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 Gα family members. In this study, we investigated whether Gα₁₂ 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 Gα₁₂ but not Gα₁₃, Gαq, or Gαi/o. The specific role of Gα₁₂ in COX-2 induction by S1P was verified by promoter luciferase assay, Gα₁₂ transfection, and knockdown experiments. Experiments using siRNAs specifically directed against S1P₁₋₅ showed that S1P₁, S1P₃, and S1P₅ are necessary for the full activation of COX-2 induction. Gel shift, immunocytochemistry, chromatin immunoprecipitation, and NF-κB site mutation analyses revealed the role of NF-κBin COX-2 gene transcription by S1P. Gα₁₂ deficiency did not affect S1P-mediated IκBα phosphorylation but abrogated IκBα ubiquitination and degradation. Moreover, the inhibition of S1P activation of JNK abolished IκBα ubiquitination. Consistently, JNK transfection restored the ability of S1P to degrade IκBα during Gα₁₂ deficiency. S1P injection induced COX-2 in the lungs and livers of mice and increased plasma prostaglandin E₂, and these effects were prevented by Gα₁₂ deficiency. Our data indicate that, of the Gα proteins coupled to S1P receptors, Gα₁₂ specifically regulates NF-κB-mediated COX-2 induction by S1P downstream of S1P₁, S1P₃, and S1P₅, in a process mediated by the JNK-dependent ubiquitination and degradation of IκBα.