Sphingosine-1-phosphate and ceramide-1-phosphate promote migration, pro-inflammatory and pro-fibrotic responses in retinal pigment epithelium cells

Abstract

Retinal pigment epithelium (RPE) cells, essential for preserving retina homeostasis, also contribute to the development of retina proliferative diseases, through their exacerbated migration, epithelial to mesenchymal transition (EMT) and inflammatory response. Uncovering the mechanisms inducing these changes is crucial for designing effective treatments for these pathologies. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in several cell types; we recently established that they stimulate the migration of retina Müller glial cells (Simón et al., 2015; Vera et al., 2021). We here analyzed whether S1P and C1P regulate migration, inflammation and EMT in RPE cells. We cultured two human RPE cell lines, ARPE-19 and D407 cells, and supplemented them with either 5 μM S1P or 10 μM C1P, or their vehicles, for 24 h. Analysis of cell migration by the scratch wound assay showed that S1P addition significantly enhanced migration in both cell lines. Pre-treatment with W146 and BML-241, antagonists for S1P receptor 1 (S1P1) and 3 (S1P3), respectively, blocked exogenous S1P-induced migration. Inhibiting sphingosine kinase 1 (SphK1), the enzyme involved in S1P synthesis, significantly reduced cell migration and exogenous S1P only partially restored it. Addition of C1P markedly stimulated cell migration. Whereas inhibiting C1P synthesis did not affect C1P-induced migration, inhibiting S1P synthesis strikingly decreased it; noteworthy, addition of C1P promoted the transcription of SphK1. These results suggest that S1P and C1P stimulate RPE cell migration and their effect requires S1P endogenous synthesis. Both S1P and C1P increase the transcription of pro-inflammatory cytokines IL-6 and IL-8, and of EMT marker α-smooth muscle actin (α-SMA) in ARPE-19 cells. Collectively, our results suggest new roles for S1P and C1P in the regulation of RPE cell migration and inflammation; since the deregulation of sphingolipid metabolism is involved in several proliferative retinopathies, targeting their metabolism might provide new tools for treating these pathologies.