Immunohistochemical and molecular characterization of cultured keratinocytes after dispase-mediated detachment from the growth substratum

Keratinocyte activation comprises changes in protein and gene expression pa ttern resulting in phenotypic and functional changes necessary for re-epith elialization such as the expression of urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPA-R; CD87). As uPA and uPA-R are ra pidly induced after dispase-mediated detachment of cultured normal human ep idermal keratinocytes (NHEK) we hypothesized that dispase-mediated detachme nt may cause a similar "activation" of keratinocytes with uPA and uPA-R bei ng only one aspect of a complex "activation reaction". To test this hypothe sis we have comparatively analysed adherent versus detached keratinocyte sh eets for selected indicators of keratinocyte activation by immunohistochemi stry. Furthermore we have identified genes via subtraction cloning which ar e up-regulated upon dispase-induced detachment. The analyses provided evide nce for an increased transcriptional and translational activity in detached keratinocytes, as indicated by over-expression of several ribosomal compon ents (L3 and S10 ribosomal protein) and transcription factors (initiation f actor 4A, elongation factor Icr). Increased proliferative activity was indi cated by increased expression of the proliferation markers Ki67, keratin 6 and keratin 17. Finally, several markers of keratinocyte activation such as the integrin chain alpha(v), psoriasin, glutathion-S-transferase and hepar in-binding epidermal growth factor-like growth factor were up-regulated. Fu rthermore mevalonate kinase, a molecule as yet unknown to be expressed in k eratinocytes, was identified. The findings provide evidence that dispase-me diated detachment in cultured keratinocytes induces a reaction, which compr ises the up-regulation of a complex array of proliferation- and migration-r elated molecules. The pattern of which resembles the activation reaction ob served in the re-epithelializing keratinocytes in vivo.