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The G Protein–Coupled Receptor S1P₂ Regulates Rho/Rho Kinase Pathway to Inhibit Tumor Cell Migration

¹ Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford and ² Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut

Requests for reprints: Fernando Ferrer, Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, CT. Phone: 860-545-9658; Fax: 860-545-9545; E-mail: fferrrer{at}ccmckids.org/.

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_1-5), 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 S1P₂ receptor expression. FTY720-P, an S1P analogue that binds all S1P receptors except S1P₂, did not inhibit glioblastoma cell migration. Overexpression of S1P₂ further suppressed migration, and blockage of S1P₂ mRNA expression by small interfering RNA reversed the inhibitory effect. Contrary to previous reports showing bimodal regulation of Rac activity and migration by S1P₂ receptor stimulation, both Rac1 and RhoA GTPases were activated by S1P treatment in native cells and cells overexpressing S1P₂. 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 ß-galactosidase resulted in pronounced stress fiber formation that was exacerbated by S1P₂ overexpression, partially blocked by S1P₁, 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.

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