FAK induction in keratinocytes in an in vitro model of reepithelialization

During reepithelialization, keratinocytes must become activated in order to migrate over the provisional extracellular matrix of the wound. Previously we have shown that focal adhesion kinase (FAK) is induced in activated keratinocytes. The mechanisms responsible for keratinocyte activation are unknown. Here we use an organ culture system to investigate FAK up-regulation and regulation of keratinocyte activation. Normal human skin was cultured on type I collagen. Keratinocytes migrated out of the explant onto the supporting collagen. Immunostaining for FAK showed induction in the migrating epithelium and also in the center of the explant some distance from the cut edge. Cells from the center of the explant expressed FAK and showed the activated phenotype as defined by their ability to spread on collagen. Since FAK is a tyrosine kinase, the tyrosine kinase inhibitors genistein or herbimycin A were added to the explant medium for 24 h. Inhibition of tyrosine kinase activity delayed epithelial migration, but keratinocytes were able to begin migrating after removal of the inhibitors. We conclude that FAK is up-regulated in keratinocytes in this whole skin explant model. Furthermore FAK up-regulation and keratinocyte activation are not confined to the migrating cells but are found in cells some distance from the skin margin. These data suggest that (1) cell migration, contact with wound matrix molecules, loss of cell-cell contact, or loss of basement membrane contact is not necessary for FAK up-regulation or keratinocyte activation; and (2) tyrosine kinase signaling pathways are important for reepithelialization.