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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BDCN-F (02))
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Shen, Grace L
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Boston University
Schools of Medicine
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Minns, Martin S; Teicher, Gregory; Rich, Celeste B et al. (2016) Purinoreceptor P2X7 Regulation of Ca(2+) Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury. Am J Pathol 186:285-96
Derricks, Kelsey E; Trinkaus-Randall, Vickery; Nugent, Matthew A (2015) Extracellular matrix stiffness modulates VEGF calcium signaling in endothelial cells: individual cell and population analysis. Integr Biol (Camb) 7:1011-25
Sanderson, Julie; Dartt, Darlene A; Trinkaus-Randall, Vickery et al. (2014) Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland. Exp Eye Res 127:270-9
Stepp, Mary Ann; Zieske, James D; Trinkaus-Randall, Vickery et al. (2014) Wounding the cornea to learn how it heals. Exp Eye Res 121:178-93
Karamichos, D; Hutcheon, A E K; Rich, C B et al. (2014) In vitro model suggests oxidative stress involved in keratoconus disease. Sci Rep 4:4608
Lee, Albert; Derricks, Kelsey; Minns, Martin et al. (2014) Hypoxia-induced changes in Ca(2+) mobilization and protein phosphorylation implicated in impaired wound healing. Am J Physiol Cell Physiol 306:C972-85
Chi, Cheryl; Trinkaus-Randall, Vickery (2013) New insights in wound response and repair of epithelium. J Cell Physiol 228:925-9
Kehasse, Amanuel; Rich, Celeste B; Lee, Albert et al. (2013) Epithelial wounds induce differential phosphorylation changes in response to purinergic and EGF receptor activation. Am J Pathol 183:1841-1852
Oswald, Duane J; Lee, Albert; Trinidad, Monique et al. (2012) Communication between corneal epithelial cells and trigeminal neurons is facilitated by purinergic (P2) and glutamatergic receptors. PLoS One 7:e44574
Mankus, Courtney; Chi, Cheryl; Rich, Celeste et al. (2012) The P2X(7) receptor regulates proteoglycan expression in the corneal stroma. Mol Vis 18:128-38

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