Controlling morphology and monolayer integrity in retina pigment epithelial cells: a role for sphingolipids?

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

Ciudad Autónoma de Buenos Aires

Simposio; Simposio Frontiers in Bioscience 4; 2023

IBIOBA

Controlling morphology and monolayer integrity in retina pigment epithelial cells: a role for sphingolipids?Torlaschi 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.Retinal pigment epithelium (RPE) cells are crucial for retina homeostasis. However, multiple injuries trigger their reactive response, termed epithelial mesenchymal transition (EMT), and inflammation. We have shown that sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in RPE cells. Using two human RPE cell lines (ARPE-19 and D407), we here analyzed whether S1P and C1P regulate cell morphology and RPE monolayer integrity. Inhibiting S1P synthesis with PF543, a selective sphingosine kinase 1 inhibitor, markedly decreased ARPE-19 cell migration in confluent cultures. Using 50% confluent cultures, to improve observation of cell morphology, we determined that PF543 provoked a remarkable cell retraction. Highly elongated cells, very few in controls, augmented to 30% in PF543-treated cultures, while the cell length/width ratio increased from 1.6 to 5.3. Similar changes were observed when treating D407 cells with PF543. Noteworthy, S1P addition after 1 h of PF543 treatment preserved cell morphology, reducing elongated cells to 9%. This suggests that S1P inside-out signaling is required for preserving cell morphology. Inhibiting C1P synthesis with NVP also induced morphological changes in these cells, increasing the percentage of round cells and promoting the loss of RPE typical cobblestone appearance, without altering the cell length/width ratio. PF543 affected the distribution and expression of vimentin, an intermediate filament known to increase in EMT. Whereas ARPE19 cells in controls showed a low, perinuclear, vimentin labeling, PF543 increased this labeling, inducing vimentin homogenous distribution throughout the cell body. Distribution of paxillin, a scaffold protein in focal adhesions, was also affected; controls showed spot-like paxillin clusters in the cell periphery, which disappeared in PF543-treated cells. These results suggest that inhibition of S1P synthesis promotes EMT and focal adhesion remodeling, while synthesis of S1P and C1P is required for preserving cell morphology and RPE monolayer integrity. Controlling morphology and monolayer integrity in retina pigment epithelial cells: a role for sphingolipids?Torlaschi 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.Retinal pigment epithelium (RPE) cells are crucial for retina homeostasis. However, multiple injuries trigger their reactive response, termed epithelial mesenchymal transition (EMT), and inflammation. We have shown that sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in RPE cells. Using two human RPE cell lines (ARPE-19 and D407), we here analyzed whether S1P and C1P regulate cell morphology and RPE monolayer integrity. Inhibiting S1P synthesis with PF543, a selective sphingosine kinase 1 inhibitor, markedly decreased ARPE-19 cell migration in confluent cultures. Using 50% confluent cultures, to improve observation of cell morphology, we determined that PF543 provoked a remarkable cell retraction. Highly elongated cells, very few in controls, augmented to 30% in PF543-treated cultures, while the cell length/width ratio increased from 1.6 to 5.3. Similar changes were observed when treating D407 cells with PF543. Noteworthy, S1P addition after 1 h of PF543 treatment preserved cell morphology, reducing elongated cells to 9%. This suggests that S1P inside-out signaling is required for preserving cell morphology. Inhibiting C1P synthesis with NVP also induced morphological changes in these cells, increasing the percentage of round cells and promoting the loss of RPE typical cobblestone appearance, without altering the cell length/width ratio. PF543 affected the distribution and expression of vimentin, an intermediate filament known to increase in EMT. Whereas ARPE19 cells in controls showed a low, perinuclear, vimentin labeling, PF543 increased this labeling, inducing vimentin homogenous distribution throughout the cell body. Distribution of paxillin, a scaffold protein in focal adhesions, was also affected; controls showed spot-like paxillin clusters in the cell periphery, which disappeared in PF543-treated cells. These results suggest that inhibition of S1P synthesis promotes EMT and focal adhesion remodeling, while synthesis of S1P and C1P is required for preserving cell morphology and RPE monolayer integrity.