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

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)/Ca(2+) 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/Ca(2+) 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-Ca(2+) system and PLD in native C6 glioma cells.