Targeted inhibition of wound-induced PAI-1 expression alters migration and differentiation in human epidermal keratinocytes.
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.
Li
F
F
Department of Surgery, Cornell University Medical College, 1300 York Avenue, New York, New York 10021, USA.
Goncalves
J
J
Faughnan
K
K
Steiner
M G
MG
Pagan-Charry
I
I
Esposito
D
D
Chin
B
B
Providence
K M
KM
Higgins
P J
PJ
Staiano-Coico
L
L
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*
D017395:Plasminogen Activator Inhibitor 1; D007544:Isopropyl Thiogalactoside