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. 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-GSK3beta 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-GSK3beta-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.

Figure 1

PAI-1 loss induces senescence-bypass in primary mouse fibroblasts. (a) Colony formation assay in primary MEFs overexpressing the indicated constructs. A knockdown vector for p53 was used as a positive control. (b) Relative PAI-1 expression analysed by QRT–PCR on extracts from the indicated post-senescent polyclonal cell lines. P3 are young passage 3 and P9 are senescent passage 9 MEFs. (c) Growth curves of PAI-1^–/–, p53^–/– and wild-type (WT) MEFs. For each genotype the mean ± s.d. of six independent cultures is shown. (d) Western blot analysis of a spontaneously immortal line (spi), six independent PAI-1^–/– passage 19 (P19), p53^kd and p16^INK4A–p19^ARF-deficient immortal NIH3T3 cell lines showing status of p53 and its targets p19^ARF and p21^CIP1 after cisplatin-induced DNA damage. CDK4 is a loading control. Uncropped full scans are shown in the Supplementary Information.

Figure 2

MEFs reduce PKB activation and exclude cyclin D1 from the nucleus during replicative senescence. (a) Western blot analysis of P3, P9, p53^kd, PAI-1^kd or uPA overexpressing MEFs for cell-cycle related proteins. PCNA and CDK4 are proliferation and loading controls, respectively. (b) Qualitative immunofluorescence microscopy analysis of serially passaged MEFs, (P1–P8), for cyclin D1 expression. The scale bar represents 50 μm. (c) Expression of phosphorylated PKB or GSK3β related to unphosphorylated fraction of the same proteins in P3, P6 and P9 MEFs and post-senescent p53^kd cells as analysed by western blot.

Figure 3

Sustained PI(3)K–PKB signalling or nuclear retention of cyclin D1 induces bypass of senescence. (a) Colony formation assay of primary MEFs infected with the indicated retroviral constructs. Immortalizing efficiency controls are p53^kd MEFs stained after 1 or 3 weeks. (b) Growth curves of various depicted immortalizing constructs versus wild-type cyclin D1 infected MEFs. Overexpression of p53^kd or control vector are positive and negative controls, respectively. The results shown are of two independent infections per construct (I, II). (c) Qualitative immunofluorescence microscopy analysis for cyclin D1 of post-senescent MEFs immortalised with the indicated constructs and control P3 and P9 MEFs. The scale bar represents 50 μm.

Figure 4

PAI-1 expression is sufficient for the induction of replicative senescence. (a) PAI-1, PTEN or GSK3β overexpression induces senescence in p53^kd MEFs as indicated by staining for senescence-associated β-galactosidase (SA-β-Gal). Control cells are mock-infected, serum depleted (starved) and wild-type senescent MEFs. The scale bar represents 400 μm. (b) Quantification of SA-β-Gal-positive p53^kd cells after retroviral overexpression of constructs as indicated in a. The mean ± s.d. of three independent plates is shown. (c) Colony formation assay in immortal PAI-1^kd MEFs after retroviral overexpression of the indicated constructs. p21^CIP1 and red fluorescent protein (RFP) are positive and negative controls, respectively. (d) Quantification of SA-β-Gal-positive PAI-1^kd MEFs after retroviral overexpression of PAI-1, PTEN, GSK3β, p21^CIP1 or an RFP control. Shown is the mean ± s.d. per plate per infection. (e) Colony formation assay in immortal p53^–/–, TA-D1 or TKO (retinoblastoma family triple knockout; pRb^–/––p107^–/––p130^–/–) fibroblasts infected with indicated retroviral overexpression constructs.

Figure 5

PAI-1 is necessary and sufficient for senescence in human BJ fibroblasts. (a) Growth curves of primary human BJ fibroblasts overexpressing the indicated constructs. The mean ± s.d. of three independent cultures per genotype is shown. (b) Relative PAI-1 and p21^CIP1 expression analysed by QRT–PCR on the indicated post-senescent BJ cell lines and control-infected young or senescent BJ fibroblasts. (c) Western blot analysis of cells from a for p53-target p21^CIP1 and cell-cycle related protein cyclin D1. CDK4 is a loading control. (d) Quantification of SA-β-Gal-positive post-senescent PAI-1^kd or p53^kd BJ fibroblasts after retroviral overexpression of PAI-1, PTEN, GSK3β, p21^CIP1 or an RFP control. The mean ± s.d. of three independent plates is shown. (e) Colony formation assay in post-senescent p53^kd or PAI-1^kd BJ fibroblasts infected with the indicated retroviral overexpression constructs. RFP is a negative control. (f) Schematic representation of a proposed model for senescence in fibroblasts. p53 induces PAI-1 and p21^CIP1 during ageing in culture. PAI-1 antagonizes uPA–GF (growth factor) signalling to cyclin D1 through PI(3)K–PKB–GSK3β and p21^CIP1 blocks cyclin D1 activity directly. The PAI-1–cyclin D1 connection is dominant over p21^CIP1 activity and controls induction of the senescence response downstream of p53 and upstream of pRb.