Control of Corneal Epithelial Cell Proliferation

角膜上皮细胞增殖的控制

• 批准号: 6744196
• 负责人: Peter S Reinach
• 金额: $ 45.8万
• 依托单位: STATE COLLEGE OF OPTOMETRY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6744196
• 关键词: biological signal transduction; calcium flux; cell growth regulation; cell migration; cell morphology; cell proliferation; confocal scanning microscopy; corneal epithelium; enzyme activity; epidermal growth factor; genetic transcription; growth factor receptors; immunoprecipitation; kinase inhibitor; light scattering; membrane transport proteins; mitogen activated protein kinase; protein protein interaction; protein structure function; receptor expression; reporter genes; sodium potassium exchanging ATPase; video microscopy

DESCRIPTION (provided by applicant): Epidermal growth factor, EGF, plays an essential role in corneal epithelial renewal. It is needed for proper corneal epithelial healing following wounding of this layer, which is required for normal vision. Our long-term goal is to further understand how EGF receptor-linked signaling elicits its effects on corneal epithelial homeostasis. We have found that initiation by EGF of cell cycle progression is dependent on the stimulation of ion transporters activity; EGF stimulates Na:K:2Cl cotransporter 1 (NKCC1) activity, and subsequently stimulates K+ and Cl- conductance activity, which results in a transient increase in cell volume. On the other hand, cell cycle progression is known to be associated with increases in cell volume. Therefore, we now hypothesize that in rabbit corneal epithelial cells (RCEC), EGF-induced changes in NKCC1 activity and expression is a requisite for cell cycle progression from early G1 to mitosis. We further hypothesize that modulation of activity of all ion transporters and channels involved in cell volume regulation is needed for cell proliferation and migration. Our three aims entail: 1) determining during cell cycle progression whether there is differential activity and expression of ion transporters (NKCC1, Na/K-ATPase, K+ and Cl- conductances) underlying cell volume regulation; 2) determining the interactions between the cell signaling pathways mediating EGF receptor control of the aforementioned ion transporters; 3) dissecting how EGF controls cell proliferation and migration. Cell volume and regulatory volume behavior will be characterized with video, confocal microscopy and low angle light scattering, respectively. NKCC1 gene transcription will be characterized with a reporter gene assay. NKCC1 activity will be evaluated based on measurements of 86Rb+ influx. EGF-induced activation of mitogen activated protein kinase (MAPK) signaling and crosstalk will be determined with kinase assays. Interactions of specific MAPK components with NKCC1 will be evaluated using coimmunoprecipitation. EGF mediated signaling control of epithelial proliferation and migration will be investigated using kinase inhibitors and stable MAPK gene (constitutively active and tetracycline-inducible dominant negative) expression. Rates of epithelial proliferation and migration will be characterized in an in vitro wound healing model system. These studies could provide new strategies for hastening corneal epithelial wound healing and reducing severity of losses in vision. In addition, they may provide insights on how to stimulate healing bypassing growth factor receptor control.

描述（由申请人提供）：表皮生长因子EGF在角膜上皮更新中起着至关重要的作用。这层受伤后需要适当的角膜上皮愈合，这是正常视力所必需的。我们的长期目标是进一步了解EGF受体互联信号如何引起其对角膜上皮稳态的影响。我们发现，EGF的启动细胞周期进程取决于离子转运蛋白活性的刺激。 EGF刺激Na：K：2Cl共转运蛋白1（NKCC1）活性，然后刺激K+和Cl-电导活性，从而导致细胞体积的短暂增加。另一方面，已知细胞周期进程与细胞体积增加有关。因此，我们现在假设在兔角膜上皮细胞（RCEC）中，EGF诱导的NKCC1活性变化和表达是从早期G1到有丝分裂的细胞周期进程的必要条件。我们进一步假设，需要调节所有离子转运蛋白的活性和参与细胞体积调节的通道的调节，以进行细胞增殖和迁移。我们的三个目的需要：1）在细胞周期进程中确定离子转运蛋白（NKCC1，Na/k-ATPase，K+和Cl-电导率）是否存在差异活性和表达。 2）确定介导上述离子转运蛋白的EGF受体控制的细胞信号通路之间的相互作用； 3）解剖EGF如何控制细胞增殖和迁移。细胞体积和调节体积行为将分别以视频，共聚焦显微镜和低角度光散射为特征。 NKCC1基因转录将以报告基因测定法进行表征。 NKCC1活性将根据86RB+涌入的测量值进行评估。 EGF诱导的有丝分裂原活化蛋白激酶（MAPK）信号传导和串扰的激活将用激酶测定确定。特定MAPK组件与NKCC1的相互作用将使用共免疫沉淀评估。 EGF介导的上皮增殖和迁移的信号传导控制将使用激酶抑制剂和稳定的MAPK基因（组成型活性和四环素诱导的显性负阴性）进行研究。上皮增殖和迁移的速率将在体外伤口愈合模型系统中进行表征。这些研究可以提供新的策略，以加快角膜上皮伤口愈合并减少视力损失的严重程度。此外，它们可能会提供有关如何刺激绕过生长因子受体控制的愈合的见解。