Cell signal transduction in corneal wound healing

描述（由申请人提供）：该提案是我们正在进行的角膜脂质和蛋白质磷酸化研究的延续，重点关注伤口闭合和上皮细胞增殖期间的细胞信号传导事件。我们将测试这一假设，即角膜损伤后释放的两种旁分泌生长因子，肝细胞生长因子（HGF）和角质形成细胞生长因子（KGF），通过激活-失活与磷脂酰肌醇-3激酶（PI-3K）和丝裂原活化蛋白激酶（MAPK）通路相关的特异性激酶来调节角膜修复。我们提出实验来测试HGF如何通过激活PI-3K-蛋白激酶B/Akt-1来保护角膜上皮细胞免于凋亡。我们将研究三种特异性蛋白激酶C（PKC）亚型，PKCa，PKCe和PKCu作为细胞增殖和迁移调节剂的作用，以及两种MAPK级联反应，ERK 1/2和p38在伤口愈合调节中的关系。研究提出了调查行动的具体蛋白酪氨酸磷酸酶（PTPs）在关闭PI-3K信号后，创伤过程已被激活。角膜上皮层清创术将用作伤口愈合模型，并辅以体外迁移和增殖试验。不同信号通路的选择性抑制剂将用于将我们的发现与伤口闭合相关联。将通过特异性抗体的蛋白质印迹法、酶免疫复合物的激酶测定法和使用标记至PKC亚型的绿色荧光蛋白的活细胞荧光显微镜检查法完成组分的鉴定。所获得的结果将使我们能够更好地确定PI-3K和MAPK激活的信号，以及特定的PKC和PTP在角膜伤口愈合中的参与。这些知识将使我们能够表征针对信号通路的选择性步骤的药物。

DESCRIPTION (provided by applicant): This proposal is a continuation of our ongoing studies on lipid and protein phosphorylation in the cornea, focusing on cell signaling events during wound closure and epithelial cell proliferation. We will test the hypothesis that two paracrine growth factors released after corneal injury, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), modulate corneal repair through activation-deactivation of specific kinases linked to the phosphatidylinositol-3 kinase (PI-3K) and the mitogen-activated protein kinase (MAPK) pathways. We propose experiments to test how HGF protects corneal epithelial cells from apoptosis through activation of PI-3K-protein kinase B/Akt-1 activation. We will investigate the role of three specific protein kinase C (PKC) isoforms, PKCa, PKCe, and PKCu, as modulators of cell proliferation and migration and the relation between two MAPK cascades, ERK1/2 and p38 in the regulation of wound healing. Studies are proposed to investigate the action of specific protein tyrosine phosphatases (PTPs) in the switching-off of PI-3K signals after the wound process has been activated. The debridement of the corneal epithelial layer will be used as a model of wound healing, complemented by in vitro migration and proliferation assays. Selective inhibitors of the different signal pathways will be used to correlate our findings with wound closure. Identification of components will be accomplished by Western blot with specific antibodies, kinase assays using enzyme immunocomplexes, and fluorescence microscopy in live cells using green fluorescent protein tagged to PKC isoforms. The results obtained will allow us to better define the signals activated by PI-3K and MAPK and the involvement of specific PKCs and PTP in corneal wound healing. This knowledge will allow us to characterize drugs that target selective steps of the signaling pathways.