alpha-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 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.

Hepatocyte growth factor protects RPE cells from apoptosis induced by glutathione depletion.

PURPOSE: To study the mechanism of the protective effect of hepatocyte growth factor (HGF) in oxidative injury to RPE cells induced by glutathione (GSH) depletion. METHODS: RPE cells were treated with HGF for 24 hours (20 ng/mL) and then were treated with DL-buthionine-(S,R)-sulfoximine (BSO) for an additional 24 hours. Cell death, apoptosis, and GSH levels were measured. Levels of intracellular reactive oxygen species (ROS) and their cellular localization were assessed by confocal microscopy. Expression of Bcl-2 and release of cytochrome c from mitochondria were quantified. The effect of BSO on caspase-3 activation and expression was determined. Gene expression of key enzymes of GSH metabolism by real-time PCR and regulation and translocation of the transcription factor NF-E2-related factor (Nrf2) by BSO were examined. RESULTS: Treatment with BSO-induced apoptosis in RPE caused a significant decrease in intracellular GSH and in GSH/GSSG ratios. Marked increases in lipid peroxidase (LPO), ROS, and mitochondrial cytochrome c release and a decrease in Bcl-2 expression were observed. Elevated GSH/GSSG ratio (especially in mitochondria), decreased LPO and ROS, attenuation of apoptosis, and partial restoration of Bcl-2 expression were found in the HGF-pretreated cells. BSO activated caspase-3, and this effect was significantly blocked by HGF. Both HGF and BSO induced anti-oxidant gene expression. Nrf2 translocated to the nuclear region after treatment with BSO, whereas HGF did not induce such translocation. CONCLUSIONS: The protective effect of HGF may be attributed in part to the elevation of mitochondrial GSH. BSO and HGF act in concert to enhance GSH-related gene expression in stressed RPE cells.

Endoplasmic reticulum stress induced by oxidative stress in retinal pigment epithelial cells.

BACKGROUND: Induction of glucose-regulated protein (GRP)-78 in the endoplasmic reticulum (ER) is a protective mechanism cells use to adapt to ER stress. We evaluated the expression of GRP-78 and its regulation by an oxidant tert-butyl hydroperoxide (tBH) in human retinal pigment epithelium (RPE) cells. METHODS: We used a carboxy-H2-DCFDA staining method to detect tBH-induced accumulation of reactive oxygen species (ROS) in RPE cells, and analyzed the expression of GRP-78 in normal human fetal and adult retinas and in cultured human RPE cells by immunohistochemistry. The effects of tBH (10-100 microM) on GRP-78 and on growth arrest and DNA damage inducible genes 153 (GADD153) protein and mRNA expression were studied using Western blot and real-time polymerase chain reaction. RESULTS: Sections of fetal retinas were negative for GRP-78. Adult retinas showed moderate cytoplasmic GRP-78 staining in the RPE and choroid. tBH-induced ROS accumulation in RPE cells showed partial colocalization with the ER. GRP-78 and GADD153 mRNA and protein expression in cultured RPE cells were significantly upregulated by treatment with tBH. CONCLUSION: tBH increases oxidative stress, increases accumulation of ROS in the ER, and upregulates expression of GRP-78 and GADD153. This supports the connection between oxidative stress and ER stress, and suggests that GRP-78 may serve a protective role in the RPE response to oxidative stress.

Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia.

This study evaluated the role of crystallins in retinal degeneration induced by chemical hypoxia. Wild-type, alphaA-crystallin (-/-), and alphaB-crystallin (-/-) mice received intravitreal injection of 12 nmol (low dose), 33 nmol (intermediate dose) or 60 nmol (high dose) cobalt chloride (CoCl(2)). Hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) stains were performed after 24 h, 96 h, and 1 week post-injection, while immunofluorescent stains for alphaA- and alphaB-crystallin were performed 1 week post-injection. The in vitro effects of CoCl(2) on alphaB-crystallin expression in ARPE-19 cells were determined by real time RT-PCR, Western blot, and confocal microscopy and studies evaluating subcellular distribution of alphaB-crystallin in the mitochondria and cytosol were also performed. Histologic studies revealed progressive retinal degeneration with CoCl(2) injection in wild-type mice. Retinas of CoCl(2) injected mice showed transient increased expression of HIF-1alpha which was maximal 24h after injection. Intermediate-dose CoCl(2) injection was associated with increased retinal immunofluorescence for both alphaA- and alphaB-crystallin; however, after high-dose injection, increased retinal degeneration was associated with decreased levels of crystallin expression. Injection of CoCl(2) at either intermediate or high dose in alphaA-crystallin (-/-) and alphaB-crystallin (-/-) mice resulted in much more severe retinal degeneration compared to wild-type eyes. A decrease in ARPE-19 total and cytosolic alphaB-crystallin expression with increasing CoCl(2) treatment and an increase in mitochondrial alphaB-crystallin were found. We conclude that lack of alpha-crystallins accentuates retinal degeneration in chemically induced hypoxia in vivo.

Protection from oxidative stress by methionine sulfoxide reductases in RPE cells.

We investigated the role of methionine sulfoxide reductases (Msrs) in oxidant-stress-induced cell death in retinal pigmented epithelial (RPE) cells. In RPE cells exposed to varying doses of H(2)O(2), gene expression of MsrA and hCBS-1 (the human analog of MsrB2) increased in a dose-dependent and time-dependent manner with maximal increase with 150 microM H(2)O(2) in 24h. H(2)O(2) treatment resulted in the generation of reactive oxygen species and activation of caspase 3. Confocal microscopic and protein analysis showed an increase in MsrA expression in cytosol and mitochondria. Silencing of MsrA resulted in caspase 3 induction and accentuated cell death from H(2)O(2). Focal, strong immunoreactivity for MsrA was observed in sub-RPE macular drusen from patients with age-related macular degeneration. In summary, our data show that MsrA and hCBS-1 are up-regulated in oxidative stress to counteract injury to RPE.