Previous data from our laboratory indicate that the lipid-metabolizing enzyme phospholipase D2 (PLD2) and the glycerol transporter aquaporin-3 (AQP3) physically and functionally interact in epidermal keratinocytes. AQP3 transports glycerol into the cell, making it available to the associated PLD2, which converts it to phosphatidylglycerol (PG). Our results further demonstrate that PG acts as a lipid second messenger to promote epidermal wound healing. Verkman and colleagues have generated an AQP3 knockout mouse model, which shows a profound epidermal phenotype including delayed skin wound healing. Interestingly, and of direct significance to this application, these AQP3 knockout mice also exhibit impaired corneal wound healing. Inspection of the literature suggests that epidermal keratinocytes and corneal epithelial cells show a number of similarities and may be regulated by common mechanisms. In exciting preliminary data we have found that PLD2 and AQP3 also associate in corneal epithelial cells and that the product of this interaction, PG, enhances scratch wound closure of corneal epithelial cells in culture as well as corneal wound healing in wild-type and AQP3 knockout mice in vivo. These findings lead us to hypothesize that the PLD2/AQP3/PG signaling module is important in the cornea. Because these effects of PG are reminiscent of the actions of epidermal growth factor receptor (EGFR) ligands on corneal epithelium, we specifically hypothesize that PLD2 and AQP3 associate in corneal epithelial cells to produce PG in response to EGFR ligands and that this PG acts as a signaling molecule to accelerate wound healing in the cornea by increasing epithelial cell migration and/or proliferation. In the research proposed, we will test this hypothesis and define the role of the PLD2/AQP3 signaling module in corneal cell biology and wound healing. Data in the literature and our own initial results in epidermal keratinocytes indicate that PG can activate protein kinase C?II (PKC?II). Based on these data, as well as reports that PKC? inhibition or silencing can inhibit corneal proliferation and proliferative signaling, we further hypothesize that the mechanism by which the PLD2/AQP3 model functions is through PG-activated PKC?II activation. We will test this idea using knockout animals, as well as overexpression, RNA interference, and pharmacological inhibition approaches to increase and decrease the levels of PLD2, AQP3 and PKC?II; we will then monitor corneal epithelial cell migration and proliferation, PG levels, PKC?II autophosphorylation (activation) and corneal wound healing in vivo. If our hypothesis proves correct, the research will lead to a better understanding of the corneal epithelial wound healing process and may lead to the identification of novel targets for the development of treatments to promote corneal would healing in patients after trauma, infection or ophthalmic surgery.
Proposal Narrative Corneal wounds resulting from trauma (including ophthalmic surgery), infection, and other etiologies are extremely painful, oftentimes compromise vision, and can lead to blindness. The events that regulate the healing of corneal wounds are not entirely known; however, mice that have been genetically engineered to lack a glycerol channel called aquaporin-3 exhibit delayed corneal wound healing. The hypothesis that we will test is that aquaporin-3 in combination with the lipid-metabolizing enzyme phospholipase D2 allows for the formation of phosphatidyglycerol, a signaling molecule that increases corneal cell migration and proliferation thereby accelerating corneal wound healing.