A Novel Inhibitor of 5-Lipoxygenase (5-LOX) Prevents Oxidative Stress-Induced Cell Death of Retinal Pigment Epithelium (RPE) Cells.

PURPOSE: 5-Lipoxygenase (5-LOX) oxygenates arachidonic acid to form 5-hydroperoxyeicosatetraenoic acid, which is further converted into biologically detrimental leukotrienes, such as leukotriene B4 (LTB4). The RPE and retina express the PNPLA2 gene for pigment epithelium-derived factor receptor (PEDF-R), a lipase involved in cell survival. The purpose here was to investigate the role of PEDF-R on the 5-LOX pathway in oxidative stress of RPE.

METHODS: Lipoxygenase activity assays were performed with soybean and potato lipoxygenase. Binding was evaluated by peptide-affinity chromatography and pull-down assays with PEDF-R-derived synthetic peptides or recombinant protein. Oxidative stress was induced in human ARPE-19 and primary pig RPE cells with indicated concentrations of H2O2/TNF-α. Reverse transcription-PCR of ALOX5 and PNPLA2 genes was performed. Cell viability and death rates were determined using respective biomarkers. Leukotriene B4 levels were measured by ELISA.

RESULTS: Among five peptides spanning between positions Leu159 and Met325 of human PEDF-R polypeptide, only two overlapping peptides, E5b and P1, bound and inhibited lipoxygenase activity. Human recombinant 5-LOX bound specifically to peptide P1 and to His6/Xpress-tagged PEDF-R via ionic interactions. The two inhibitor peptides E5b and P1 promoted cell viability and decreased cell death of RPE cells undergoing oxidative stress. Oxidative stress decreased the levels of PNPLA2 transcripts with no effect on ALOX5 expression. Exogenous additions of P1 peptide or overexpression of the PNPLA2 gene decreased both LTB4 levels and death of RPE cells undergoing oxidative stress.

CONCLUSIONS: A novel peptide region of PEDF-R inhibits 5-LOX, which intersects with RPE cell death pathways induced by oxidative stress.