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 Galpha family members. In this study, we investigated whether Galpha(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 Galpha(12) but not Galpha(13), Galpha(q), or Galpha(i/o). The specific role of Galpha(12) in COX-2 induction by S1P was verified by promoter luciferase assay, Galpha(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 immunoprecipitation, and NF-kappaB site mutation analyses revealed the role of NF-kappaBin COX-2 gene transcription by S1P. Galpha(12) deficiency did not affect S1P-mediated IkappaBalpha phosphorylation but abrogated IkappaBalpha ubiquitination and degradation. Moreover, the inhibition of S1P activation of JNK abolished IkappaBalpha ubiquitination. Consistently, JNK transfection restored the ability of S1P to degrade IkappaBalpha during Galpha(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 Galpha(12) deficiency. Our data indicate that, of the Galpha proteins coupled to S1P receptors, Galpha(12) specifically regulates NF-kappaB-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 IkappaBalpha.