Ceramide: A potential mediator of apoptosis in human retinal pigment epithelial cells

PURPOSE. To investigate the signal transduction mechanisms involved in the cell death of human retinal pigment epithelial (RPE) cells after their exposure to either hydrogen peroxide (H₂O₂) or tri-butyl hydroxperoxide (tBH). METHODS. Cultured human RPE (hRPE) cells were treated with the chemical oxidants tBH and H₂O₂ as well as with the synthetic ceramide analogs C₂, C₆, and dihydroceramide for different time periods. Apoptosis was determined by TUNEL staining and annexin-V labeling of phosphatidylserine exposure. Ceramide levels were quantified by the diacylglycerol kinase assay using thin-layer chromatography. RESULTS. H₂O₂ and tBH caused a high level of apoptosis in the hRPE cells. At the same time, both of these oxidants induced an early and late increase in the intracellular production of ceramide, a lipid second messenger. Moreover, addition of C₂ and C₆ synthetic ceramides caused a high level of apoptosis in these hRPE cells. In contrast, treatment with the immediate precursor of ceramide, dihydroceramide, resulted in no apoptotic response. CONCLUSIONS. The results demonstrate that H₂O₂ and tBH induce apoptosis in hRPE cells and suggest that the underlying signaling mechanism involves ceramide generation.