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-κB. 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 ATPγS. We found that ATPγS-induced COX-2 and cPLA₂ expression and PGE₂ release was attenuated by the pharmacological inhibitors or transfection with siRNA against PKCδ, c-Src, EGFR, PI3-K, Akt, p44/p42 MAPK or Elk-1. Moreover, ATPγS-stimulated phosphorylation of PKCδ, c-Src, EGFR, Akt, p42/p44 MAPK and Elk-1, suggesting the participation of PKCδ/c-Src/EGFR/PI3-K/Akt/p42/p44 MAPK cascade in mediating Elk-1 activities in VSMCs. In addition, migration assay revealed that ATPγS 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 ATPγS up-regulated the expression of COX-2 and cPLA₂ through transactivation of PKCδ/c-Src/EGFR/PI3K/Akt/Elk-1 pathway. Newly synthesized PGE₂ acted on its receptors to promote cell motility of ATPγS-stimulated VSMCs.