The Aquaporin-3/Phospholipase D2 Signaling Pathway in Corneal Wound Healing

角膜伤口愈合中的 Aquaporin-3/磷脂酶 D2 信号通路

• 批准号: 10201519
• 负责人: Wendy B Bollag
• 金额: --
• 依托单位: CHARLIE NORWOOD VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10201519
• 关键词: Address; Affect; Blindness; Cell Cycle Progression; Cell Proliferation; Cell physiology; Cells; Cellular biology; Cornea; Corneal Injury; Data; Enzymes; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Epithelial Cell Proliferation; Epithelial Cells; Etiology; Event; Exhibits; Family; Genetic Engineering; Glycerol; Human; Impaired wound healing; Impairment; In Situ; In Vitro; Individual; Infection; Injury; Knockout Mice; Laboratories; Lead; Leukemic Cell; Ligands; Lipids; Literature; Modeling; Monitor; Mus; Operative Surgical Procedures; PKC-betaII; Pain; Patients; Pharmacology; Phenotype; Phosphatidylglycerols; Phospholipase; Process; Production; Protein Kinase; Protein Kinase C; RNA Interference; Reporting; Research; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Molecule; Skin; Skin wound healing; System; Testing; Trauma; Vision; Work; aquaporin 3; base; cell motility; cell type; corneal epithelial wound healing; corneal epithelium; healing; in vivo; inhibitor/antagonist; keratinocyte; knockout animal; limbal; migration; mouse model; novel; overexpression; phospholipase D2; protein E; protein activation; protein kinase C beta; response; therapy development; wound closure; wound healing

Project Summary/Abstract 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 CβII (PKCβII). Based on these data, as well as reports that PKCβ 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 PKCβII 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 PKCβII; we will then monitor corneal epithelial cell migration and proliferation, PG levels, PKCβII 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.

项目摘要/摘要 我们实验室的先前数据表明，脂量代谢磷脂酶D2（PLD2） 甘油转运蛋白水通道蛋白-3（AQP3）在表皮角质形成细胞中的物理和功能上相互作用。 AQP3将甘油传输到细胞中，使其可用于关联的PLD2，将其转换为 磷脂酰甘油（Pg）。我们的结果进一步表明，PG充当脂质的第二使者 促进表皮伤口愈合。 Verkman及其同事已经生成了AQP3敲除鼠标模型， 显示出深刻的表皮表型，包括延迟皮肤伤口愈合。有趣的是，以及 这些AQP3敲除小鼠对此应用的直接意义也暴露了角膜伤口愈合受损。 文献检查表明表皮角质形成细胞和角膜上皮细胞显示了许多 相似性，可以通过共同的机制来调节。在令人兴奋的初步数据中，我们发现 PLD2和AQP3还与角膜上皮细胞相关联，并且这种相互作用的乘积PG增强了 培养中角膜上皮细胞的刮擦闭合以及野生型和角膜伤口愈合 AQP3淘汰小鼠在体内。这些发现使我们假设PLD2/AQP3/PG信号传导模块 在角膜中很重要。因为PG的这些影响提醒表皮生长的作用 因子受体（EGFR）角膜上皮上的配体，我们特别假设PLD2和AQP3 角膜上皮细胞中的辅助对EGFR配体产生PG，并且该Pg充当 通过增加上皮细胞迁移和/或 增殖。在提出的研究中，我们将检验这一假设并定义PLD2/AQP3的作用 角膜细胞生物学和伤口愈合中的信号传导模块。文献中的数据和我们自己的初始结果 表皮角质形成细胞表明PG可以激活蛋白激酶CβII（PKCβII）。基于这些数据，为 以及报道PKCβ抑制或沉默可以抑制角膜增殖和增殖信号，我们 进一步假设PLD2/AQP3模型函数通过PG激活的机制 PKCβII激活。我们将使用基因敲除动物以及过表达，RNA干扰， 和药物抑制方法，以增加和降低PLD2，AQP3和PKCβII的水平； 然后，我们将监测角膜上皮细胞迁移和增殖，PG水平，PKCβII自磷酸化 （激活）和角膜伤口在体内愈合。如果我们的假设证明是正确的，该研究将导致 更好地了解角膜上皮伤口愈合过程，并可能导致新颖的鉴定 创伤后患者的促进角膜治疗以促进角膜的靶标将愈合 或眼科手术。