Gα12 specifically regulates COX-2 induction by sphingosine 1-phosphate -: Role for JNK-dependent ubiquitination and degradation of IκBα

Cyclooxygenase-2 (COX-2) plays a critical role in vasodilatation and local inflammatory responses during platelet aggregation and thrombosis. Sphingosine 1-phosphate (S1P), a sphingolipid released from activated platelets, stimulates COX-2 induction and activates G-protein-coupled receptors coupled to G alpha family members. In this study, we investigated whether G alpha(12) family regulates COX-2 induction by S1P and investigated the molecular basis of this COX-2 regulation. Gene knock-out and chemical inhibitor experiments revealed that the S1P induction of COX-2 requires G alpha(12) but not G alpha(13), G alpha(q), or G alpha(i/o). The specific role of G alpha(12) in COX-2 induction by S1P was verified by promoter luciferase assay, G alpha(12) transfection, and knockdown experiments. Experiments using siRNAs specifically directed against S1P(1-5) showed that S1P(1), S1P(3), and S1P(5) are necessary for the full activation of COX-2 induction. Gel shift, immunocytochemistry, chromatin immuno-precipitation, and NF-kappa B site mutation analyses revealed the role of NF-kappa B inCOX-2 gene transcription by S1P. G alpha(12) deficiency did not affect S1P-mediated I kappa B alpha phosphorylation but abrogated I kappa B alpha ubiquitination and degradation. Moreover, the inhibition of S1P activation of JNK abolished I kappa B alpha ubiquitination. Consistently, JNK transfection restored the ability of S1P to degrade I kappa B alpha during G alpha(12) deficiency. S1P injection induced COX-2 in the lungs and livers of mice and increased plasma prostaglandin E-2, and these effects were prevented by G alpha(12) deficiency. Our data indicate that, of the G alpha proteins coupled to S1P receptors, G alpha(12) specifically regulates NF-kappa B-mediated COX-2 induction by S1P downstream of S1P(1), S1P(3), and S1P(5), in a process mediated by the JNK-dependent ubiquitination and degradation of I kappa B alpha.