Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells: role of ERK1/2, p38 MAPK, and NF-κB

Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in inflammatory diseases such as atherosclerosis and angiogenesis. As extracellular nucleotides such as ATP have been shown to act via activation of P-2 purinoceptors implicated in various inflammatory diseases, we hypothesized that extracellular nucleotides contribute to vascular diseases via upregulated expression of inflammatory proteins, such as cyclooxygenase (COX-2) and cytosolic phospholipase A(2) (cPLA(2)) in VSMCs. Western blotting, promoter assay, RT-PCR, and PGE(2) immunoassay revealed that ATP gamma S induced expression of COX-2 and prostaglandin (PGE(2)) synthesis through the activation of p42/p44 MAPK (mitogen-activated protein kinase), p38 MAPK, and nuclear factor-kappa B (NF-kappa B). These responses were attenuated by inhibitors of MAPK/ERK kinase (MEK1/2; U0126), p38 MAPK (SB202190), and NF-kappa B (helenalin), or by tranfection with dominant negative mutants of p42, p38, I kappa B kinase (IKK)alpha, and IKK beta. Furthermore, the ATP gamma S-stimulated translocation of NF-kappa B into the nucleus and degradation of I kappa B alpha was blocked by U0126 and helenalin. In addition, the ATP gamma S-stimulated cPLA(2) expression was inhibited by U0126, SB202190, helenalin, celecoxib (a selective COX-2 inhibitor), and PGE(2) receptor antagonists (AH6809, GW627368X, and SC-19220). However, the inhibitory effect of celecoxib on cPLA(2) expression was reversed by addition of exogenous PGE(2). Our results suggest that in VSMCs, activation of p42/p44 MAPK, p38 MAPK, and NF-kappa B is essential for ATP gamma S-induced COX-2 expression and PGE(2) synthesis. Newly synthesized PGE(2) was observed to act as an autocrine signal contributing to cPLA(2) expression, which may be implicated in inflammatory responses. Collectively, our findings provide insights into the correlation between COX-2 and cPLA(2) expression in ATP gamma S-stimulated VSMCs with similar molecular mechanisms and functional coupling to amplify the occurrence of vessel disease-related vascular inflammation.