Cell signal transduction in corneal wound healing

描述(由申请人提供)：这项建议是我们正在进行的关于角膜中脂质和蛋白质磷酸化的研究的继续，重点是伤口闭合和上皮细胞增殖过程中的细胞信号事件。我们将验证这样一种假设，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/去激活与磷脂酰肌醇-3激酶(PI-3K)和丝裂原激活的蛋白激酶(MAPK)途径相关的特定激酶来调节角膜修复。我们提出了实验来测试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受凋亡的影响。我们将研究三种特定的蛋白激酶C(PKC)亚型，即PKCA、PKCE和PKCu，作为细胞增殖和迁移的调节因子的作用，以及两个MAPK级联信号通路ERK1/2和p38在调控伤口愈合中的关系。有人建议研究在创伤过程被激活后，特定蛋白酪氨酸磷酸酶(PTPs)在PI-3K信号关闭中的作用。角膜上皮层的清创将作为伤口愈合的模型，并辅之以体外迁移和增殖分析。不同信号通路的选择性抑制剂将被用来将我们的发现与伤口闭合联系起来。成分的鉴定将通过使用特定抗体的Western印迹、使用酶免疫复合体的激酶分析以及使用标记到PKC亚型的绿色荧光蛋白在活细胞中的荧光显微镜来完成。这些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PKCs和PTP参与角膜伤口愈合的过程。这一知识将使我们能够确定针对信号通路选择性步骤的药物的特征。

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.