Kirwin M. Providence

PAI-1 expression is required for epithelial cell migration in two distinct phases of in vitro wound repair.

Several proteases and their specific inhibitors modulate the interdependent processes of cell migration and matrix proteolysis as part of the global program of trauma repair. Expression of plasminogen activator inhibitor type‐1 (PAI‐1), a serine protease inhibitor (SERPIN) important in the control of barrier proteolysis and cell‐to‐matrix adhesion, for example, is spatially‐temporally regulated following epithelial denudation injury in vitro as well as in vivo. PAI‐1 mRNA/protein synthesis was induced early after epidermal monolayer scraping and restricted to keratinocytes comprising the motile cohort closely recapitulating, thereby, similar events during cutaneous healing. The time course of PAI‐1 promoter‐driven PAI‐1‐GFP fusion “reporter” expression in wound‐juxtaposed cells approximated that of the endogenous PAI‐1 gene confirming the location‐specificity of gene regulation in this model. ERK activation was evident within 5 min after injury and particularly prominent in cells residing at the scrape‐edge (suggesting a possible role in PAI‐1 induction and/or the motile response) as was myosin light chain (MLC) phosphorylation. Indeed, MEK blockade with PD98059 or U0126 attenuated keratinocyte migration (by ≥60%), as did transient transfection of a dominant‐negative ERK1 construct (40% decrease in monolayer repair), and completely inhibited PAI‐1 transcript expression. Anti‐sense down‐regulation of PAI‐1 synthesis (by 80–85%), or addition of PAI‐1 neutralizing antibodies also inhibited injury site closure over a 24 h period establishing that PAI‐1 was required for efficient long‐term planar motility in this system. PAI‐1 anti‐sense transfection or actinomycin D transcriptional blockade, in contrast, did not affect the initial migratory response suggesting that residual PAI‐1 protein levels (at least in transfectant cells and actinomycin D‐treated cultures) may be sufficient to support early cell movement. Pharmacologic inhibition of keratinocyte MEK signaling effectively ablated scrape‐induced PAI‐1 mRNA expression but failed to attenuate wound‐associated increases in cellular PAI‐1 protein levels soon after monolayer injury. Collectively, these data suggest that basal PAI‐1 transcripts may be mobilized for initial PAI‐1 synthesis and, perhaps, the early motile response while maintenance of the normal rate of migration requires the prolonged PAI‐1 expression that typically accompanies the repair response. To assess this possibility, scrape site closure studies were designed using keratinocytes isolated from PAI‐1−/− mice. PAI‐1−/− keratinocytes, in fact, had a significant wound healing defect evident even within the first 6 h following monolayer denudation injury. Addition of active PAI‐1 protein to PAI−/− keratinocytes rescued the migratory phenotype that that approximating wild‐type cells. These findings validate use of the present keratinocyte model to investigate injury‐related controls on PAI‐1 gene regulation and, collectively, implicate participation of PAI‐1 in two distinct phases of epidermal wound repair.

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 major negative regulator of pericellular plasmin generation, accompanies wound repair in vitro and in vivo. Since transcriptional control of the PAI‐1 gene 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 potentially functional relationships between PAI‐1 synthesis and subpopulations of cells that emerge during the process of injury repair in T2 renal epithelial cells. Specific cohorts of migratory and proliferating cells induced in response to monolayer trauma were spatially as well as temporally distinct. Migrating cells did not divide in the initial 12 to 20 h postinjury. After 24 h, S‐phase cells were generally restricted to a region 1 to 2 mm from, and parallel to, the wound edge. Proliferation of wound bed cells occurred subsequent to wound closure, whereas the distal contact‐inhibited monolayer remained generally quiescent. Hydroxyurea blockade indicated, however, that proliferation (most likely of cells immediately behind the motile “tongue”) was necessary for maintenance of cell‐to‐cell cohesiveness in the advancing front, although the ability to migrate was independent of proliferation. PAI‐1 mRNA expression was rapidly up‐regulated in response to wounding with inductive kinetics approximating that of serum‐stimulated cultures. Differential harvesting of T2 cell subpopulations, based on proximity to the injury site, prior to Northern assessments of PAI‐1 mRNA abundance indicated that PAI‐1 transcripts were restricted to cells immediately bordering the wound or actively migrating and not expressed by cells in the distal contact‐inhibited monolayer regions. Such cell location–specific distribution of PAI‐1‐producing cells was confirmed by immunocytochemistry. PAI‐1 synthesis in cells that locomoted into the wound field continued until injury closure. Down‐regulation of PAI‐1 synthesis and matrix deposition in renal epithelial cells, stably transfected with a PAI‐1 antisense expression vector, significantly impaired wound closure. Transfection of the wound repair–deficient R/A epithelial line with a sense PAI‐1 expression construct restored both approximately normal levels of PAI‐1 synthesis and repair ability. These 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. J. Cell. Physiol. 182:269–280, 2000.

PLASMOLYSIS, HECHTIAN STRAND FORMATION, AND LOCALIZED MEMBRANE‐WALL ADHESIONS IN THE DESMID, CLOSTERIUM ACEROSUM (CHLOROPHYTA)1

Closterium acerosum Ehrenberg (Chlorophyta) produced a distinct network of thin cytoplasmic strands, or Hechtian strands, upon controlled plasmolysis in a sucrose solution. The strands persisted for 30 min or longer and could be visualized with both LM and EM. Near the plasma membrane of the polar zones of plasmolyzing protoplasts, the strands formed a “lattice”‐like arrangement with interstrand spacing of 120–130 nm. The strands terminated at the fibrous zone of the inner cell wall stratum. Although actin cables could be found attached to the plasma membrane upon rhodamine phalloidin labeling of membrane ghosts, neither microfilaments nor microtubules were found in Hechtian strands at any stage of development. The formation of strands was not disrupted by centrifugation at 8000 g or by repeated cycles of plasmolysis‐deplasmolysis. Application of microtubule‐ or microfilament‐affecting agents or various proteolytic/polysaccharide‐degrading enzymes did not disrupt the formation of strands. Cold treatment of cells resulted in the formation of Hechtian strands.