Differential growth state‐dependent regulation of plasminogen activator inhibitor type‐1 expression in senescent IMR‐90 human diploid fibroblasts

Abstract

The type-1 inhibitor of plasminogen activator (PAI-1) regulates pericellular proteolytic activity functioning, thereby to control matrix integrity, cell growth, and morphology. Subconfluent late-passage IMR-90 human fibroblasts and normal rat kidney (NRK) cells, both at the stage of replicative senescence accumulated 15- to 30-fold more undersurface PAI-1 protein compared to early-passage, actively-proliferating, cultures. Senescence-associated elevations in PAI-1 expression by IMR-90 cells reflected corresponding 11-fold increases in the 3.0- and 2.2-kb PAI-1 mRNA species. The 2.2-kb transcript exhibited a greater age-dependent increase (7.2-fold) compared to the 3.0-kb mRNA (3.7-fold). Since PAI-1 expression is coupled to growth activation in serum-deprived cultures (Ryan and Higgins, 1993, J. Cell. Physiol., 155:376-384), it was important to determine if PAI-1 gene regulation was altered as a function of cellular aging. In contrast to early-passage cultures, senescent IMR-90 fibroblasts did not down-regulate either PAI-1 protein expression or steady-state levels of PAI-1 mRNA transcripts upon serum-deprivation. Late-passage human fibroblasts at their proliferative end-stage, thus, appear to regulate PAI-1 mRNA levels through different mechanisms than do young, actively-proliferating, cells. PAI-1 overexpression during in vitro cellular aging, therefore, may contribute to the acquisition of specific senescence-associated phenotypic traits (e.g., enlarged cell morphology; increased adhesivity) by altering the pericellular proteolytic balance influencing, in turn, the formation or stability of cell-to-substrate attachment complexes.

E6AP is required for replicative and oncogene-induced senescence in mouse embryo fibroblasts

“…Two other lines of evidence support the impaired ability of E6AP-deficient MEFs to undergo senescence. First, we measured the expression of two known markers of senescence: p21 and the PAI-1 (Mu and Higgins, 1995;Serrano et al, 1997;Kortlever et al, 2006). The protein and mRNA levels of p21 were higher in WT than in E6AP KO MEFs as measured by western blot analysis and by quantitative real-time PCR (qPCR), respectively ( Figure 2d).…”

“…The mechanism by which p53 promotes senescence is only partially understood. P53 target genes have been implicated in the induction of senescence, these include the CDK inhibitor p21, the plasminogen activator inhibitor-1 (PAI-1) (Mu and Higgins, 1995;Serrano et al, 1997) and MIC-1, a cytokine inducing senescence (Sherman et al, 2007).…”

Adult Sox2+ stem cell exhaustion in mice results in cellular senescence and premature aging

“…To confirm this result, we used an alternative substrate that produces a luminescent reaction, Galacton (Bassaneze, Miyakawa, & Krieger, 2008), instead of a color reaction obtaining very similar results (Figure 1g). In addition, we measured the expression levels of several senescence marker genes: cell cycle inhibitor Ink4a (Krishnamurthy et al, 2004), matrix metalloprotease Mmp3 (Komosinska‐Vassev et al, 2011), and the serine protease inhibitor Serpine1 (Goldstein, Moerman, Fujii, & Sobel, 1994; Mu & Higgins, 1995). The mRNA levels of these genes were elevated in Sox2‐TK mice after GCV treatment, although in the case of Ink4a the increase did not reach statistical significance (Figure 1h), corroborating the induction of senescence in Sox2‐TK tissues.…”

“…In agreement with the X‐Gal data, Galacton revealed a massive induction of senescence in the kidneys of GCV‐treated mice (Figure 2f). We also measured the mRNA expression of different genes linked to the senescent cell response, such as Ink4a (Krishnamurthy et al, 2004), Ink4b (Malumbres et al, 2000), Il6 (Acosta et al, 2008; Kuilman et al, 2008), Mmp1 (Benanti, Williams, Robinson, Ozer, & Galloway, 2002), Serpine1 (Goldstein et al, 1994; Mu & Higgins, 1995), and Timp1 (Komosinska‐Vassev et al, 2011). In all cases, we observed a statistically significant increase in the levels of mRNA for these genes, in agreement with the notion of a strong induction of senescence, a response typically associated with advanced aging (Figure 2g).…”

Senescence, Wound Healing, and Cancer: the PAI-1 Connection

“…[45][46][47][48] (2) PAI-1 is a senescence marker in fibroblasts, both in vivo and in vitro. 46,49 (3) p53 is critically involved in the senescence response of fibroblasts, 3 and progressively upregulates PAI-1 expression during prolonged culturing in serum.…”