Role of FAK in keratinocyte adhesion

在伤口愈合过程中，角化细胞必须被激活，以便在填充伤口的临时细胞外基质分子上扩散和迁移。这种行为依赖于细胞外基质受体的整合素家族。整合素不仅是重要的结构粘附分子。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局灶黏附激酶（FAK）。FAK似乎参与了细胞的扩散和迁移，并可能在整合素依赖信号的转导中发挥其他重要作用。在以前的工作中，我们发现正常人类皮肤的角质形成细胞具有无法检测到的FAK水平。当这些细胞在培养中被激活时，它们开始表达FAK。为了评估FAK在调节角化细胞行为中的重要性，我们引入了FAK的激酶缺陷突变体，作为显性阴性抑制剂。我们发现FAK的抑制导致90%的角质细胞附着于胶原蛋白的抑制。在目前的建议中，我们的目的是研究局灶黏附激酶（FAK）在角化细胞黏附中的作用。瞬时转染人原代角质形成细胞不适合用于下游效应的生化评价。为了克服在原代细胞中研究FAK功能的局限性，我们提出在诱导启动子的控制下，建立稳定表达显性阴性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。如果没有，我们会问FAK的抑制是否会减少β 1整合素的表达。我们建议创建的细胞系对于进行FAK在角质形成细胞中的作用的下一层次的研究是必要的。

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.