Role of phospholipase D pathway in the inflammatory response of the retinal pigment epithelium cells

Abstract

The retinal pigment epithelium (RPE) plays an important immunological role in the retina and it is involved in many ocular inflammatory diseases that may end in loss of vision and blindness. In this work the role of the phospholipase D (PLD) pathway in the inflammatory response of the human RPE cells (ARPE-19) exposed to bacterial lipopolysaccharide (LPS) was studied. ARPE-19 were exposed to LPS (10 pg/ml) for 24 and 48 h. LPS treatment reduced cell viability by 15% after 48 h and increased NO production by 1 and 1.8 fold after 24 and 48 h, respectively. Furthermore, 24 h LPS treatment strongly induced ciclooxygenase-2 (COX-2) expression and the activation of protein kinase C (PKCa/pII) and extracellular signal-regulated kinase (ERK1/2). Transfected cells with EGFP-PLD plasmids showed the typical subcellular localization of PLD1 (perinuclear) and PLD2 (plasma membrane). Untransfected cells expressed both PLD isoforms. LPS treatment increased PLD activity (measured as phosphatidylethanol formation) by 80% with respect to the control. The presence of the PLD1 inhibitor (EVJ 0.15 pM) or the PLD2 inhibitor (APV 0.5 pM) reduced LPS- induced PKCa/pII activation and COX-2 induction but only PLD2 inhibition reduced ERK1/2 activation. Moreover, the inhibition of PLD2 and ERK1/2 restored cell viability to control levels. Our results demonstrate for the first time the participation of the PLD pathway in the inflammatory response of RPE cells exposed to LPS.