Data from The G Protein–Coupled Receptor S1P<sub>2</sub> Regulates Rho/Rho Kinase Pathway to Inhibit Tumor Cell Migration

Abstract

<div>Abstract<p>Sphingosine 1-phosphate (S1P) is a lysophospholipid that exerts a variety of responses in cells such as proliferation, migration, and survival. These effects are mediated by G protein–coupled receptors on the cell surface (S1P<sub>1-5</sub>), which activate downstream signaling intermediates such as Rac and Rho GTPases. Mechanisms of S1P action in human glioblastoma cells are not well defined. S1P receptors (1–5) and S1P-metabolizing enzymes were expressed in three human glioblastoma cell lines. S1P had a profound and differential effect on glioblastoma cell migration. U87 cells treated with S1P showed a significant increase in migration, whereas U118 and U138 cell lines were strongly inhibited. S1P-mediated inhibition correlated with S1P<sub>2</sub> receptor expression. FTY720-P, an S1P analogue that binds all S1P receptors except S1P<sub>2</sub>, did not inhibit glioblastoma cell migration. Overexpression of S1P<sub>2</sub> further suppressed migration, and blockage of S1P<sub>2</sub> mRNA expression by small interfering RNA reversed the inhibitory effect. Contrary to previous reports showing bimodal regulation of Rac activity and migration by S1P<sub>2</sub> receptor stimulation, both Rac1 and RhoA GTPases were activated by S1P treatment in native cells and cells overexpressing S1P<sub>2</sub>. Treatment of U118 cells with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 restored migration suggesting that ROCK-dependent mechanisms are important. Actin staining of S1P stimulated U118 cells overexpressing β-galactosidase resulted in pronounced stress fiber formation that was exacerbated by S1P<sub>2</sub> overexpression, partially blocked by S1P<sub>1</sub>, or totally abolished by pretreatment with Y-27632. These data provide evidence of a novel mechanism of S1P inhibition of tumor cell migration via Rho kinase–dependent pathway.</p></div>