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

Abstract

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 prolifer- ative retinopathies. However, the underlying causes of this disrup- tion 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 synthe- sis with PF543, a sphingosine kinase 1 (SphK1) inhibitor, markedly decreased ARPE-19 cell migration in confluent cultures, without af- fecting cell survival. Using 50% confluent cultures, to better observe morphological changes, we determined that PF543 treatment pro- moted 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 af- ter 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 addi- tion 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 establish- ing focal adhesions.