PLD1 and PLD2 mediate retinal pigment epithelium cell damage induced by high glucose concentrations. Chamonix, France, desde el 4 al 8 de Septiembre de 2016

TENCONI, S; SALVADOR, G; GIUSTO, N.; MATEOS, M

Chamonix

Congreso; 57th International Conference on the Bioscience of Lipids,; 2016

ICBL

PLD1 and PLD2 mediate retinal pigment epithelium cell damage induced by high glucose concentrations.Paula E. Tenconi*, Gabriela A. Salvador*, Norma M. Giusto*, and Melina V. Mateos**Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Universidad Nacional del Sur (UNS) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 8000 Bahía Blanca, Argentina. Diabetic retinopathy (DR) is one of the most serious complication of diabetes and the main cause of blindness among working-age people. Hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of DR. The retinal pigment epithelium (RPE) is essential for the integrity and function of the retina thus elucidating the effects of inflammatory processes in these cells could lead to the discovery of new therapeutic targets for the treatment of retinal degenerative diseases. The objective of the present work was to describe the effects and signaling events elicited by high glucose (HG) concentrations in RPE cells. In particular, we wanted to study the role of classical phospholipases D (PLD1 and PLD2). Classical PLDs hydrolyze phosphatidylcholine (PC) to generate phosphatidic acid (PA), and choline. PA can be further dephosphorylated by lipid phosphate phosphatases (LPPs) in order to generate diacylglycerol (DAG). Thus, PLD activation can modulate the activity of several PA and DAG-responding proteins.ARPE-19 cells (human RPE cell line) were exposed to HG (16.5 and 33 mM) or to normal glucose (5 mM, NG) concentrations for 4 (16.5 mM glucose) or 72 h (33mM glucose) in order to mimic a peak of and a sustained hyperglycemia. Osmotic controls were performed with mannitol. After 72 h of incubation with HG, cell viability (evaluated by the MTT reduction assay) was reduced by 30% and reactive oxygen species (ROS) generation was increased 5 times in HG conditions with respect to NG. Western blot (WB) assays showed that sustained exposure to HG reduced peroxiredoxin (PRX) expression but did not affect superoxide dismutase 1 (SOD1) expression. After 4 h exposure to HG WB and immunocytochemistry assays showed activation of the extracellular signal-regulated kinase (ERK1/2) and translocation of the transcription factor nuclear factor κ B (NFκB) to the nucleus. Moreover, 4 h exposure to HG increased PLD activity (measured as the generation of [3H ]-Phosphatidylethanol) by 80% with respect to cells exposed to NG. The HG-induced ERK1/2 activation was completely abolished by the pre-incubation with 0.15 µM EVJ (PLD1 inhibitor) or 0.5 µM APV(PLD2 inhibitor) as well as HG-induced NFκB nuclear translocation, which was prevented by the pre-incubation with both PLD inhibitors and also by the MEK-ERK1/2 pathway inhibitor (10 µM U0126). Furthermore, the HG-induced loss in cell viability was prevented by both PLD inhibitors, U0126 and by the cyclooxygenase-2 (COX-2) inhibitor (10 µM celecoxib).Our results demonstrate that in an in vitro model of DR, the subsequent activation of PLD1 and 2, ERK1/2 and NFκB mediates RPE cell damage, possibly though COX-2 induction as well as other inflammatory cytokines regulated by NFκB. Our findings point to the potential use of classical PLDs as therapeutic targets for the treatment of ocular inflammatory diseases, such as DR.