Abstract
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 (S1P1-5), 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 beta-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.
Highlights
Sphingosine 1-phosphate (S1P) is a bioactive lipid produced by the breakdown of the membrane phospholipid, sphingomyelin
We measured the mRNA expression of the five S1P receptors (S1P1/EDG-1, S1P2/ EDG-5, S1P3/EDG-3, S1P4/EDG-6, and S1P5/EDG-8) in three adult glioblastoma cell lines (U87, U118, and U138,) using quantitative reverse transcription-PCR (RT-PCR)
Transcripts for all receptors were detected at varying levels in all cell lines examined, except S1P4/EDG-6 in U138 cells (Supplemental Fig. S1A)
Summary
Sphingosine 1-phosphate (S1P) is a bioactive lipid produced by the breakdown of the membrane phospholipid, sphingomyelin. S1P exerts a variety of responses in cells such as proliferation, differentiation, migration, and survival [1]. These effects are mediated by the EDG family of extracellular G protein–coupled receptors, some evidence suggests that unknown intracellular targets may exist [2, 3]. Five members of the EDG family bind with high affinity to S1P: S1P1/EDG-1, S1P2/EDG-5, Note: Supplementary data for this article are available at Cancer Research Online (http://canres.aacrjournals.org). S1P3/EDG-3, S1P4/EDG-6, and S1P5/EDG-8 [4] Each of these receptors activates different intracellular signaling pathways depending on which Ga protein they couple to intracellularly [5]. The ability of S1P to bind to multiple S1P receptors, which in turn activate multiple Ga protein–coupled pathways, creates a complex signaling system downstream of S1P receptors
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