Increased transcription and modified growth state-dependent expression of the plasminogen activator inhibitor type-1 gene characterize the senescent phenotype in human diploid fibroblasts.

Abstract

The type-1 inhibitor of plasminogen activator (PAI-1) is a major physiologic regulator of pericellular proteolytic activity and, as such, influences matrix integrity, cell-to-substrate adhesion, and cellular proliferation. Excessive accumulation of both PAI-1 mRNA and protein correlates with the progressive acquisition of morphological and growth traits characteristic of the senescent phenotype (Mu and Higgins, 1995, J. Cell. Physiol., 165:647-657). Compared to early-passage IMR-90 human diploid fibroblasts, a late-passage senescence-associated 11-fold elevation in steady-state PAI-1 mRNA content reflected a 15-fold increase in constitutive PAI-1 gene transcription. Differential mRNA stability was not a factor in age-associated PAI-1 overexpression in IMR-90 cells. Upon removal of serum, early-passage human fibroblasts enter into a state of growth arrest with marked down-regulation of PAI-1 synthesis. Rapid induction of both the 3.0- and 2.2-kb PAI-1 mRNA species was evident upon serum-induced "activation" of quiescent early-passage fibroblasts; induced PAI-1 transcripts were maximal at 2 hr post-serum stimulation and declined in late G1 prior to entry into S phase. In contrast, late-passage (p32) fibroblasts maintained a significant level of PAI-1 expression under serum-free culture conditions. Although the PAI-1 gene was further responsive to serum in senescent cells, transcript abundance remained elevated and actually increased over the 12 to 16 hr post-serum addition period (a time when early-passage fibroblasts down-regulate PAI-1 mRNA content). Development of the senescent phenotype in human fibroblasts is associated, therefore, with significant changes in PAI-1 gene regulation. Such reprogramming involves predominantly 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.