The importance of Bcl-xL in the survival of human RPE cells.

PURPOSE: In normal eyes and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cell survival is critically important. Bcl-x(L) has been shown to be among the most highly expressed survival factors in cultured human RPE cells. In the current study the effect of Bcl-x(L) blockade on human RPE cell survival was determined under normal conditions and after induced oxidative stress. METHODS: Cultured human RPE cells from three different donors were transfected with modified, 2'-O-methoxyethoxy Bcl-x(L)-mismatched control antisense oligonucleotides (ASOs), Bcl-x(L)-specific ASOs, and Bcl-x(L) splice switching oligonucleotides (SSOs), which shift the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) into Bcl-x(S), a proapoptotic factor. RNA and protein were harvested at various time points after transfection. Bcl-x(L) and Bcl-x(S) mRNA transcript levels were analyzed using gene-specific primers with reverse transcription-polymerase chain reaction. Bcl-x(L) protein levels were analyzed using Western blot. Cell viability was measured by WST-1 and lactate dehydrogenase (LDH) assays. The mode of cell death was determined with a cell death ELISA and an M30 assay. To study the effects of oxidative stress, the cells were stimulated after transfection with various concentrations of H(2)O(2.) Cell viability was analyzed by WST-1 (Roche, Indianapolis, IN) and LDH assays. RESULTS: After Bcl-x(L)-specific ASO and SSO transfections, Bcl-x(L) mRNA and protein levels were significantly reduced. Bcl-x(S) levels were increased after transfection with SSO. By day 8 after plating, the cells transfected with Bcl-x(L)-specific ASO had significantly decreased viability, which was further reduced by day 10. The SSO had an even more potent effect. Cell viability was reduced on day 4 after plating and by day 10, less than 10% of the cells were viable. Apoptotic cell death occurred as early as day 4 after plating. H(2)O(2), used as a model oxidant, further enhanced cell death induced by Bcl-x(L)-specific ASO and SSO. CONCLUSIONS: Bcl-x(L) plays an important role in human RPE cell survival under normal conditions and when cells are exposed to oxidative stress. Treatment strategies that enhance Bcl-x(L) expression and/or prevent conversion of Bcl-x(L) to Bcl-x(S) may be useful in preventing RPE cell death in AMD. Treatments that reduce Bcl-x(L) and enhance Bcl-x(S) may be useful in inhibiting unwanted RPE cell proliferation in PVR.

Caspase-8-mediated apoptosis in human RPE cells.

PURPOSE: Tumor necrosis factor (TNF)-alpha is an important cytokine associated with age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). TNF-alpha activates the extrinsic apoptotic pathway. In many cells, nuclear transcription factor (NF)-kappaB upregulates antiapoptotic proteins and prevents TNF-alpha-mediated apoptosis. However, retinal pigment epithelial (RPE) cells are resistant to TNF-alpha-induced apoptosis, even after specific NF-kappaB blockade. Herein, the authors investigated the role of caspase-8 in RPE cell resistance to TNF-alpha-mediated cell death. METHODS: Caspase-8 mRNA and protein expression were measured in human RPE cells, human lens epithelial cells, human trabecular meshwork (TM) cells, human choroidal endothelial cells, human uveal melanoma cells (OCM-1, 92.1 and MKT-BR), T-98G, OVCAR-3, HCT116, and Jurkat cancer cells by real-time reverse transcription-polymerase chain reaction and Western blot, respectively. RPE cells were coinfected with adenovirus encoding caspase-8 and Cre. RPE and T-98G cells were infected with adenovirus encoding mutant inhibitory (I)-kappaB and then were treated with media alone or with TNF-alpha. Cell viability was determined by WST-1 assay, and apoptosis was evaluated with DNA fragmentation assay and M30 assay. Caspase-3, -7, -9 expression and Bid protein expression after caspase-8 overexpression were examined by Western blot. RESULTS: Human RPE cell caspase-8 mRNA and protein levels were low compared with levels in nonneoplastic ocular cells and cancer cells. Overexpression of caspase-8 significantly decreased cell number, caused caspase-8 and caspase-3 activation, decreased full-length Bid, caspase-9, and caspase-7, and significantly increased DNA fragmentation and M30-positive RPE cells. Without TNF-alpha treatment, NF-kappaB blockade had no effect on caspase-8-mediated RPE cell death. In the presence of TNF-alpha, NF-kappaB blockade slightly but significantly enhanced caspase-8-mediated RPE cell death. CONCLUSIONS: RPE cell caspase-8 protein levels are low compared with levels for other cell types and may be regulated posttranscriptionally. Low caspase-8 levels may protect RPE cells from apoptosis normally and in diseases such as AMD and may promote the survival of abnormal cells in PVR. Introduction of caspase-8 into RPE cells may be a potential strategy to treat PVR.

Oxidant-mediated Akt activation in human RPE cells.

PURPOSE: To determine whether a model oxidant, hydrogen peroxide (H2O2), influences Akt activation and, if so, whether Akt activation promotes retinal pigment epithelial (RPE) cell survival. METHODS: Cultured human RPE cells were pretreated with medium alone, with LY294002 (LY), an inhibitor of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt, or with Akt/protein kinase B signaling inhibitor (API)-2, a specific Akt inhibitor, and then were stimulated with H2O2 at different doses for various times. Akt phosphorylation was evaluated by Western blot using antibody against phosphorylated Akt (Ser473). The effect of Akt blockade on RPE cell viability was assessed by tetrazolium salt (WST-1) assay and a lactate dehydrognease (LDH) release assay. Caspase-mediated cytokeratin cleavage, an early apoptosis marker, was assessed by M30 antibody staining. Caspase-independent apoptosis was determined by nuclear translocation of apoptosis-inducing factor (AIF). RPE cell morphology was evaluated by electron microscopy. The effect of H2O2 on downstream Akt targets was examined by Western blot using antibody against phosphorylated forkhead in rhabdomyosarcoma (FKHR) and phosphorylated glycogen synthase kinase (GSK)-3beta. RESULTS: H2O2 induced Akt phosphorylation in a dose-dependent manner and also induced the phosphorylation of downstream effectors FKHR and GSK-3beta. LY markedly inhibited H2O2-mediated Akt phosphorylation and significantly enhanced caspase-associated and caspase-independent RPE cell death. CONCLUSIONS: A model oxidant, H2O2, induces PI3K and thereby activates Akt. Akt activation enhances RPE cell survival and thus may protect RPE cells from oxidant-induced cell death under normal circumstances and in disease states such as age-related macular degeneration (AMD).

Human RPE expression of cell survival factors.

PURPOSE: To determine basal and tumor necrosis factor (TNF)-alpha-regulated expression of retinal pigment epithelial (RPE) cell survival factors and whether regulation is dependent on nuclear transcription factor (NF)-kappaB. METHODS: Cultured human RPE cells were infected with adenovirus encoding either mutant inhibitory (I)-kappaB or beta-galactosidase and treated with TNF-alpha for various times. Freshly prepared RPE/choroid and RPE samples were isolated from human donor eyes. Real-time reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used to determine survival factor gene expression, cellular protein levels, and localization, respectively. RESULTS: Multiple survival factor genes, including cellular inhibitor of apoptosis protein (c-IAP1), c-IAP2, TNF receptor-associated factor-1 (TRAF-1), TRAF-2, B-cell leukemia/lymphoma-2 (Bcl-2), Bcl-x, A1, and cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme-like inhibitory protein (c-FLIP), were expressed in basal conditions in both cultured RPE cells and RPE cells in situ, whereas survivin was expressed only by cultured cells. TNF-alpha upregulated expression of TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1. TRAF-1, c-FLIP, and to a lesser extent c-IAP2 protein levels were increased by TNF-alpha in a time-dependent manner, whereas c-IAP1, survivin, Bcl-x(L), and TRAF-2 protein levels were not influenced by TNF-alpha treatment at any time point tested. In contrast, Bcl-2 and A1 proteins were not detected under basal conditions or after TNF-alpha treatment. Overexpression of mutant IkappaB blocked TNF-alpha-induced TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1 gene expression and downregulated TRAF-1 protein levels. TRAF-1 and Bcl-x(L) proteins were localized diffusely in RPE cytoplasm. CONCLUSIONS: Multiple RPE cell survival factors are expressed by human RPE cells. TNF-alpha regulates expression of some of these factors in an NF-kappaB-dependent manner, whereas others are not influenced by NF-kappaB. RPE cell survival factors may protect RPE cells from apoptosis normally and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR).