title: 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 alpha/H2O2-mediated oxidative stress in nonspecific cells (controls), whereas in silenced cells, negligible amounts of NPD1, 12(S)- and 15(S)-hydroxyeicosatetraenoic acid, and lipoxin A4 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.
authors.affiliation: Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, School of Medicine, New Orleans, Louisiana 70112, USA.
authors.forename: Jorgelina M
authors.initials: JM
authors.lastname: Calandria
authors.forename: Victor L
authors.initials: VL
authors.lastname: Marcheselli
authors.forename: Pranab K
authors.initials: PK
authors.lastname: Mukherjee
authors.forename: Jasim
authors.initials: J
authors.lastname: Uddin
authors.forename: Jeremy W
authors.initials: JW
authors.lastname: Winkler
authors.forename: Nicos A
authors.initials: NA
authors.lastname: Petasis
authors.forename: Nicolas G
authors.initials: NG
authors.lastname: Bazan
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D044045:Lipoxins / Q000378:metabolism; D016859:Lipoxygenase Inhibitors; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D044045:Lipoxins; D016859:Lipoxygenase Inhibitors; D016877:Oxidants; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; C040527:lipoxin A4; C487679:protectin D1; D004281:Docosahexaenoic Acids; D006861:Hydrogen Peroxide; C458528:ALOX15B protein, human; D001093:Arachidonate 15-Lipoxygenase