SPHINGOSINE-1-PHOSPHATE SIGNALING IS ESSENTIAL FOR PRESERVING MORPHOLOGY AND FOCAL ADHESIONS OF RETINA PIGMENT EPITHELIAL CELLS

TORLASCHI CAMILA; GUTIERREZ JOFRE GABRIELA; ROTSTEIN NORA PATRICIA; SIMON MARIA VICTORIA

Congreso; LXVIII ANNUAL MEETING OF SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2023

Cell-cell interactions between retinal pigment epithelium (RPE) cells provide theretina with a physical and metabolic barrier, the disruption of which is a hallmarkof many retina inflammatory and proliferative diseases. However, the underlyingcauses of this disruption are still ill-defined. We showed that the bioactivesphingolipids sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P)promote migration and inflammation in RPE cells. Using the human RPE cellline ARPE-19, we now analyzed whether S1P regulates cell morphology andRPE monolayer integrity. Inhibiting S1P synthesis with PF543, a sphingosinekinase 1 (SphK1) inhibitor, markedly decreased ARPE-19 cell migration inconfluent cultures, without affecting cell survival. Using 50% confluent cultures,to better observe morphological changes, we determined that PF543 treatmentpromoted a remarkable cell retraction; highly elongated cells, absent in controls,augmented to 34±2% (p>0.01), their cell length/width ratio increasing to 5.3,from 1.6 in controls. S1P addition, 1 h after PF543 treatment, restored cellmorphology, reducing elongated cells to 8±1.4% (p>0.01), suggesting that S1Pinside-out signaling is required for preserving cell morphology. In contrast, C1Paddition did not restore cell morphology in PF543-treated cells. When wepreincubated cells with PF543 and JTE-013, a S1P2 receptor (S1P2)antagonist, before S1P addition, JTE-013 partially blocked S1P restoration ofcell morphology. To analyze the mechanisms involved in cell adhesion, wedetermined distribution of paxillin, a scaffold protein in focal adhesions. Whilecontrols showed spot-like paxillin clusters in the cell periphery, these clustersdisappeared in PF543-treated cells and were restored after S1P addition. Theseresults suggest that inhibiting S1P synthesis leads to morphological changesand focal adhesion remodeling, and activation of the S1P/S1P2 axis is requiredfor preserving cell morphology and establishing focal adhesions.