Targeted inhibition of wound-induced PAI-1 expression alters migration anddifferentiation in human epidermal keratinocytes

In the adult epidermis, keratinocytes do not normally express the type-1 in hibitor of plasminogen activator (PAI-1), Basal epithelial cell-specific PA I-1 synthesis, however, accompanies epidermal wound repair in vivo in which PAI-1 transcripts and immunoreactive protein are confined to epithelial ce lls in the migrating tongue and the hyperproliferative zone. A model system using human keratinocytes (HaCaT cells) was developed to assess functional relationships between epithelial growth state transitions and PAI-1 expres sion, PAI-1 synthesis was maximal in low population density, exponentially growing HaCaT cultures; relative PAI-1 mRNA and protein levels progressivel y declined as cells attained, and were maintained in, a postconfluent condi tion. While the fraction of PAI-1(+) keratinocytes remained stable (at appr oximately 85-90% of the population) throughout the culture period, both PAI -1 mRNA abundance and mean cell-associated PAI-1 protein declined by >90% d uring prolonged (i.e., 8-day) growth arrest. Similar to epidermal trauma in vivo, scrape wounding of HaCaT monolayers resulted in the rapid and locati on-specific induction of PAI-1 protein (an increase of 11- to 16-fold relat ive to unwounded cultures) in cells immediately bordering the injury site, PAI-1 expression was evident in keratinocytes that comprised the opposed mi grating fronts and remained elevated until wound closure, Down-regulation o f PAI-1 synthesis in HaCaT cells transfected with an inducible LacSwitch-ba sed antisense vector system markedly impaired both the rate and the extent of wound closure. All injuries created in antisense PAI-1 monolayers remain ed unhealed at day 8 postinjury compared to the 3-day complete repair typic al of control cultures. Vector-driven modulation of PAI-1 synthesis was als o associated with an increase in the percentage of suprabasal-type keratino cytes within the wound held, PAI-1 expression by migrating HaCaT cells appe ars necessary to maintain the basal epidermal phenotype and/or appropriate cell-to-substrate adhesion during injury repair. (C) 2000 Academic Press.