Injury-induced P2Y receptor stimulation leads to epidermal growth factor receptor activation and corneal epithelial wound healing

Abstract

The cornea plays a major role in the refraction of light to the retina. Therefore, the integrity and transparency of the corneal epithelium are critical to vision. Following injury, a combination of rapid signal transduction and long-term cell migration is essential for wound closure. Previous studies demonstrated that injury to corneal epithelial cells resulted in the release of nucleotides that induce the propagation of a Ca²⁺ wave to neighboring cells. The hypotheses tested are that specific purinergic (P2Y) receptors are responsible for this wave and that P2Y stimulation is necessary to activate signals, including epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) phosphorylation, that lead to cell migration and wound healing. It was demonstrated that pretreatment of primary corneal epithelial cells with ATP and UTP inhibited the immediate wound response, while 3'-O-(4-benzoyl)benzoylATP (BzATP), ADP, and UDP did not. The role of specific P2Y receptors in a corneal epithelial cell line was demonstrated by using siRNA to knockdown the P2Y₂ and P2Y ₄ receptors. This downregulation inhibited the injury and UTP induced Ca²⁺ waves. Additionally, we hypothesized that purinergic receptor stimulation could mediate cell migration. ATP, UTP, ADP, and BzATP induced cell migration, and integrins and Src were necessary for ATP-induced migration. Reactive Blue-2, a general purinergic receptor inhibitor, and P2Y₂ receptor knockdown reduced injury- and nucleotide-induced phosphorylation of ERK. Further studies established a role for EGFR in purinergic signaling. Heparin binding (HB)-EGF induced cell migration, and inhibition of HE-EGF signaling reduced both cell migration and EGFR phosphorylation. Moreover, injury and trinucleotide pretreatment reduced the EGF induced Ca ²⁺ wave, and EGF enhanced ATP- and UTP-induced migration. Injury and nucleotides induced phosphorylation of the EGFR on specific tyrosine residues. Porcine aortic endothelial cells (PAE) cells lacking the EGFR showed reduced ERK phosphorylation upon injury and nucleotide stimulation compared to PAE cells stably transfected with wild-type EGFR (E1-PAE). E1-PAE cells showed increased migration upon stimulation with UTP, while EGFR mutants Y1068F and Y1086F reduced this migration. These data indicate that EGFR phosphorylation is essential for P2Y mediated migration and that P2Y₂ is a central player in corneal epithelial repair.