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  • Title: PAI-1 gene expression is growth state-regulated in cultured human epidermal keratinocytes during progression to confluence and postwounding.
    Author: Staiano-Coico L, Carano K, Allan VM, Steiner MG, Pagan-Charry I, Bailey BB, Babaar P, Rigas B, Higgins PJ.
    Journal: Exp Cell Res; 1996 Aug 25; 227(1):123-34. PubMed ID: 8806459.
    Abstract:
    Growth of human keratinocytes (NHKs) in submerged cultures approximates a "wound" response generally considered equivalent to regenerative maturation. Within this context, PAI-1 expression in cultured NHKs appears to be growth state-regulated and is associated with specific NHK subpopulations undergoing migration and proliferation in response to wounding; NHKs transit through specific phases during growth to confluence. Basal layer keratinocytes comprise several classes of nucleated epidermal cells (designated "A," "B," and "C") which are distinguishable on the basis of RNA content, population generation time, and expression of basal cell marker proteins. "A" substrate cells initially give rise to expanding proliferating keratinocyte colonies in vitro, but are rapidly replaced (at the stage of 50-75% culture confluence) by transient amplifying "B" cells and, eventually, the larger "C" subpopulation which subsequently differentiates into suprabasal spinous cells during the postconfluent growth period. Expression of plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor and member of the serpin gene family which is synthesized by "activated" or migrating keratinocytes in vivo, was restricted to the preconfluent stages of cell growth in vitro, a condition equivalent to wound regeneration in situ. PAI-1 mRNA and protein expression was maximal in 50-75% confluent cultures correlating, thereby, with the emergence of the transient amplifying "B" cell population. Flow cytometric analysis revealed that PAI-1 is detected early in expanding NHK colonies, during the initial recruitment of basal cells from the "A" state into the "B" compartment in the absence of significant proliferation, suggesting that PAI-1 may be active in regulating early NHK migratory events, independent of cell proliferation. Thereafter, these PAI-1-expressing "A" cells are recruited into the "B" compartment, where they continue to migrate, proliferate, and enlarge, eventually giving rise to PAI-1-expressing "C" cells. By the time NHK cultures reach exponential growth, virtually no small "A" cells contain immunoreactive PAI-1. PAI-1 expression peaks during preconfluent growth and remains confined to larger basal cell phenotypes. Cellular accumulation of both PAI-1 mRNA and protein appeared to be cell cycle phase-specific and characteristic of progression through an activated G1 growth phase. Induced expression, moreover, was restricted to a "window" in G1 with PAI-1 mRNA evident within 2 h after serum addition to growth-arrested cells and attaining maximal levels (50-fold) at 10 h poststimulation. Induced PAI-1 expression, thus, appears to be a general characteristic of the activated epidermal phenotype in vitro as well as in vivo.
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