Modulation of RPE cell-cell interactions and extracellular matrix adhesion: a role for sphingosine-1-phospate.

Abstract

Introduction Interactions between retinal pigment epithelium (RPE) cells provide the retina with a physical and metabolic barrier, the disrup- tion of which contributes to the onset of inflammatory and proliferative retinopathies. We shave shown that the bioactive sphingo- lipids sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) promote migration and inflammation in RPE cells (Simón et al., Exp. Eye Res. 2022). Objectives Our objective was to evaluate whether S1P modulates RPE monolayer integrity and morphological changes. Methods Human RPE cell line (ARPE-19) cultures were treated with PF543, a sphingosine kinase 1 (SphK1) inhibitor, with S1P or C1P, or with antagonists for S1P receptors S1P1, S1P2 and S1P3, W146, JTE013 and BML-241, respectively. Cell migration was analyzed by the scratch wound assay and cell death by MTT assay. Cell morphology was determined with phalloidin, and expression of paxillin, talin and fibronectin, focal adhesion proteins, and ZO1, a tight junction protein, by immunocytochemistry. Results PF543 markedly decreased cell migration in confluent cultures. In 50% confluent cultures PF543 promoted cell retraction; highly elongated cells, absent in controls, increased to 34±2% (p>0.01), their cell length/width ratio rising from 1.6 in controls to 5.3 in PF543-treated cultures. Addition of S1P, but not of C1P, preserved cell morphology, reducing elongated cells to 8±1.4%. W146 and JTE-013 partially blocked S1P preservation of cell morphology, whereas BML-241 did not, implying activation of S1P1 and S1P2 was required for S1P effect. These antagonists did not modify cell morphology when added to controls, suggesting that in- side-out signaling was not involved in endogenous S1P effect. Paxillin and talin clusters were present along the cell periphery in controls, and virtually disappeared in PF543-treated cells; S1P addition preserved these clusters in spite of PF543 treatment. Fibronectin expression decreased in RPE cells treated with PF543 and increased compared to controls with addition of S1P. ZO-1, which localized in the plasma membrane in controls, showed a discontinuous presence and internalization in PF543-treated cultures; noteworthy, S1P addition prevented this loss. Conclusion Our results suggest that S1P, both endogenous and exogenous, was required to preserve RPE cell morphology and for the assem- bly of focal adhesions and tight junctions, sharing intracellular signaling pathways for these effects