Abstract
Purpose: :
Retinal pigment epithelial cells (RPE) support rods and cones. Failure to accomplish their function leads to the death of photoreceptors and blindness. Apoptotic cell death of photoreceptors takes place in degenerative diseases including retinitis pigmentosa and macular degeneration. Oxidative stress injury triggers apoptotic damage on RPE cells enhancing at the same time, the enzymatic production of NPD1, a DHA-oxygenation product (Mukherjee et al, 2004, PNAS 101(22): 8491-8496). NPD1 is a potent neuroprotective lipid mediator that inhibits the expression of pro-inflammatory proteins and enhances the expression of anti-apoptotic Bcl2 family (Bazan NG, 2007, IOVS 48(11):4866-81) and hence promoting cell survival. The biosynthetic pathway leading to the oxygenation of DHA into NPD1 is presently unknown. We hypothesize that 15-Lipoxygenase-1 modulates the synthesis of NPD1 in response to injury in RPE cells.
Methods: :
shRNA technology was used to knock-down 15LOX-1 in ARPE-19 cells. The oxidative stress-induced apoptosis was assessed using Hoechst staining. The biosynthesis of NPD1 and other 15LOX-1 products were measured using liquid chromatography- tandem mass spectrometry. Immunocytochemistry was used to define cellular localization of 15LOX-1 and its expression was quantified by Western blot analysis. Rescue experiments were performed treating 15LOX-1 deficient cells and non-specific shRNA control cells with H2O2/TNFα and 15(S)HETE, 12(S)HETE, Lipoxin A4, DHA/PEDF and NPD1 50nM.
Results: :
A stable cell line was established in which the expression of 15LOX-1 was decreased in a 68% and 15LOX-2, another isoform, was not affected. 15LOX-1 showed a cytoplasm localization. Upon exposure to oxidative stress, 15(S) HETE and 12(S) HETE, two products of the enzyme, were depleted in the silenced cell line. In contrast with non deficient cells, 15LOX-1 deficient cells failed to activate NPD1 synthesis after being challenged with oxidative stress indicating the 15LOX-1 involvement in the synthesis of NPD1. Furthermore, the 15LOX-1 deficient cell line showed to be more susceptible to oxidative stress-induced apoptosis than the control cells. 15(S) HETE, 12(S) HETE and lipoxinA4 as well as DHA/PEDF treatment failed to rescue the cell line from damaged caused by oxidative stress. On the contrary, NPD1 was the only mediator that fully rescued the cells from apoptosis.
Conclusions: :
Here we demonstrate that 15LOX-1 is directly involved in the production of NPD1 and hence a key enzyme in RPE cell survival regulation.
Keywords: neuroprotection • retinal pigment epithelium • second messengers