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Vol. 55, Issue 1, 126-133, January 1999
Laboratory of Signal Transduction, The early signaling mechanism of sphingosine 1-phosphate (S1P) on
extracellular signal-regulated kinase (ERK) activation was investigated
in C6 glioma cells. S1P activated the enzyme in association with a
shift in the mobility on electrophoresis reflecting phosphorylation of
both ERK1/ERK2 at as low as 10 nM. The lipid-induced ERK1/2 activation
was partially inhibited by treatment of the cells with either phorbol
12-myristate 13-acetate (a long-term treatment to desensitize protein
kinase C) or pertussis toxin (PTX) and was completely inhibited by a
simultaneous treatment with both agents. Similarly, either calphostin
C, an inhibitor of protein kinase C, or U73122, an inhibitor of
phospholipase C, partially inhibited the S1Pinduced ERK1/2
activation in the nontreated cells with PTX and completely in the
toxin-treated cells. On the other hand, the S1P-induced ERK activation
was hardly affected by ethanol, which switched the product of
phospholipase D from phosphatidic 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 activation in
spite of a marked elevation of intracellular S1P accumulation. This
indicates that intracellular increase in S1P is not necessary for the
S1P-induced ERK activation, and hence suggests the extracellular action
mechanism of S1P. Supporting this idea, mRNAs of recently identified
S1P specific receptors, Edg-1 and AGR16/H218, were expressed in C6
cells. Taken together, these results suggested that S1P acts on C6
cells extracellularly possibly through S1P receptors which are linked
to at least two signaling pathways, i.e., the PTX-sensitive
Gi/Go protein pathway and the toxin-insensitive
Gq/G11-phospholipase C-PKC pathway, resulting in the activation of ERK.
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
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