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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Center Core Grants (P30)
Project #
5P30AR041940-09
Application #
6318465
Study Section
Special Emphasis Panel (ZAR1-AAA-C (J2))
Project Start
1997-06-01
Project End
2001-05-31
Budget Start
Budget End
Support Year
9
Fiscal Year
2000
Total Cost
$55,151
Indirect Cost
City
Dallas
State
TX
Country
United States
Zip Code
75390
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