Sphingosine-1-phosphate signaling is essential for preserving morphology and focal adhesions of retina pigment epithelial cells

TORLASCHI C.; GUTIÉRREZ JOFRÉ G.; ROTSTEIN N.P.; SIMÓN M.V.

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencias; 2023

SAIC-SAI-AAFE-AACYTAL

Sección: NeurocienciasSphingosine-1-phosphate signaling is essential for preserving morphology and focal adhesions of retina pigment epithelial cellsTorlaschi C., Gutiérrez Jofré G., Rotstein N.P. and Simón M.V. Instituto de Investigaciones Bioquímicas, Depto. de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur-CONICET, 8000 Bahía Blanca, Buenos Aires, Argentina.Cell-cell interactions between retinal pigment epithelium (RPE) cells provide the retina with a physical and metabolic barrier, the disruption of which characterizes many inflammatory and proliferative retinopathies. However, the underlying causes of this disruption are still ill-defined. We showed that the bioactive sphingolipids sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) promote migration and inflammation in RPE cells. Using the human RPE cell line ARPE-19, we now analyzed whether S1P regulates cell morphology and RPE monolayer integrity. Inhibiting S1P synthesis with PF543, a sphingosine kinase 1 (SphK1) inhibitor, markedly decreased ARPE-19 cell migration in confluent cultures, without affecting cell survival. Using 50% confluent cultures, to better observe morphological changes, we determined that PF543 treatment promoted 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 cell morphology, reducing elongated cells to 8±1.4% (p>0.01), suggesting that S1P inside-out signaling is required for preserving cell morphology. In contrast, C1P addition did not restore cell morphology in PF543-treated cells. When we preincubated cells with PF543 and JTE-013, a S1P2 receptor (S1P2) antagonist, before S1P addition, JTE-013 partially blocked S1P restoration of cell morphology. To analyze the mechanisms involved in cell adhesion, we determined distribution of paxillin, a scaffold protein in focal adhesions. While controls showed spot-like paxillin clusters in the cell periphery, these clusters disappeared in PF543-treated cells and were restored after S1P addition. These results suggest that inhibiting S1P synthesis leads to morphological changes and focal adhesion remodeling, and activation of the S1P/S1P2 axis is required for preserving cell morphology and establishing focal adhesions.