Sphingosine-1-phosphate stimulates human glioma cell proliferation through Gi-coupled receptors: role of ERK MAP kinase and phosphatidylinositol 3-kinase β
Introduction
Sphingosine-1-phosphate (S1P) is a bioactive lipid that is mitogenic for a wide variety of cell types [1]. S1P is present at high levels in brain tissue [2], and stimulates signaling pathways in C6 glioma cells including increased [Ca2+]i [3], [4] and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases [5]. However, the effects of S1P on glioma cell proliferation have not been established.
S1P signals through several members of the endothelial differentiation gene (EDG) family of G protein-coupled receptors [6], [7], which have recently been renamed S1P receptors by the IUPHAR Nomenclature Committee [8]. S1P binds with high affinities to S1P1/EDG-1, S1P3/EDG-3 and S1P2/EDG-5, S1P4/EDG-6, and S1P5/EDG-8 [9], [10], [11], [12], [13]. These receptors activate a variety of signal transduction pathways, several of which may be involved in mitogenic signaling, including ERK MAP kinases [9], [11], [14], [15], decreased cAMP levels [12], [16], increased inositol phosphate production and [Ca2+]i [14], [15], [17], and regulation of gene expression through the serum response element [18]. S1P receptors activate this broad variety of signaling pathways by coupling to Gi, Gq, and G12/13 types of G proteins [7].
In addition to its role as a ligand for S1P receptors, S1P is formed within cells in response to platelet-derived growth factor (PDGF) or serum. Inhibition of sphingosine kinase, the enzyme which forms S1P, partially inhibits the mitogenic effect of PDGF or serum [19], suggesting that S1P acts intracellularly as a second messenger to mediate the effects of these mitogens. Thus it has been proposed that S1P plays dual roles as a second messenger and an extracellular mediator [16]. Therefore, it is important to determine whether a given cellular response to S1P is mediated through cell surface receptors or intracellularly.
Several members of the S1P receptor subfamily are expressed in the brain [20], [21], [22]. Thus, it is possible that S1P and it's receptors play important physiologic and/or pathologic roles in the brain. In this study the mitogenic effect of S1P on human glioma cell lines was investigated. We show here that U-373 MG cells respond mitogenically to nanomolar concentrations of S1P, and that this response is mediated through G protein-coupled receptors. In addition, S1P receptors are expressed in human glioblastoma multiforme tissue.
Section snippets
Materials
S1P (dihydro-S1P) and sphingosine were purchased from Avanti Polar Lipids (Birmingham, AL). Sphingosylphosphorylcholine (SPC) was from Matreya (Pleasant Gap, PA). Serum and medium were obtained from Biofluids (Rockville, MD). Pertussis toxin (PTX) was from Research Biochemicals International (Natick, MA). PD98059 and wortmannin were from Calbiochem (La Jolla, CA).
Analysis of cell growth
Human glioma cells were grown in Eagle's minimum essential medium (EMEM) containing Earl's Balanced Salt Solution, non-essential
Stimulation of human glioma cell proliferation by S1P
U-373 MG human glioblastoma cells were treated with S1P ranging from 10 nM to 10 μM and DNA synthesis was measured by [3H]thymidine incorporation. U-373 MG cells responded to 10 nM S1P with a potent increase in DNA synthesis (Fig. 1A). The effect of S1P on DNA synthesis in U-373 MG cells peaked at 100 nM. Interestingly, slightly less stimulation was apparent at micromolar concentrations of S1P. We also treated U-373 MG cells with the structurally related lipids dihydro-S1P and SPC. Dihydro-S1P,
Discussion
In this study, S1P was shown to be mitogenic for U-373 MG human glioma cells at concentrations as low as 10 nM. The affinities of the known S1P receptors, S1P1, S1P3, S1P2, S1P4 and S1P5 for S1P are all in the nanomolar range [9], [10], [11], [12], [13]. In contrast, putative, intracellular effects of S1P are usually seen at micromolar concentrations [16]. Thus, our results are consistent with the mitogenic effect of S1P on U-373 MG cells being mediated by a cell surface receptor. Moreover,
Acknowledgements
The authors thank Drs Sarah Spiegel and Richard W. Burry for helpful comments on this manuscript. This work was supported by Grant #IRG-98-278-01 from the American Cancer Society and the Department of Pathology, The Ohio State University.
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