PAI-1 GENE-EXPRESSION IS GROWTH STATE-REGULATED IN CULTURED HUMAN EPIDERMAL-KERATINOCYTES DURING PROGRESSION TO CONFLUENCE AND POSTWOUNDING

Growth of human keratinocytes (NHKs) in submerged cultures approximate s a ''wound'' response generally considered equivalent to regenerative maturation. Within this context, PAI-1 expression in cultured NHKs ap pears to be growth state-regulated and is associated with specific NHK subpopulations undergoing migration and proliferation in response to wounding; NHRs transit through specific phases during growth to conflu ence. Basal layer keratinocytes comprise several classes of nucleated epidermal cells (designated ''A,'' ''B,'' and ''C'') which are disting uishable on the basis of RNA content, population generation time, and expression of basal cell marker proteins. ''A'' substrate cells initia lly give rise to expanding proliferating keratinocyte colonies in vitr o, 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 spin ous cells during the postconfluent growth period. Expression of plasmi nogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor and member of the serpin gene family which is synthesized by ''activat ed'' or migrating keratinocytes in vivo was restricted to the preconfl uent stages of cell growth in vitro, a condition equivalent to wound r egeneration 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 analys is revealed that PAI-1 is detected early in expanding NHK colonies, du ring the initial recruitment of basal cells from the ''A'' state into the ''B'' compartment in the absence of significant proliferation, sug gesting that PAI-1 may be active in regulating early NHK migratory eve nts, independent of cell proliferation. Thereafter, these PAI-1-expres sing ''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 expone ntial growth, virtually no small ''A'' cells contain immunoreactive PA I-1. PAI-1 expression peaks during preconfluent growth and remains con fined to larger basal cell phenotypes. Cellular accumulation of both P AI-1 mRNA and protein appeared to be cell cycle phase-specific and cha racteristic of progression through an activated G(1) growth phase. Ind uced expression, moreover, was restricted to a ''window'' in G(1) 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 characteristi c of the activated epidermal phenotype in vitro as well as in vivo. (C ) 1996 Academic Press, Inc.