title: Targeted inhibition of wound-induced PAI-1 expression alters migration and differentiation in human epidermal keratinocytes.
abstract: In the adult epidermis, keratinocytes do not normally express the type-1 inhibitor of plasminogen activator (PAI-1). Basal epithelial cell-specific PAI-1 synthesis, however, accompanies epidermal wound repair in vivo in which PAI-1 transcripts and immunoreactive protein are confined to epithelial cells 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 expression. PAI-1 synthesis was maximal in low population density, exponentially growing HaCaT cultures; relative PAI-1 mRNA and protein levels progressively declined as cells attained, and were maintained in, a postconfluent condition. While the fraction of PAI-1(+) keratinocytes remained stable (at approximately 85-90% of the population) throughout the culture period, both PAI-1 mRNA abundance and mean cell-associated PAI-1 protein declined by >90% during prolonged (i.e., 8-day) growth arrest. Similar to epidermal trauma in vivo, scrape wounding of HaCaT monolayers resulted in the rapid and location-specific induction of PAI-1 protein (an increase of 11- to 16-fold relative to unwounded cultures) in cells immediately bordering the injury site. PAI-1 expression was evident in keratinocytes that comprised the opposed migrating fronts and remained elevated until wound closure. Down-regulation of PAI-1 synthesis in HaCaT cells transfected with an inducible LacSwitch-based antisense vector system markedly impaired both the rate and the extent of wound closure. All injuries created in antisense PAI-1 monolayers remained unhealed at day 8 postinjury compared to the 3-day complete repair typical of control cultures. Vector-driven modulation of PAI-1 synthesis was also associated with an increase in the percentage of suprabasal-type keratinocytes within the wound field. PAI-1 expression by migrating HaCaT cells appears necessary to maintain the basal epidermal phenotype and/or appropriate cell-to-substrate adhesion during injury repair.
authors.affiliation: Department of Surgery, Cornell University Medical College, 1300 York Avenue, New York, New York 10021, USA.
authors.forename: F
authors.initials: F
authors.lastname: Li
authors.forename: J
authors.initials: J
authors.lastname: Goncalves
authors.forename: K
authors.initials: K
authors.lastname: Faughnan
authors.forename: M G
authors.initials: MG
authors.lastname: Steiner
authors.forename: I
authors.initials: I
authors.lastname: Pagan-Charry
authors.forename: D
authors.initials: D
authors.lastname: Esposito
authors.forename: B
authors.initials: B
authors.lastname: Chin
authors.forename: K M
authors.initials: KM
authors.lastname: Providence
authors.forename: P J
authors.initials: PJ
authors.lastname: Higgins
authors.forename: L
authors.initials: L
authors.lastname: Staiano-Coico
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D000078404:Epidermal Cells; D015870:Gene Expression; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D014945:Wound Healing / Q000502:physiology*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D007544:Isopropyl Thiogalactoside