ATP and UTP Mediated Epithelial Injury Response Is Associated With Transactivation of EGF Receptors

Abstract

Abstract: : Purpose:We have shown that injury to corneal epithelial cells releases nucleotides and that a calcium wave is propagated to neighboring cells. Furthermore we have elucidated which P2Y receptors are expressed. Our goal is to determine which P2Y receptors play a role in the wound response and elucidate the cross–talk that occurs with the EGF pathway to generate cellular signaling cascades. Methods: Experiments were performed on both primary corneal epithelial cells and HCE–Ts. Calcium signaling was monitored using live cell confocal imaging with a perfusion system. Localization of the receptors was assessed using confocal microscopy. Cell migration was determined using scratch assays and modified Boyden chambers. Expression and phosphorylation of PLCgamma1 and EGFR were performed using western blot analysis. Assays were performed to determine levels of diacylglycerol. Results: We have demonstrated that there is a dose dependent response to the nucleotides where ATP and UTP show saturation at lower concentrations than ADP or UDP. To determine which are involved in the wound response, cells were pre–treated and then injured. When cells were pretreated with ATP or UTP the injury induced calcium wave was inhibited indicating desensitization of receptors. Desensitization of receptors was not detected when cells were pretreated with ADP and UDP, as the injury induced calcium wave was present. In addition, only pretreatment with ATP and UTP decreased the EGF induced Ca2+ response. Migration induced by either ATP or UTP was significantly greater than that induced by ADP or UDP and the latter were enhanced by EGF. Furthermore the integrin peptide, RGD, diminished trinucleotide induced migration. Cross–talk was exhibited when ATP attenuated the ability of EGF to activate phospholipase C gamma1. In adition, while ATP and UTP transactivated ErbB1 and ErbB2, the trinucleotides decreased the ability of EGF to induce autophosphorylation of the EGFR. Other experiments using CRM 197 and TIMP showed that specific parts of the signaling pathway could be inhibited. Conclusions:These results indicate that the injury response is mediated by P2Y2 and P2Y4 receptors and we hypothesize that cross–talk may occur via a triple–membrane pass mechanism.

Keywords: signal transduction • cell-cell communication • wound healing