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

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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY006000-26
Application #
8268453
Study Section
Special Emphasis Panel (ZRG1-BDCN-F (02))
Program Officer
Mckie, George Ann
Project Start
1986-06-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
26
Fiscal Year
2012
Total Cost
$386,100
Indirect Cost
$148,500
Name
Boston University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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
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
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
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
Chi, Cheryl; Trinkaus-Randall, Vickery (2013) New insights in wound response and repair of epithelium. J Cell Physiol 228:925-9
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
Boucher, Ilene; Kehasse, Amanuel; Marcincin, Meredith et al. (2011) Distinct activation of epidermal growth factor receptor by UTP contributes to epithelial cell wound repair. Am J Pathol 178:1092-105
Mankus, Courtney; Rich, Celeste; Minns, Martin et al. (2011) Corneal epithelium expresses a variant of P2X(7) receptor in health and disease. PLoS One 6:e28541
Boucher, Ilene; Rich, Celeste; Lee, Albert et al. (2010) The P2Y2 receptor mediates the epithelial injury response and cell migration. Am J Physiol Cell Physiol 299:C411-21
Ren, Ruiyi; Hong, Zhenning; Gong, Haiyan et al. (2010) Role of glycosaminoglycan sulfation in the formation of immunoglobulin light chain amyloid oligomers and fibrils. J Biol Chem 285:37672-82

Showing the most recent 10 out of 22 publications