Elsevier

Molecular Brain Research

Volume 85, Issues 1–2, 28 December 2000, Pages 151-160
Molecular Brain Research

Research report
Differential roles of Edg-1 and Edg-5, sphingosine 1-phosphate receptors, in the signaling pathways in C6 glioma cells

https://doi.org/10.1016/S0169-328X(00)00262-XGet rights and content

Abstract

Several cDNA encoding G-protein-coupled receptors, i.e. Edg-1,-3,-5,-6 and -8, have recently been identified as sphingosine 1-phosphate (S1P) receptors. However, the role of the respective receptor subtype has not been well defined. In C6 glioma cells, exogenous S1P induced expression of fibroblast growth factor-2 (FGF-2), a potent neurotrophic factor, which was associated with the stimulation of extracellular signal-regulated kinase (ERK) and the expression of early growth response-1 (Egr-1). S1P also stimulated phospholipase C (PLC)/Ca2+ system and phospholipase D (PLD). In this study, we sought to identify S1P receptors responsible for these S1P-induced actions. Of five S1P receptor subtypes, Edg-1 and Edg-5 are expressed in the glioma cells, as evidenced by Northern blotting. We therefore prepared the cells overexpressing these S1P receptor subtypes and compared the intrinsic activities to stimulate these signaling pathways and their sensitivity to pertussis toxin (PTX). The potency of S1P and dihydrosphingosine 1-phosphate (DHS1P), another S1P receptor agonist, to stimulate the Edg-1 and Edg-5 receptors was also examined. We found that the intrinsic activity that stimulated ERK/Egr-1/FGF-2 system was much higher in Edg-1 than in Edg-5. Furthermore, DHS1P was as potent as S1P in activating ERK in control C6 cells, a pattern also observed in cells overexpressing Edg-1. On the other hand, the stimulation of the PLC/Ca2+ system and PLD induced by S1P was PTX-insensitive, and the potency of S1P in activating PLD was roughly one order higher than that of DHS1P in control C6 cells; similar responsiveness to such pharmacological tools were observed in Edg-5-overexpressing cells. Taken together, these results suggest that Edg-1 may be the main receptor mediating the stimulation of ERK/Egr-1/FGF-2 system but that Edg-5 may be responsible for the stimulation of PLC–Ca2+ system and PLD in native C6 glioma cells.

Introduction

Sphingosine 1-phosphate (S1P) has recently been thought to be involved in the regulation of a variety of cellular processes, including cell proliferation [10], [28], [37], [43], metabolic regulation [14], [25], morphological change [29], [35], [45], [46] and cell motility [13], [31]. This novel lipid mediator functions as not only an intracellular signaling molecule but also an extracellular signaling molecule [8], [11], [37]. Actually, exogenously applied S1P stimulates many early signaling events including phospholipase C (PLC) [10], [14], [25], [33], [34], increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) [14], [21], [25], [26], [36], [40], [46], extracellular signal-regulated kinase (ERK) activation [36], [37], [40] and phospholipase D (PLD) activation [2], [4]. Several cDNA encoding G-protein-coupled receptors have recently been identified as S1P receptors, i.e. Edg-1, Edg-3, Edg-5, Edg-6 and Edg-8 [1], [15], [20], [33], [44].

In neural cells, S1P has been shown to induce morphological and functional responses [29], [35]. Thus, S1P induces the rapid retraction of neurites and the transient rounding of the cell body in some neuronal cells, such as PC12 cells [35] and N1E115 cells [29]. This S1P-induced action was likely to be mediated through cell surface receptors, possibly Edg-5 [29], [35], [39]. In regard to astroglia cells, we have recently shown that S1P induces the expression of fibroblast growth factor-2 (FGF-2), a potent neurotrophic factor, in association with the activation of ERK and the expression of early growth response-1 (Egr-1), an essential transcription factor for FGF-2 expression [32]. We have also shown that the stimulation of the ERK/Egr-1/FGF-2 system may be mediated by two signaling pathways, i.e. PTX-sensitive Gi/Go pathway and the toxin-insensitive PLC pathway [32]. S1P has also been shown to activate PLD, but this lipase activation is unlikely to be involved in the regulation of ERK/Egr-1/FGF-2 system [36]. These S1P-induced early signaling events and functional responses in C6 glioma cells seem to be mediated by extracellular S1P receptors [32], [36]; however, it remains to be clarified which receptor subtype or subtypes are responsible for these S1P actions.

In C6 glioma cells, Edg-1 and Edg-5, but not Edg-3, have been shown to be expressed [36]. In our preliminary results, the expression of Edg-6 and Edg-8 was not detected. In order to characterize the receptor subtype or subtypes involved in the S1P-induced actions in C6 cells, we prepared C6 cells which overexpress Edg-1 or Edg-5. If the respective receptor was responsible for the given responses, we could expect an enhancement of the responses with a similar specificity to the pharmacological tools, as observed in native C6 cells. Our results suggest that Edg-1 may be more important than Edg-5 for the S1P-induced stimulation of the ERK/Egr-1/FGF-2 system. On the other hand, Edg-5 may be responsible for the S1P-induced activation of PLC–Ca2+ system and PLD.

Section snippets

Materials

Sphingosine and 1,2-diacyl-sn-glycero-3-phosphate (l-α-phosphatidic acid or PA) were purchased from Sigma Chemical Co.; sphingosine 1-phosphate (S1P) was from Cayman Chemical Co.; dihydrosphingosine 1-phosphate (DHS1P) and 1-palmitoyl-2-oleoyl-sn-3-phosphoethanol (phosphatidylethanol or PEt) were from Biomol Research Labs. Inc.; FuGene 6 transfection reagent was from Boehringer Mannheim. Plasmid pTB784 containing a rat FGF-2 cDNA [19] was generously provided by Dr T. Kurokawa (Takeda Chemical

Preparation of C6 cells overexpressing Edg-1 or Edg-5

In order to prepare C6 cells which overexpress Edg-1 or Edg-5, the cells were transfected with a pEFneo eukaryotic expression vector containing Edg-1 cDNA or Edg-5 cDNA. As a control, a pEFneo empty vector was transfected. The G418-resistant C6 cells were then selected. As shown in Fig. 1, although mRNAs for Edg-1 and Edg-5 were expressed and a significant S1P binding activity was observed at higher concentrations of S1P (50–100 nM) in empty vector-transfected cells, we could not detect a

Discussion

There are at least two mechanisms by which exogenous S1P stimulates the intracellular signaling pathways: an intracellular mechanism through unidentified targets and an extracellular mechanism through S1P receptors [8], [11], [37]. In the previous 3T3 fibroblast studies [4], [37], [38], [40], ERK activation and PLD activation induced by exogenous S1P have been proposed to be mediated by an intracellular mechanism, although the intracellular target(s) for S1P has not yet been identified. On the

Acknowledgements

We thank Dr. T. Kurokawa of Takeda Chemical Industries Ltd., Tsukuba, Ibaraki, for providing the plasmid pTB784 containing rat FGF-2 cDNA. This work was supported in part by a research grant from the Ministry of Education, Science, and Culture of Japan.

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