Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase β -: Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways

Sphingosine 1-phosphate (SIP) is a platelet-derived sphingolipid that elicits diverse biological responses, including angiogenesis, via the activation of G protein-coupled EDG receptors. SIP activates the endothelial isoform of nitric-oxide synthase (eNOS), associated with eNOS phosphorylation at Ser-1179, a site phosphorylated by protein kinase Akt. We explored the proximal signaling pathways that mediate Akt activation and eNOS regulation by S1P/EDG receptors. Akt is regulated by the lipid kinase phosphoinositide 3-kinase (PI3-K). We found that bovine aortic endothelial cells (BAEC) express both alpha and beta isoforms of PI3-K, while lacking the gamma isoform. SIP treatment led to the rapid and isoform-specific activation of PI3-K beta in BAEC. PI3-K beta can be regulated by G protein beta gamma subunits (G beta gamma). The overexpression of a peptide inhibitor of G beta gamma attenuated S1P-induced eNOS enzyme activation, as well as S1P-induced phosphorylation of eNOS and Akt. In contrast, bradykinin, a classical eNOS agonist, neither activated any PI3-K isoform nor induced eNOS phosphorylation at Ser-1179, despite activating eNOS in BAEC. Vascular endothelial growth factor activated both PI3-K alpha and PI3-K beta via tyrosine kinase pathways and promoted eNOS phosphorylation that was unaffected by G beta gamma inhibition. These findings indicate that PI3-K beta (regulated by G beta gamma) may represent a novel molecular locus for eNOS activation by EDG receptors in vascular endothelial cells. These studies also indicate that different eNOS agonists activate distinct signaling pathways that diverge proximally following receptor activation but converge distally to activate eNOS.