Sphingosine-1-phosphate induces COX-2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. SIP increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results Suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by SIP in VSMCs. In accordance with these findings, SIP stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappa B inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappa B into the nucleus was revealed by immunofluorescence staining. Moreover, SI P-stirnulated activation of NF-kappa B promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, Suggesting that involvement of PI3K/Akt in the activation of NF-kappa B. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappa B. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappa B pathways was essential for S1P-induced COX-2gene expression. Understanding the mechanisms involved in SIP-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.