Yaung Jennifer, Jin Manlin, Barron Ernesto, Spee Christine, Wawrousek Eric F, Kannan Ram, Hinton David R
Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Mol Vis. 2007 Apr 4;13:566-77.
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.
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.
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.
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.
研究αA（-/-）和αB（-/-）小鼠视网膜色素上皮（RPE）对氧化应激的易感性，以及氧化应激下αA和αB晶状体蛋白的亚细胞变化。
通过TUNEL和AnnexinV/碘化丙啶检测评估过氧化氢（H₂O₂）对αA（-/-）、αB（-/-）和野生型（wt）小鼠RPE细胞凋亡的影响。测定H₂O₂诱导的半胱天冬酶-3活性变化和线粒体通透性转换（MPT）。将早期传代（2-4代）的人RPE细胞在含1%胎牛血清的杜氏改良 Eagle培养基（DMEM）中饥饿培养，并用H₂O₂处理24小时。通过实时PCR定量基因表达。共聚焦显微镜用于检查α晶状体蛋白的分区。通过透射电子显微镜（TEM）和蛋白质免疫印迹分析检测全细胞和线粒体α晶状体蛋白的含量。
αA（-/-）、αB（-/-）小鼠的RPE细胞对H₂O₂诱导的凋亡敏感性增加，半胱天冬酶-3激活增加，MPT增加。用H₂O₂处理人RPE细胞导致αB晶状体蛋白mRNA表达呈剂量依赖性下降。共聚焦显微镜和RPE细胞亚细胞分级分离显示，H₂O₂处理降低了αB晶状体蛋白的胞质和线粒体池，但αA晶状体蛋白含量没有变化。TEM证实了氧化应激下αA和αB晶状体蛋白表达的变化。
缺乏α晶状体蛋白使RPE细胞更容易受到氧化应激诱导的凋亡。线粒体α晶状体蛋白可能在保护RPE细胞免受氧化应激增加的易感性方面发挥重要作用。
