Possible involvement of cell surface receptors in sphingosine 1-phosphate-induced activation of extracellular signal-regulated kinase in C6 glioma cells

The early signaling mechanism of sphingosine I-phosphate (SIP) on extracell ular signal-regulated kinase (ERK) activation was investigated in C6 glioma cells. S1P activated the enzyme in association with a shift in the mobilit y on electrophoresis reflecting phosphorylation of both ERK1/ERK2 at as low as 10 nM. The lipid-induced ERK1/2 activation was partially inhibited by t reatment of the cells with either phorbol S1P-myristate 13-acetate (a long- term treatment to desensitize protein kinase C) or pertussis toxin (PTX) an d was completely inhibited by a simultaneous treatment with both agents. Si milarly, either calphostin C, an inhibitor of protein kinase C, or UT3122, an inhibitor of phospholipase C, partially inhibited the S1P-induced ERK1/2 activation in the nontreated cells with PTX and completely in the toxin-tr eated cells. On the other hand, the SIP-induced ERK activation was hardly a ffected by ethanol, which switched the product of phospholipase D from phos phatidic acid to metabolism-resistant phosphatidylethanol. S1P was able to activate ERK1/2 without a detectable increase in the intracellular content of the lipid, but sphingosine, a substrate of sphingosine kinase, which is an enzyme for S1P generation in the cells, hardly affected the ERK1/2 activ ation in spite of a marked elevation of intracellular SIP accumulation. Thi s indicates that intracellular increase in S1P is not necessary for the SIP -induced ERK activation, and hence suggests the extracellular action mechan ism of S1P. Supporting this idea, mRNAs of recently identified S1P specific receptors, Edg-1 and AGR16/H218, were expressed in C6 cells. Taken togethe r, these results suggested that S1P acts on C6 cells extracellularly possib ly through S1P receptors which are linked to at least two signaling pathway s, i.e., the PTX-sensitive G(i)/G(o) protein pathway and the toxin-insensit ive G(q)/G(11)-phospholipase C-PKC pathway, resulting in the activation of ERK.