Stable knock-down of the sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells

Objectives - Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein - coupled receptors S1P(1-5) and as a second messenger. Because S1P(1) knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P(1) in endothelial cells is needed. Methods and Results - All human endothelial cells analyzed expressed abundant S1P(1) transcripts. We permanently silenced ( by RNA interference) the expression of S1P(1) in the human endothelial cell lines AS-M. 5 and ISO-HAS.1. The S1P(1) knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress - mediated injury. New S1P(1)-dependent gene targets were identified in human endothelial cells. S1P(1) silencing decreased the expression of platelet - endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P(1) silencing. Conclusions - Long-term silencing of S1P(1) enabled us for the first time to demonstrate the involvement of S1P(1) in key functions of endothelial cells and to identify new S1P(1)-dependent gene targets.