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The Importance of Bcl-x_L in the Survival of Human RPE Cells

¹From the Department of Ophthalmology, Duke University, Durham, North Carolina; and the ²Lineberger Comprehensive Cancer Center and the ³Department of Pharmacology, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina.

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 H₂O_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. H₂O₂, 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.

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				Y. Chen, P. Sternberg, and J. Cai Characterization of a Bcl-XL-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1721 - 1727. [Abstract] [Full Text] [PDF]