The G protein-coupled receptor S1P2 regulates Rho/Rho kinase pathway to inhibit tumor cell migration

Sphingosine 1-phosphate (SIP) 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 (SIP1-5), which activate downstream signaling intermediates such as Rac and Rho GTPases. Mechanisms of SIP action in human glioblastoma cells are not well defined. SIP receptors (1-5) and SIP-metabolizing enzymes were expressed in three human glioblastoma cell lines. SIP had a profound and differential effect on glioblastoma cell migration. U87 cells treated with SIP showed a significant increase in migration, whereas U118 and U138 cell lines were strongly inhibited. SIP-mediated inhibition correlated with SIP2 receptor expression. FTY720-P, an SIP analogue that binds all SIP receptors except SIP2, did not inhibit glioblastorna cell migration. Overexpression of SIP2 further suppressed migration, and blockage Of SIP2 mRNA expression by small interfering RNA reversed the inhibitory effect. Contrary to previous reports showing bimodal regulation of Rac activity and migration by SIP2 receptor stimulation, both Rac1 and RhoA GTPases were activated by SIP treatment in native cells and cells over-expressing S1P(2). 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 SIP stimulated U118 cells overexpressing beta-galactosidase resulted in pronounced stress fiber formation that was exacerbated by SIP2 overexpression, partially blocked by SIP,, or totally abolished by pretreatment with Y-27632. These data provide evidence of a novel mechanism of SIP inhibition of tumor cell migration via Rho kinase-dependent pathway.