Transactivation of EGFR/PI3K/Akt involved in ATP-induced inflammatory protein expression and cell motility

Phenotype transition of vascular smooth muscle cells (VSMCs) is important in vascular diseases, such as atherosclerosis and restenosis. Once released, ATP may promote activation of VSMCs by stimulating cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2) expression and prostaglandin (PG)E2 synthesis via activation of MAPKs and NF-?B. However, whether alternative signaling pathways participated in regulating COX-2 and cPLA2 expression associated with cell migration were investigated in rat VSMCs. Western blot analysis, RT-PCR, promoter assay and PGE2 ELISA were used to determine expression of COX-2, cPLA2 and PGE2. Specific inhibitors and siRNAs against various protein kinases or transcription factors were used to investigate the related signaling components in inflammatory protein induction by ATP?S. We found that ATP?S-induced COX-2 and cPLA2 expression and PGE2 release was attenuated by the pharmacological inhibitors or transfection with siRNA against PKCd, c-Src, EGFR, PI3-K, Akt, p44/p42 MAPK or Elk-1. Moreover, ATP?S-stimulated phosphorylation of PKCd, c-Src, EGFR, Akt, p42/p44 MAPK and Elk-1, suggesting the participation of PKCd/c-Src/EGFR/PI3-K/Akt/p42/p44 MAPK cascade in mediating Elk-1 activities in VSMCs. In addition, migration assay revealed that ATP?S promoted cell mobility through up-regulation of COX-2 and cPLA2 expression and PGE2 release, which was attenuated by pretreatment with PGE2 receptor antagonists. Taken together, these data showed that ATP?S up-regulated the expression of COX-2 and cPLA2 through transactivation of PKCd/c-Src/EGFR/PI3K/Akt/Elk-1 pathway. Newly synthesized PGE2 acted on its receptors to promote cell motility of ATP?S-stimulated VSMCs. J. Cell. Physiol. 227: 1628-1638, 2012. (C) 2011 Wiley Periodicals, Inc.