PAI-1 gene expression is regionally induced in wounded epithelial cell monolayers and required for injury repair

Induced expression of plasminogen activator inhibitor type-1 (PAI-1), a maj or negative regulator of pericellular plasmin generation, accompanies wound repair in vitro and in vivo. Since transcriptional control of the PAI-1 ge ne is superimposed on a growth state-dependent program of cell activation ( Kutz et al., 1997, J Cell Physiol 170:8-18), it was important to define pot entially functional relationships between PAI-1 synthesis and subpopulation s of cells that emerge during the process of injury repair in T2 renal epit helial cells. Specific cohorts of migratory and proliferating cells induced in response to monolayer trauma were spatially as well as temporally disti nct. Migrating cells did not divide in the initial 12 to 20 h postinjury. A fter 24 h, S-phase cells were generally restricted to a region 1 to 2 mm fr om, and parallel to, the wound edge. Proliferation of wound bed cells occur red subsequent to wound closure, whereas the distal contact-inhibited monol ayer remained generally quiescent. Hydroxyurea blockade indicated, however, that proliferation (most likely of cells immediately behind the motile "to ngue") was necessary for maintenance of cell-to-cell cohesiveness in the ad vancing front, although the ability to migrate was independent of prolifera tion. PAI-1 mRNA expression was rapidly up-regulated in response to woundin g with inductive kinetics approximating that of serum-stimulated cultures. Differential harvesting of T2 cell subpopulations, based on proximity to th e injury site, prior to Northern assessments of PAI-1 mRNA abundance indica ted that PAI-1 transcripts were restricted to cells immediately bordering t he wound or actively migrating and not expressed by cells in the distal con tact-inhibited monolayer regions. Such cell location-specific distribution of PAI-1-producing cells was confirmed by immunocytochemistry. PAI-1 synthe sis in cells that locomoted into the wound field continued until injury clo sure. Down-regulation of PAI-1 synthesis and matrix deposition in renal epi thelial cells, stably transfected with a PAI-1 antisense expression vector, significantly impaired wound closure. Transfection of the wound repair-def icient R/A epithelial line with a sense PAI-1 expression construct restored both approximately normal levels of PAI-1 synthesis and repair ability. Th ese data indicate that PAI-1 induction is an early event in creation of the wound-activated phenotype and appears to participate in the regulation of renal epithelial cell motility during in vitro injury resolution. (C) 2000 Wiley-Liss, Inc.