Selective Survival Rescue in 15-Lipoxygenase-1-deficient Retinal Pigment Epithelial Cells by the Novel Docosahexaenoic Acid-derived Mediator, Neuroprotectin D1

Abstract

The integrity of the retinal pigment epithelial (RPE) cell is essential for the survival of rod and cone photoreceptor cells. Several stressors, including reactive oxygen species, trigger apoptotic damage in RPE cells preceded by an anti-inflammatory, pro-survival response, the formation of neuroprotectin D1 (NPD1), an oxygenation product derived from the essential omega-3 fatty acid family member docosahexaenoic acid. To define the ability of NPD1 and other endogenous novel lipid mediators in cell survival, we generated a stable knockdown human RPE (ARPE-19) cell line using short hairpin RNA to target 15-lipoxygenase-1. The 15-lipoxygenase-1-deficient cells exhibited 30% of the protein expression, and 15-lipoxygenase-2 remained unchanged, as compared with an ARPE-19 cell line control established using nonspecific short hairpin RNA transfected cells. NPD1 synthesis was stimulated by tumor necrosis factor ␣/H 2 O 2 -mediated oxidative stress in nonspecific cells (controls), whereas in silenced cells, negligible amounts of NPD1, 12(S)-and 15(S)-hydroxyeicosatetraenoic acid, and lipoxin A 4 were found under these conditions. Neither control nor the deficient cells showed an increase in 15-lipoxygenase-1 protein content after 16 h of oxidative stress, suggesting that the increased activity of 15-lipoxygenase-1 is due to activation of pre-existing proteins. 15-Lipoxygenase-silenced cells also displayed an exacerbated sensitivity to oxidative stress-induced apoptosis when compared with the control cells. NPD1 selectively and potently rescued 15-lipoxygenase-silenced cells from oxidative stress-induced apoptosis. These results demonstrate that 15-lipoxygenase-1 is activated by oxidative stress in ARPE-19 cells and that NPD1 is part of an early survival signaling in RPE cells. Retinal pigment epithelial (RPE)2 cells are essential for the survival of rod and cone photoreceptors. RPE cells mediate the renewal of photoreceptor outer segments (1, 2); synthesis and secretion of neurotrophins (3); recycling of bleached visual pigments; and transport of vitamin A (4), docosahexaenoic acid (DHA), and other nutrients, and ions and fluids between photoreceptors and the choriocapillaries (5). Failure of RPE cells to accomplish their functions leads to photoreceptor damage or death and, as a consequence, decreased vision and eventually blindness. Apoptotic cell death of RPE cells takes place in retinal degenerative diseases, including retinitis pigmentosa and age-related macular degeneration; as a result, photoreceptors degenerate (6, 7).Cell fate decisions made at the ectoderm yield either neuronal progeny or RPE cells. Thus RPE cells display similarities to neuronal lineages, even when differentiated (8), that make them suitable for conversion into neurons for therapeutic purposes (9). For example, recent studies revealed mechanisms for the transdifferentiation of RPE cells that depend on the expression status of certain genes (10). These studies have created a new interest in RPE cells caused by the potential applications not on...

Significance of lipid mediators in corneal injury and repair

“…NPD1 also inhibits PMN infi ltration and pro-infl a mmatory gene expression in brains damaged by ischemia-reperfusion ( 132 ). Synthesis of NPD1 occurs after free DHA is converted by a 15-LOX-1 and further epoxydation ( 131,141 ). In the cornea, DHA is a minor component of membrane phospholipids ( 14 ), but synthesis of NPD1 has been reported in mice fed with -3 enriched diets ( 78 ).…”

Lipoxin A4 impairment of apoptotic signaling in macrophages: implication of the PI3K/Akt and the ERK/Nrf-2 defense pathways

“…9 For instance, an apoptotic effect on fibroblasts after treatment with LXA 4 at micromolar concentrations has been described, 10 whereas it has been also reported that this compound inhibits peroxynitrite formation in leukocytes, 11 reduces colonocyte apoptosis 12 and promotes survival of retinal pigment epithelial cells. 13 Therefore, the precise role of LXs as modulators of apoptosis remains elusive and seems to be dependent on the cell type and on the LXA 4 concentration. Given the lack of studies regarding the role of LXA 4 in macrophage apoptosis and considering the importance of this immune cell in inflammation, we have investigated the effect of native LXA 4 on macrophage apoptosis using staurosporine, etoposide and GSNO as strong apoptosis inducers in many cell types, 3,14,15 and LPS/IFNg challenge as a more pathophysiological condition.…”

Rescue and repair during photoreceptor cell renewal mediated by docosahexaenoic acid-derived neuroprotectin D1

“…2 ). Recent evidence shows 15-LOX-1 as the enzyme that oxygenates DHA into NPD1 ( 91 ). In ARPE-19 cells where 15-LOX-1 protein expression was knocked down by 70% posttranscriptionally, the production of NPD1 was not increased, in contrast with normal cells ( Fig.…”