INCREASED TRANSCRIPTION AND MODIFIED GROWTH STATE-DEPENDENT EXPRESSION OF THE PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 GENE CHARACTERIZE THE SENESCENT PHENOTYPE IN HUMAN-DIPLOID FIBROBLASTS

The type-1 inhibitor of plasminogen activator (PAI-1) is a major physi ologic regulator of pericellular proteolytic activity and, as such, in fluences matrix integrity, cell-to-substrate adhesion, and cellular pr oliferation. Excessive accumulation of both PAI-1 mRNA and protein cor relates with the progressive acquisition of morphological and growth t raits characteristic of the senescent phenotype (Mu and Higgins, 1995, J. Cell. Physiol., 165:647-657). Compared to early-passage IMR-90 hum an diploid fibroblasts, a late-passage senescence-associated 11-fold e levation in steady-state PAI-1 mRNA content reflected a 15-fold increa se in constitutive PAI-1 gene transcription. Differential mRNA stabili ty was not a factor in age-associated PAI-1 overexpression in IMR-90 c ells. Upon removal of serum, early-passage human fibroblasts enter int er a state of growth arrest with marked down-regulation of PAI-1 synth esis. Rapid induction of both the 3.0- and 2.2-kb PAI-T mRNA species w as evident upon serum-induced ''activation'' of quiescent early-passag e fibroblasts; induced PAI-1 transcripts were maximal at 2 hr post-ser um stimulation and declined in late G(1) prior to entry into S phase. In contrast, late-passage (p32) fibroblasts maintained a significant l evel of PAI-1 expression under serum-free culture conditions. Although the PAI-1 gene was further responsive to serum in senescent cells, tr anscript abundance remained elevated and actually increased over the 1 2 to 16 hr post-serum addition period (a time when early-passage fibro blasts down-regulate PAI-1 mRNA content). Development of the senescent phenotype in human fibroblasts is associated, therefore, with signifi cant changes in PAI-1 gene regulation. Such reprogramming involves pre dominantly transcriptional events and results in a marked increase in steady-state PAI-1 transcript abundance involving both the 3.0- and 2. 2-kb mRNA species. (C) 1998 Wiley-Liss, Inc.