Purinergic P2Y1 receptor signaling mediates wound stimuli-induced cyclooxygenase-2 expression in intestinal subepithelial myofibroblasts

Abstract

Intestinal subepithelial myofibroblasts (ISMFs) are crucial for barrier formation against inflammatory stimuli. Physical injury induces cyclooxygenase-2 (COX-2) expression, which accelerates wound healing by ISMFs. However, the mechanism of COX-2 induction remains unclear. Physically damaged cells release ATP. Here, we investigate the role of ATP-purinergic signaling in wound-induced COX-2 induction in ISMFs. By 24h post-injury, bovine ISMFs had migrated to and closed the wounded area. A COX inhibitor, indomethacin or a purinergic P2 receptor antagonist, suramin, inhibited wound healing. However, additional treatment with indomethacin did not influence wound healing in suramin-treated ISMFs. RT-PCR showed an increase in COX-2 mRNA expression 2h post-injury, which was inhibited by suramin. These results suggest that ATP mediates wound-induced COX-2 elevation. We next assessed the contribution of various purinergic receptors in COX-2 induction. An ATP analog, ATPγS and a purinergic P2Y1, 11–13 receptors agonist, ADP, were among the agents tested which increased COX-2 expression. ATPγS-induced COX-2 mRNA expression was suppressed by suramin or a purinergic P2Xs, P2Y1, 4, 6, and 13 receptors antagonist, PPADS. These data suggest the involvement of Gq-coupled purinergic P2Y1 receptor or Gi-coupled purinergic P2Y13 receptor in COX-2 induction. U73122, an inhibitor of phospholipase C, which is a downstream signal of Gq protein, showed suppression of COX-2 mRNA expression. However, pertussis toxin, a Gi inhibitor, did not show suppression. We also revealed that inhibitors of p38 MAPK and PKC inhibited ATPγS-induced COX-2 mRNA expression. Collectively, purinergic P2Y1 receptor signaling mediates wound-induced COX-2 expression through p38 MAPK and PKC pathways in ISMFs.