ATP stimulates PGE2/cyclin D1-dependent VSMCs proliferation via STAT3 activation: Role of PKCs-dependent NADPH oxidase/ROS generation

Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in cardiovascular diseases. 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 up-regulation of inflammatory proteins, including cyclooxygenase-2 (COX-2) and cytosolic phospholipase A(2) (cPLA(2)) in VSMCs. However, the mechanisms of ATP-induced cPLA(2) and COX-2 expression and PGE(2) synthesis remain largely unclear. We showed that pretreatment with the inhibitors of STAT3 (CBE), NADPH oxidase [diphenyleneiodonium chloride (DPI) or apocynin (APO)], ROS [N-acetyl-L-cysteine (NAC)], and PKC (Ro-318220, G66983, or Rottlerin) or transfection with siRNAs of STAT3 and p47(phox) markedly inhibited ATP gamma S-induced cPLA(2) and COX-2 mRNA/protein expression and promoter activity and PGE(2) secretion. ATP gamma S further stimulated PKC, p47(phox), and STAT3 translocation. Moreover, ATP gamma S-induced STAT3 phosphorylation and translocation was inhibited by pretreatment with the inhibitors of PKC, NADPH oxidase, and ROS. ATP gamma S enhanced NADPH oxidase activity and ROS generation in VSMCs, which were reduced by pretreatment with Ro-318220, G66983, or Rottlerin. Finally, we found that ATP gamma S significantly induced cyclin D1 expression and VSMCs proliferation, which were inhibited by pretreatment with NAC, APO, DPI, Ro-318220, G66983, Rottlerin, or CBE or transfection with siRNAs of COX-2 and cyclin D1. We also demonstrated that ATP gamma S induced cyclin D1 expression via a PGE(2)-dependent pathway. These results suggested that ATRyS-induced cPLA(2)/COX-2 expression and PGE(2) secretion is mediated through a PKC/NADPH oxidase/ROS/STAT3-dependent pathway in VSMCs. (C) 2013 Elsevier Inc. All rights reserved.