Cell Cycle of Corneal Endothelium

描述(申请人提供)：角膜内皮负责维持角膜的清晰度，但这些细胞不会在一生中分裂。我们的长期目标是开发方法来刺激增殖并增加因低内皮细胞计数而导致视力丧失风险的个体的细胞密度。人角膜内皮细胞在器官培养中可以分裂，提示多种因素共同作用在体内具有较强的抗增殖压力。接触依赖的增殖抑制似乎是一个重要的抗增殖因子，它部分地受磷酸酪氨酸磷酸酶(PTPs)的调节。受体酪氨酸激酶的激活，如EGF受体，导致酪氨酸磷酸化(Tyr-PO4)诱导的细胞周期进入。细胞间的接触可以被钙粘蛋白相关蛋白的Tyr-PO4破坏。PTPs抑制这两个Tyr-PO4事件，表明PTP活性有助于维持接触依赖性的增殖抑制。在没有丝裂原的情况下，融合内皮中细胞-细胞接触的释放不会刺激细胞周期进入。原钒酸钠(SOV)是一种PTP抑制剂，可以在没有丝裂原的情况下释放细胞接触并促进细胞周期进入，提示抑制PTP活性可能会增强EGF等丝裂原对融合内皮细胞的增殖作用。我们假设，通过下调内皮细胞中蛋白的表达或抑制特定PTPs的活性，我们将能够使细胞对有丝分裂原刺激更有反应，从而为因内皮细胞计数低而有视力丧失风险的个体提供一种增加细胞密度的方法。为验证这一假说，提出的具体目标包括：1.确定在角膜内皮细胞中表达的特定PTPs；2.确定每个PTP在调节EGF驱动的有丝分裂信号中的重要性；3.确定钙粘素-连环蛋白复合体是否是角膜内皮中PTP调节的靶标；以及4.测试下调特定PTPs的表达或抑制其活性是否会促进原位人角膜内皮细胞的增殖和增加细胞密度。免疫沉淀、Western blotting、RT-PCR、免疫细胞化学、流式细胞术、反义方法和显性-阴性突变体的表达将被用于实现这一提议的目标。

DESCRIPTION (provided by applicant): The corneal endothelium is responsible for maintaining corneal clarity, but these cells do not divide throughout life. Our long-term goal is to develop methods to stimulate proliferation and increase cell density in individuals at risk for vision loss due to low endothelial cell counts. Human corneal endothelial cells CAN divide in organ culture, suggesting that multiple factors together exert a strong anti-proliferative pressure in vivo. Contact-dependent inhibition of proliferation appears to be an important anti-proliferative factor, which is regulated, in part, by phosphotyrosine phosphatases (PTPs). Activation of receptor tyrosine kinases, such as the EGF receptor, leads to tyrosine phosphorylation (Tyr-PO4)-induced cell cycle entry. Cell-cell contacts can be disrupted by Tyr-PO4 of cadherin-associated proteins. PTPs inhibit both Tyr-PO4 events, suggesting that PTP activity helps maintain contact-dependent inhibition of proliferation. Release of cell-cell contacts in confluent endothelium does NOT stimulate cell cycle entry in the absence of mitogens. Sodium orthovanadate (SOV), a PTP inhibitor, releases cell contacts and promotes cell cycle entry without mitogens, suggesting that inhibition of PTP activity might augment the proliferative effect of mitogens, such as EGF, in confluent endothelium. We hypothesize that, by down-regulating the protein expression or inhibiting the activity of specific PTPs in the endothelium, we will be able to make the cells more responsive to mitogenic stimulation, thus providing a method to increase cell density in individuals at risk for vision loss due to low endothelial cell counts. Specific Aims proposed to test this hypothesis include: I. Identify specific PTPs expressed in corneal endothelial cells; II. Determine the importance of each PTP in regulating EGF-driven mitogenic signaling; III. Determine whether the cadherin-catenin complex is a target for PTP regulation in corneal endothelium; and IV. Test if down-regulating the expression or inhibiting the activity of specific PTPs will promote proliferation and increase cell density in human corneal endothelial cells in situ. Immunoprecipitation, Western blotting, RT-PCR, immunocytochemistry, flow cytometry, antisense methods, and expression of dominant-negative mutants will be used to accomplish the objectives of this proposal.