Abstract
Abstract: :
Purpose: To investigate the cellular mechanisms involved in ceramide–induced apoptosis in RPE cells and whether hepatocyte growth factor (HGF) modulates this response. Methods: Confluent fetal human RPE cells were treated with varying concentrations of C2 ceramide for 24 h. Apoptosis was quantified by TUNEL assay. Dose–dependent production of reactive oxygen species (ROS), localization to mitochondria, and effect of HGF on ROS were studied by dual staining. C2 ceramide–induced changes in membrane permeability transition (MPT) were assessed by flow cytometry. Activation of caspase 3 by ceramide and effect of pretreatment with HGF on caspase 3 activation were examined by immunofluorescence confocal microscopy. Expression and activity of catalase in C2 ceramide–induced apoptosis and its potentiation by H2O2 were studied by western blot and flow cytometric analyses. Results: RPE incubated with C2 ceramide accumulated ROS in mitochondria, and underwent apoptosis in a dose–dependent manner. Ceramide treated cells showed increased caspase 3 activation and an increase in MPT. Low doses of H2O2 (100 µM) alone induced negligible apoptosis; however, ceramide–induced apoptosis was significantly enhanced by co–incubation with H2O2 (100 µM). Ceramide treatment significantly decreased catalase enzymatic activity and protein expression. HGF pretreatment (20 ng/ml) significantly inhibited ceramide–induced apoptosis, and reduced the accumulation of ROS, the activation of caspase–3, and the increase in MPT, as well as blocking the reduction in catalase activity. Conclusions: Ceramide exposure induces apoptosis in RPE which is potentiated by H2O2 and is associated with decreased catalase activity, suggesting that catalase plays a central role in regulating the apoptotic response. The ability of HGF to attenuate the above effects demonstrates its effectiveness as an antioxidant growth factor.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • apoptosis/cell death