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-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate 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 consistent 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 permissive signal transduction pathway linked to DNA synthesis.
Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, Scotland, UK.
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S
Rakhit
A M
AM
Conway
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Tate
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T
Bower
N J
NJ
Pyne
S
S
Pyne
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006168:Guinea Pigs; D017874:Immediate-Early Proteins / Q000235:genetics*; D008246:Lysophospholipids; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D017978:Receptors, Growth Factor / Q000235:genetics; D049312:Receptors, Lysophospholipid; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D015533:Transcriptional Activation; D019061:src-Family Kinases / Q000378:metabolism*
D017874:Immediate-Early Proteins; D008246:Lysophospholipids; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D017978:Receptors, Growth Factor; D049312:Receptors, Lysophospholipid; C060506:sphingosine 1-phosphate; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013110:Sphingosine