Role of FAK in keratinocyte adhesion

FAK 在角质形成细胞粘附中的作用

• 批准号: 6318465
• 负责人: LAWRENCE Thomas KIM
• 金额: $ 5.52万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6318465
• 关键词: apoptosis; biological signal transduction; cell adhesion; cell line; enzyme inhibitors; enzyme mechanism; flow cytometry; focal adhesion kinase; gel electrophoresis; integrins; keratinocyte; wound healing

During wound healing keratinocytes must become activated in order to spread and migrate on the provisional extracellular matrix molecules that fill the wound. This behavior is dependent on the integrin family of extracellular matrix receptors. Integrins are not only important as structural adhesion molecules. They are able to generate a complex signaling cascade. One of the key molecules associated with integrin signaling is focal adhesion kinase (FAK). FAK appears to be involved in cell spreading and migration and may have other important roles in transducing integrin-dependent signals. In previous work we found that keratinocytes from normal human skin had undetectable levels of FAK. As these cells become activated in culture they began to express FAK. In order to assess the importance of FAK in regulating keratinocyte behavior we introduced kinase deficient mutants of FAK that act as dominant-negative inhibitors. We found that inhibition of FAK resulted in a 90% inhibition of keratinocyte attachment to collagen. In the current proposal we aim to investigate the role of focal adhesion kinase (FAK) in keratinocyte adhesion. Transient transfection of primary human keratinocytes is not suitable for biochemical evaluation of down-stream effects. To overcome the limitations of studying FAK function in primary cells we propose to create cell lines that stably- express dominant-negative FAK mutants under control of an inducible promoter. We will use these cell lines to determine the effects of FAK inhibition in epithelial cells. First we will determine if inhibition of FAK reduces adhesion by inducing apoptosis. If not we will ask whether or not inhibition of FAK reduces expression of beta1 integrins. The cell lines that we propose to create are necessary to proceed to the next level of investigation of the role of FAK in keratinocytes.

在伤口愈合过程中，角质形成细胞必须被激活，以便在填充伤口的临时细胞外基质分子上扩散和迁移。这种行为取决于细胞外基质受体的整合素家族。整合素不仅作为结构粘附分子很重要。它们能够产生复杂的信号级联。与整合素信号传导相关的关键分子之一是粘着斑激酶（FAK）。 FAK 似乎参与细胞扩散和迁移，并且可能在转导整合素依赖性信号方面具有其他重要作用。在之前的工作中，我们发现正常人类皮肤的角质形成细胞中的 FAK 水平无法检测到。当这些细胞在培养物中被激活时，它们开始表达 FAK。为了评估 FAK 在调节角质形成细胞行为中的重要性，我们引入了 FAK 的激酶缺陷突变体，其充当显性失活抑制剂。我们发现抑制 FAK 会导致角质形成细胞与胶原蛋白的附着被抑制 90%。在当前的提案中，我们的目标是研究粘着斑激酶（FAK）在角质形成细胞粘附中的作用。原代人角质形成细胞的瞬时转染不适合下游效应的生化评估。为了克服在原代细胞中研究 FAK 功能的局限性，我们建议创建在诱导型启动子控制下稳定表达显性失活 FAK 突变体的细胞系。我们将使用这些细胞系来确定 FAK 抑制对上皮细胞的影响。首先我们将确定抑制 FAK 是否通过诱导细胞凋亡来减少粘附。如果不是，我们会问抑制 FAK 是否会降低 β1 整合素的表达。我们建议创建的细胞系对于进一步研究 FAK 在角质形成细胞中的作用是必要的。