Sphingosine-1-Phosphate Enhancement of Cortical Actomyosin Organization in Cultured Human Schlemm's Canal Endothelial Cell Monolayers

Abstract

Perfusion of sphingosine-1-phosphate (S1P) in whole eye organ culture models decreases outflow facility, whereas S1P promotes stress fiber formation and contractility in cultured trabecular meshwork (TM) cells. Because of S1P's known effect of increasing barrier function in endothelial cells, the authors hypothesized that Schlemm's canal (SC) cells in culture respond to S1P by increasing actomyosin organization at the cell cortex. Using primary cultures of human SC cells, the authors determined S1P activation of the GTP-binding proteins, RhoA and Rac (1,2,3). Time- and dose-dependent myosin light chain (MLC) phosphorylation in response to S1P and total expression of MLC were determined. Immunocytochemistry after S1P treatment was used to monitor filamentous actin (F-actin) and phospho-MLC organization and the localization of β-catenin, a component of adherens junctions. TM and human umbilical vein endothelial cell monolayers were used as controls. S1P (1 μM) activated RhoA and Rac after 5- and 30-minute treatments. S1P increased MLC phosphorylation with a similar time- and dose-dependent response in SC (EC(50) = 0.83 μM) compared with TM (EC(50) = 1.33 μM), though MLC expression was significantly greater in TM. In response to 1 μM S1P treatment, phospho-MLC concentrated in the SC cell periphery, coincident with cortical actin assembly and recruitment of β-catenin to the cell periphery. Results obtained in this study support the hypothesis that S1P increases actomyosin organization at the SC cell cortex and promotes intercellular junctions at the level of the inner wall of SC to increase transendothelial resistance and in part explains the S1P-induced decrease of outflow facility in organ culture.