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P.K. Mukherjee, S.G. Barreiro, N.G. Bazan; NF-B and Oxidative Stress Induced Apoptosis in Human Retinal Pigment Epithelium (RPE) Cells Differentially Inhibited by PAF Antagonists . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4547.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Nuclear transcription factor NF-ΚB plays a major role in cell signaling. Up-regulation of NF-ΚB is associated with inflamation. Platelet activating factor(PAF), Interleukine 1beta (IL-1ß) and TNF- α a pro-inflammatory agents that cause gene expression. To understand the role of PAF antagonists in NF-ΚB and oxidative stress induced apoptosis, we tranfected human retinal pigment epithelium (ARPE-19) cells with a NF–ΚB enhancer sequences (four tandem repeats) linked to a luciferase reporter gene, induced with PAF, IL-1ß and TNF-α and challenged with either intracellular or extracellular PAF antagonists. Apoptotic cell death induced by NF-ΚB and oxidative stress induced by TNF and H2O2 were also measured under similar treatment. Methods: ARPE-19 cells were transected with NF–ΚB -luciferase construct using DOTAP . Promoterless beta-galactosidase construct was co-transfected to measure the transfection efficiency. Four hours later, cells were fed normal medium and incubated 12 hours. Then transected cells were serum starved for 8 hours before the addition of inducers. The cells were induced with PAF(100nM), IL-1ß (10ng/ml), TNF-α (10 ng/ml) for 8 hours. Oxidative stress was induced in the RPE cells by TNF(10ng/ml) and H2O2 (0.6uM) for 8 hours. In some experiments BN 50727(extracellular PAF antagonist), LAU 8080(intracellular PAF antagonist) at a concentration of 100nM, DHA (100uM), Arachidonic acid(100uM) and PGH2(100uM) were added together with the inducers. Luciferase assays were measured in ALL Luminometer using Luciferin as substrate and apoptotic cell was monitored by Hoechst staining with confocal microscopy. Results: PAF, IL-1ß and TNF-α enhance the activation of NF-ΚB in RPE cells. This up-regulation of NF-ΚB was substantially inhibited by LAU 8080, but not by BN 50727.Contrary to this, other bio-active lipids DHA, AA and PGH2 stabilized the IL-1b and TNF-a induced activation of NF-ΚB and was not affected by either of the PAF antagonists. The intracellular PAF antagonist LAU 8080 exhibited a profound inhibition of apoptotic cell death induced by NF-ΚB and oxidative stress. On the other hand, BN 50727, the extracellular antagonist was unable to protect the apoptotic cell death. Conclusion: The finding that inhibition of apoptotic cell death by LAU 8080, an intracellular PAF receptor but not by an extracellular receptor indicated that NF-ΚB and oxidative stress induced apoptosis may involve PAF signaling cascade and PAF antagonists may modulate this decision making process between survival and death.
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