The P2Y₂ receptor mediates the epithelial injury response and cell migration

Abstract

Injury to epithelial cells results in the release of ATP and stimulation of purinergic receptors and is thought to alter cell migration and wound repair. Medium from the injured cells triggers Ca(2+) mobilization and phosphorylation of ERK, both of which are inhibited if the medium is pretreated with apyrase. To understand the wound repair mechanism that occurs with injury, our goal was to determine which purinergic receptor(s) was the critical player in the wound response. We hypothesize that the P2Y(2) receptor is the key player in the response of corneal epithelial cells to cell damage and subsequent repair events. Cells transfected with short interfering RNA to either P2Y(2) or P2Y(4) were stimulated either by injury or addition of UTP and imaged using fluo 3-AM to monitor changes in fluorescence. When cells with downregulated P2Y(2) receptors were injured or stimulated with UTP, the intensity of the Ca(2+) release was reduced significantly. However, when cells with downregulated P2Y(4) receptors were stimulated, only the UTP-induced Ca(2+) response was reduced significantly. In addition, downregulation of the P2Y(2) receptor inhibited wound closure compared with unstimulated cells or cells transfected with nontargeting sequence. This downregulation resulted also in an attenuation in phosphorylation of Src and ERK. Together, these data indicate that the P2Y(2) receptor plays a major biological role in the corneal injury response and repair mechanisms.

Purinoreceptor P2X7 Regulation of Ca2+ Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury

“…However, unlike other purinoreceptors, such as P2Y2, we found that P2X7 does not influence the distance or rate of Ca 2þ wave propagation from the site of injury. 3,4 Because the region of cells in which P2X7 modulates calcium dynamics correlates with the region of wounded epithelium in which P2X7 expression increases after injury, we speculate that localized activation of P2X7 acts in a feed-forward manner to up-regulate its expression at the leading edge.…”

“…To further determine specificity, cells were also stimulated with either UTP or ADP, agonists for P2Y2 and P2Y4 or P2Y1, respectively, three other G-proteinecoupled purinoreceptors that are expressed in HCLE cells. 4,18 Treatment with oxATP, A438079, or siRNA to P2X7 did not significantly inhibit the UTP-or ADP-stimulated Ca 2þ response (Figure 3, C and D).…”

“…4,17 Previously, we demonstrated that P2X7 stimulation with BzATP, a P2X7 agonist, increases the migration rate in both a modified Boyden chamber and scratch-wound assay model and induces a prototypical mobilization of Ca 2þ in HCLE cells. 15 To further determine the role of P2X7 in Ca 2þ mobilization, we examined the effects of P2X7 antagonism on BzATP-stimulated Ca 2þ influxes.…”

“…4,15 Briefly, complete medium was replaced with keratinocyte serum free media lacking antibiotics and supplements. The siRNA sequences directed against P2X 7 were as follows: 5 0 -GGAUCCAGAGCAUGAAUUAUU-3 0 (sense) and 5 0 -UAAUUCAUGCUCUGGAUCCUU-3 0 (antisense; Dharmacon product number J-003728-07; Dharmacon, Lafayette, CO).…”

“…This has been demonstrated with apyrase, an ectonucleotidase, which abolishes the Ca 2þ response to injury. 3 Although downregulation of specific P2 receptors decreases Ca 2þ mobilization in corneal epithelial cells, 4 activation of P2 purinergic receptors by nucleotides causes increased phosphorylation of adaptor proteins and focal adhesion kinases. 5,6 These changes mediate signaling events that regulate migration, demonstrating the critical role of nucleotide signaling in wound repair.…”