S1P Activation of Rho/ROCK Pathway in Human Schlemm’s Canal Endothelial Cells in Culture

Abstract

Purpose: : Sphingosine 1-phosphate (S1P) decreases outflow facility in perfused human eyes and S1P₁ and S1P₃ receptors are expressed in cultured trabecular meshwork (TM) and Schlemm’s canal (SC) endothelial cells. The signaling pathways activated by S1P in the two cell types that contribute to outflow resistance regulation are unknown. Since S1P robustly stimulates the Rho/Rho-kinase (ROCK) pathway leading to downstream myosin light chain (MLC) phosphorylation in TM cells, the aim of this study was to assess coupling of S1P receptors to the Rho/ROCK pathway in cultured SC cells.

Methods: : Primary cultures of SC and TM cells from human donor eyes were used in all experiments, with TM cells functioning as controls. Rho activation following 1µM S1P treatments for 5 and 30 minutes was determined in pull-down assays with immobilized rhotekin Rho-binding domain; activated Rho-GTP was compared to total Rho through immunoblot analysis. Total Rho blots were reprobed for phospho-MLC to determine the concurrent MLC phosphorylated status. Additional S1P dose-response treatments for MLC phosphorylation were used to compare coupling efficacy of SC to TM cells.

Results: : Rho activation in SC cells by S1P occurred within 5 minutes, along with a 3-fold increase in phospho-MLC compared to control. SC cells displayed a comparable EC₅₀ value (~1µM) to TM cells for S1P-induced MLC phosphorylation. However, MLC protein expression was greater (~5-fold) in TM compared to SC cells. The ROCK inhibitor Y-27632 blocked S1P-mediated stimulation of MLC phosphorylation in SC cells.

Conclusions: : S1P activates Rho in cultured SC cells, inducing a ROCK-dependent MLC phosphorylation that is similar to that observed in TM cells. Due to greater MLC expression in TM compared to SC cells, S1P-induced Rho/ROCK activation may dominate whereas SC cells may behave similarly to other vascular endothelia and primarily couple to Rac following S1P stimulation.

Keywords: receptors: pharmacology/physiology • lipids • signal transduction: pharmacology/physiology