Research reportDifferential roles of Edg-1 and Edg-5, sphingosine 1-phosphate receptors, in the signaling pathways in 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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