Sphingosine-1-phosphate stimulates motility and invasiveness of human glioblastoma multiforme cells
Introduction
Sphingosine-1-phosphate (S1P) is a bioactive lipid which regulates diverse cellular processes including proliferation, survival, differentiation and motility by activating a wide variety of signal transduction pathways [1]. Although S1P was originally proposed to act intracellularly, recent evidence has shown that S1P signals through several members of a group of G protein-coupled receptors formerly known as the EDG family and now renamed S1P receptors [2]. S1P has been shown to bind and signal through S1P1/EDG-1, S1P2/EDG-5, S1P3/EDG-3, S1P4/EDG-6 and S1P5/EDG-8 [1].
We have recently shown that S1P is mitogenic for several glioblastoma cell lines [3]. S1P potently stimulated proliferation of U-373 MG cells by signaling through a Gi-coupled receptor and activation of the Extracellular Signal-Regulated Kinase (ERK) Mitogen-Activated Protein (MAP) kinase and Phosphatidylinositol (PI)-3 kinase pathways. Moreover, analysis of RNA isolated from human glioblastomas revealed that 87.5% of these tumors expressed the three S1P receptors S1P1, S1P2, and S1P3. As S1P levels are high in brain tissue [4] and rat glioma cells have been shown to make large amounts of S1P and secrete it extracellularly [5], it is possible that autocrine signaling by S1P affects glioma cell biology.
Several studies have shown that S1P regulates cell motility. S1P potently stimulates chemotaxis of endothelial cells thus leading to angiogenesis [6], [7], [8], [9]. In addition, S1P affects motility and invasiveness of malignant cells. Although one report showed that S1P enhanced invasion of T lymphoma cells [10], other studies have shown that S1P inhibits invasiveness of breast cancer [11], melanoma and fibrosarcoma [12] cells. Thus, it was of interest to determine whether S1P affected motility and invasiveness of glioma cells.
Section snippets
Materials
S1P was purchased from Avanti Polar Lipids (Alabaster, AL). Gelatin was from BioRad (Hercules, CA). Cell culture medium and fetal bovine serum was from Mediatech (Herndon, VA). Fatty acid-free BSA was from Sigma (St Louis, MO). Matrigel was from Becton Dickinson (Palo Alto, CA). Assay-On-Demand gene expression assays and real time PCR reagents were purchased from Applied Biosystems (Foster City, CA).
Cell culture
All glioma cell lines were maintained in Eagle's minimum essential medium (EMEM) containing 10%
S1P enhances glioma cell motility
The effect of S1P on U-373 MG glioma cell motility was determined using a modified Boyden chamber assay. S1P potently stimulated migration of U-373 MG cells (Fig. 1A and B). Stimulation of motility was dose-dependent, with a maximum response at 10 nM and a greatly reduced response at micromolar concentrations (Fig. 1C). This is in agreement with the high affinity of S1P for its receptors. To determine whether S1P stimulation of motility is a general characteristic of glioma cells, motility
Discussion
In this study, we show that S1P potently stimulates motility of human glioblastoma cells. We tested the motility of five different glioblastoma cell lines in response to S1P. Three of those, U-373 MG, U-87 MG and M059K responded to S1P, while A172 and U-1242 MG did not. Interestingly, U-373 MG, U-1242 MG and M059K cells are mitogenically responsive to nanomolar concentrations of S1P [3], while U-87 MG [3] and A172 (unpublished observation) are not. Thus, glioma cells which respond mitogenically
Acknowledgements
The authors thank Dr Yiwen Liu-Stratton of the Ohio State University Dorothy M. Davis Heart and Lung Research Institute Genetics/Microarray Core Lab and Ms Catherine Jackson for assistance with Real Time PCR experiments and data analysis. This work was supported by Grant # R01 NS41517 from the National Institute of Neurological Disorders and Stroke (NINDS) to JRVB.
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