The goal of our laboratory has been to understand the response of the cornea to injury and disease. In the previous application we hypothesized that epithelium released a factor that elicited a healing response, which led to our laboratory showing an interaction between the purinergic and Epidermal Growth Factor(EGF) receptor(R) signaling pathways. Our lab has demonstrated that the active components that are released upon injury are purines and pyrimidines (nucleotides), which cause the propagation of a Ca2+ wave to neighboring cells. We then identified the expression of purinergic receptors (P2Y and P2X) in both epithelium and trigeminal neuronal cells. Degradation of their ligands with ectonucleotidases inhibits early events that happen rapidly after injury such as Ca2+ waves, as well as later events such as wound repair. We found that injury (or specific agonist) induced phosphorylation of EGFR residues in vitro is distinct from that generated by EGF. Furthermore, when one of these residues is mutated, migration is altered. The goal of this proposal is to determine how epithelial debridement, along with other factors mediates the activation of purinergic receptors in the cornea. We hypothesize that downstream signaling pathways activated by different types of wounds are due to the phosphorylation of distinct residues on EGFR. Quantitative analyses of phosphorylated residues on EGFR will be performed using mass spectrometry.
Our aims are to: 1. Determine the role of the P2Y receptors and their activation on phosphorylation of EGFR in response to corneal wounds and 2. Determine the impact of hypoxia and neutrophils using WT and P2Y2 null mice on the expression of P2Y receptors, phosphorylation of EGFR and repair of corneal wounds in organ culture. The successful outcomes of this proposal will allow us to predict the downstream consequences of EGFR activation and provide a systematic method for developing therapeutic modalities.
Narrative Injury to the corneal epithelium can be painful and compromise vision. Studies have shown that different types of injuries can cause the eye to respond in distinct ways that make it difficult to develop treatment plans. Our goal is to determine what roles these proteins called purinergic receptors play and how they signal to other proteins using a technology called mass spectrometry.
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