but caused no change in αA-crystallin content. TEM confirmed changes in expression of αA and αB-crystallins with oxidative stress. CONCLUSIONS: Lack of α-crystallins renders RPE cells more susceptible to apoptosis from oxidative stress. Mitochondrial α-crystallins may play an important role in the protection from increased susceptibility of RPE in oxidative stress.