Research Article
Roles of sphingosine-1-phosphate (S1P) receptors in malignant behavior of glioma cells. Differential effects of S1P2 on cell migration and invasiveness

https://doi.org/10.1016/j.yexcr.2007.02.009Get rights and content

Abstract

Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of five G-protein-coupled receptors, termed S1P1–5. S1P stimulates growth and invasiveness of glioma cells, and high expression levels of the enzyme that forms S1P, sphingosine kinase-1, correlate with short survival of glioma patients. In this study we examined the mechanism of S1P stimulation of glioma cell proliferation and invasion by either overexpressing or knocking down, by RNA interference, S1P receptor expression in glioma cell lines. S1P1, S1P2 and S1P3 all contribute positively to S1P-stimulated glioma cell proliferation, with S1P1 being the major contributor. Stimulation of glioma cell proliferation by these receptors correlated with activation of ERK MAP kinase. S1P5 blocks glioma cell proliferation, and inhibits ERK activation. S1P1 and S1P3 enhance glioma cell migration and invasion. S1P2 inhibits migration through Rho activation, Rho kinase signaling and stress fiber formation, but unexpectedly, enhances glioma cell invasiveness by stimulating cell adhesion. S1P2 also potently enhances expression of the matricellular protein CCN1/Cyr61, which has been implicated in tumor cell adhesion, and invasion as well as tumor angiogenesis. A neutralizing antibody to CCN1 blocked S1P2-stimulated glioma invasion. Thus, while S1P2 decreases glioma cell motility, it may enhance invasion through induction of proteins that modulate glioma cell interaction with the extracellular matrix.

Introduction

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates cellular proliferation, migration, survival, cytoskeletal rearrangement, and angiogenesis [1], [2], [3]. S1P signals both intracellularly as a second messenger [4] and through five cell surface, G-protein-coupled receptors (GPCRs) of the EDG family, S1P1/EDG-1, S1P2/EDG-5, S1P3/EDG-3, S1P4/EDG-6 and S1P5/EDG-8 [3]. Our group has been exploring the roles played by S1P in the growth and invasiveness of glioblastoma multiforme (GBM) cells. GBM is the most commonly occurring primary brain tumor in adults and is highly malignant, displaying aggressive growth and invasion into surrounding brain tissue [5] leading to a median life expectancy of only 10–12 months following diagnosis [6]. A better understanding of the molecular regulation of GBM cell growth and invasion will be necessary to develop effective molecular-based therapies.

We have shown that S1P is mitogenic for [7], and enhances motility and invasiveness of GBM cell lines [8]. As an indication of the importance of this signaling system for in vivo GBM, high expression levels of the enzyme which forms S1P, sphingosine kinase-1 (SphK1), in GBM tissue correlate with a more than 3-fold shorter survival time of GBM patients [9]. Furthermore, knockdown of SphK1 or SphK2 expression using RNA interference decreases GBM cell proliferation by preventing entry of cells in the cell cycle [9].

The mitogenic and invasive effects of S1P on GBM cells are at least partially mediated through its GPCRs, since both responses are sensitive to pertussis toxin [7], which specifically inhibits signaling through the Gi/o family of G proteins. Moreover, S1P induction of both mitogenesis and invasiveness of GBM cells occurs at nanomolar concentrations, consistent with the affinities of S1P for its receptors [7]. GBM cell lines [8] and GBM tissue [9] commonly express three S1P receptors, S1P1, S1P2 and S1P3. S1P4, which is primarily expressed in cells of hematopoietic origin [10], has not been detected in gliomas. S1P5 is expressed in normal brain in oligodendrocytes [11], however, we have detected only very low levels of S1P5 expression in a limited number of GBM cases and several glioma cell lines (unpublished observation).

Each S1P receptor subtype activates a unique set of G proteins with varying preferences [3], [12]. Therefore, the individual influence of each receptor subtype on S1P regulation of GBM cell behavior may depend on the amounts of the individual S1P receptors expressed. In this study, the effects of S1P through individual receptor subtypes on glioma cell growth, migration, invasion, and adhesion were examined by overexpressing individual S1P receptors in glioma cells with low endogenous receptor levels, and knocking down individual S1P receptor expression by RNA interference in glioma cells with high levels of expression. The results show that S1P1, S1P2 and S1P3 all contribute to glioma cell growth and invasion through distinct, but overlapping, mechanisms. Conversely, S1P5 inhibits these cellular activities when overexpressed in glioma cells. Furthermore, the S1P2 receptor subtype displays a novel enhancement of S1P-stimulated invasion while decreasing migration. The S1P2-stimulated invasiveness in these glioma cell lines correlates with enhanced cell adhesion and is mediated, at least partially, by the matricellular protein CCN1/Cyr61. Thus, this study begins to define the roles of S1P in glioma cells through its individual receptors.

Section snippets

Materials

Cell culture medium and fetal bovine serum were purchased from Mediatech (Herndon, VA). Fatty acid-free BSA was purchased from Sigma (St. Louis, MO). S1P was from Avanti Polar Lipids (Alabaster, AL). Gelatin was purchased from BioRad (Hercules, CA). 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). Antibodies to CCN1, phosphoERK and phosphoAKT were from Santa Cruz

Generation of S1P receptor over- and underexpressing glioma cell lines

U-373 MG cells express similar levels of the S1P receptors, S1P1, S1P2 and S1P3 [8]. In addition, U-373 MG cells respond to S1P treatment with both increased proliferation and motility/invasiveness [7], [8]. Therefore, this cell line was chosen to down regulate individual S1P receptors using RNA interference. In contrast, U-118 MG cells express very low levels of S1P1, S1P2, S1P3 and S1P5 by real-time PCR analysis (data not shown), and respond to S1P with only minimally increased proliferation (

Discussion

The results of this study show that the pleiotropic effects of S1P on glioma cells are mediated by a combination of S1P receptors, i.e., rather than individual receptor subtypes stimulating cell proliferation or invasion, these responses are contributed to by several receptor subtypes. Indeed, all three of the S1P receptors commonly expressed in glioma tissue contributed positively to S1P regulation of cell proliferation, as evidenced by experiments utilizing both overexpression and knockdown

Acknowledgments

This work was supported by Grant #R01 NS41517 from the National Institute of Neurological Disorders and Stroke (NINDS) to J.R.V.B.

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