Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence

Abstract

p53 limits the proliferation of primary diploid fibroblasts by inducing a state of growth arrest named replicative senescence -a process which protects against oncogenic transformation and requires integrity of the p53 tumour suppressor pathway [1][2][3] . However, little is known about the downstream target genes of p53 in this growth-limiting response. Here, we report that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference (RNAi) leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ fibroblasts. PAI-1 knockdown results in sustained activation of the PI(3)K-PKB-GSK3β pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signalling. In agreement with this, we find that the PI(3)K-PKB-GSK3β-cyclin D1 pathway is also causally involved in cellular senescence. Conversely, ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induces a phenotype displaying all the hallmarks of replicative senescence. Our data indicate that PAI-1 is not merely a marker of senescence, but is both necessary and sufficient for the induction of replicative senescence downstream of p53.Primary murine fibroblasts activate the p19 ARF -p53 tumour suppressor pathway during prolonged culturing in vitro, which induces a postmitotic state referred to as replicative senescence. Senescence can be overcome by loss of either p19 ARF , p53 or the combined loss of all three retinoblastoma family proteins 1,2 . Proliferation of fibroblasts is induced by growth factors which activate cyclin-dependent kinases (CDKs), and in turn inactivate pRb's growth-limiting ability 2 , a G1 cell-cycle checkpoint that is often deregulated in cancer 3 . It is unclear which of the many downstream p53-target genes is responsible for the p53-dependent induction of replicative senescence. An attractive candidate is the CDK inhibitor p21 CIP1. However, mouse embryo fibroblasts (MEFs) knocked out for p21 CIP1 are not immortal 4 . Here, we identify an unexpected causal role for the urokinase type plasminogen activator (uPA)-PAI-1 system in the induction of replicative senescence. The serpin and extra-cellular matrix (ECM)-associated protein PAI-1 is a direct target of p53 (ref. 5, 6), is upregulated in ageing fibroblasts in vivo and in vitro, and is considered a marker of replicative senescence 7-9 . PAI-1 inhibits the activity of the secreted protease uPA by forming a stable complex. uPA expression can cause cells to progress through G1 into S phase 10 , most likely through activating a mitogenic signalling cascade by increasing the bioavailability of growth factors.To investigate the role of PAI-1 in replicative senescence, two independent retroviral vectors were generated that targeted murine PAI-1 for suppression through RNAi (Fig. 1a). As inhibition of PAI-1 expression leads to activation of uPA 12,13 , we asked whether overexpression of uPA also caused immortalisation. Retrovirusme...

The centrosomal protein TACC3 controls paclitaxel sensitivity by modulating a premature senescence program

“…Accordingly, the p53 target gene p21 WAF , a cyclin-dependent kinase inhibitor, was efficiently induced upon TACC3 depletion, whereas expression of the p53-independent cyclin-dependent kinase inhibitor, p27 KIP , remained unchanged (Supplementary Figure 3a). Furthermore, TACC3 depletion correlated with increased levels of cyclin D1 (Supplementary Figure 3a), which was predominantly confined to the cytoplasm (Schneider et al, 2008 and data not shown) and which is consistent with its function in G 1 arrest and senescence (Kortlever et al, 2006). In contrast, protein levels of the G 2 /Massociated cyclins A and B1 were strongly reduced upon prolonged TACC3 depletion (Supplementary Figure 3a), thus excluding a major contribution of the G 2 checkpoint in the post-mitotic arrest caused by TACC3 depletion.…”

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).…”

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

“…This suggests that PAI-1 kd or uPA overexpression induces a senescence-bypass that is not strictly cell autonomous. p53 kd MEFs have higher amounts of PAI-1 compared to PAI-1 kd cells 70 and therefore potentially lower uPA activity, and it is possible that this is why p53 kd cells were not able to immortalize fibroblasts in a paracrine fashion (Fig. 1A).…”

“…It should be noted however that ectopic expression of PAI-1 in immortal fibroblasts does lead to induction of senescence, consistent with the notion that PAI-1 acts downstream of p53 to block uPA activity. 70 Furthermore, we have found there is a concentration-dependent induction of senescence in immortal cells after administration of recombinant PAI-1, showing that soluble PAI-1 in culture medium is able to induce senescence (Kortlever R and Bernards R, unpublished observations). Thus, the immortalizing effects seen of PAI-1 kd are mediated, at least in part, via paracrine signaling.…”

“…70 These data are most readily explained by a model in which suppression of PAI-1 causes activation of uPA, leading to increased bio-availability of growth factors, which in turn stimulate proliferation. Our results support the notion that the observed regulation of PAI-1 and uPA in wound healing and in serum-stimulated fibroblasts are relevant to the proliferative events that take place during these processes.…”