Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle - Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase

We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-co upled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p 44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PT X)-sensitive manner in these cells. An essential role for c-Src was confirm ed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhi bitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An esse ntial role for PI-3K was supported by experiments that showed that PI-3K ac tivity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatme nt of cells with PTX. Finally, we have shown that the co-stimulation of cel ls with platelet-derived growth factor (PDGF) and S1P (which failed to stim ulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 mi n stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demo nstrate that S1P activates c-Src and Grb-2-PI-3K (intermediates in the p42/ p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is cons istent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common perm issive signal transduction pathway linked to DNA synthesis.