Autocrine regulation of wound healing by ATP release and P2Y₂ receptor activation

Aims: Application of exogenous nucleotides can modulate wound healing via the activation of purinergic receptors. However, evidence for the release of endogenous nucleotides and the subsequent activation of purinergic receptors in this process has not been well defined. Therefore, the current study aimed to investigate wound-mediated nucleotide release and autocrine purinergic signalling during HaCaT keratinocyte wound closure following scratch injury.

Main methods: An in vitro scratch wound apparatus was employed to study wound healing over 24-h in the presence of modulators of ATP release, P2 receptors and pathways downstream of P2 receptor activation.

Key findings: Adenosine 5'-triphosphate (ATP) was released from scratched cells. The ectonucleotidase apyrase and pharmacological inhibition of the nucleotide release hemichannel, pannexin-1, decreased wound closure over time. The non-selective P2Y receptor antagonist suramin and the selective P2Y₂ receptor antagonist AR-C118925XX, but not other P2 antagonists, decreased wound closure. AR-C118925XX decreased wound closure in a concentration-dependent fashion. However, exogenous P2Y₂ receptor agonists, ATP or uridine 5'-triphosphate, did not enhance wound closure. PCR and immunoblotting confirmed P2Y₂ receptor expression in HaCaT cells. U73122, a phospholipase C antagonist, and 2-aminoethoxydiphenylborate, an inositol 1,4,5-trisphosphate receptor-sensitive Ca²⁺-release channel antagonist, decreased wound closure consistent with P2Y₂ receptor activation. Absence of extracellular or intracellular Ca²⁺ or inhibition of intracellular Ca²⁺-release also impaired wound closure.

Significance: These data describe a novel autocrine signalling mechanism in which wound-mediated release of endogenous ATP in response to mechanical scratching of HaCaT cells activates P2Y₂ receptors to facilitate wound closure.