Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jul 11;283(28):19570-80.
doi: 10.1074/jbc.M710268200. Epub 2008 May 9.

Regulation of plasminogen activator inhibitor-1 expression by tumor suppressor protein p53

Sreerama Shetty  1 , Praveenkumar Shetty, Steven Idell, Thirunavukkarasu Velusamy, Yashodhar P Bhandary, Rashmi S Shetty
Affiliations

Regulation of plasminogen activator inhibitor-1 expression by tumor suppressor protein p53

Sreerama Shetty et al. J Biol Chem. .

Abstract

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
A, expression of p53 protein and mRNA by lung-derived cells. Panel i, lysates of Beas2B and p53-deficient non-small cell carcinoma (H1299) cells treated with PBS or uPA (50 ng/ml) for 24 h were subjected to Western blotting using anti-p53 antibody. The same membrane was stripped and developed with an anti-β-actin antibody for equal loading. Panel ii, RNA isolated from Beas2B and H1299 cells treated with PBS or uPA (50 ng/ml) for 6 h was subjected to Northern blotting using 32P-labeled p53 cDNA and 32P-labeled β-actin cDNA for equal loading. B, involvement of p53 in expression of PAI-1 protein and mRNA by lung epithelial cells. Panel i, CM and total CL of Beas2B and H1299 cells treated with PBS or uPA (50 ng/ml) for 24 h were subjected to Western blotting using an anti-PAI-1 antibody. Panel ii, RNA isolated from Beas2B and H1299 cells treated with PBS or uPA (50 ng/ml) for 6 h were subjected to Northern blotting using 32P-labeled PAI-1 and β-actin cDNAs. The data shown are representative of three repetitions.
FIGURE 2.
FIGURE 2.
p53 induces PAI-1 protein and mRNA expression. A, panel i, expression of WT p53 in H1299 cells. H1299 cells were transfected with vector cDNA (pcDNA 3.1) or p53 cDNA (p53) in pcDNA 3.1. Stable cell lines expressing vector cDNA or p53 cDNA were treated with PBS or 50 ng/ml of the amino-terminal fragment (ATF) of uPA for 24 h. The cell lysates were analyzed for p53 expression by Western blotting as described in Fig. 1A. Panel ii, induction of PAI-1 expression by WT p53. H1299 cells transfected with vector pcDNA3.1 or WT p53 cDNA were treated with PBS or uPA (50 ng/ml). The CM and CL were subjected to Western blotting using an anti-PAI-1 antibody. Panel iii, induction of PAI-1 mRNA expression by WT p53. RNA isolated from H1299 cells transfected with vector cDNA or p53 cDNA treated with PBS or uPA (50 ng/ml) for 6 h was subjected to Northern blotting using 32P-labeled PAI-1 and β-actin cDNAs. B, panel i, inhibition of p53 expression by siRNA in Beas2B cells. Beas2B cells transfected with control Nsp siRNA or p53 siRNA were treated with PBS or uPA (50 ng/ml) for 24 h. Cell lysates were subjected to Western blotting using anti-p53 and β-actin antibodies. Panel ii, Beas2B cells treated with control siRNA or p53 siRNA were treated with PBS or uPA (50 ng/ml) for 24 h. The CM and CL were subjected to Western blotting using anti-PAI-1 antibody. Panel iii, Beas2B cells transfected with control siRNA or p53 siRNA were treated with PBS or uPA (50 ng/ml) for 6 h. The total RNA isolated from these cells was subjected to Northern blotting using 32P-labeled PAI-1 and β-actin cDNAs. Data are representative of four independent replications.
FIGURE 3.
FIGURE 3.
Effect of p53 on PAI-1 mRNA stability. A, expression of WT p53 in non-small cell lung carcinoma cells lacking p53 (p53-/-) stabilizes PAI-1 mRNA. Naive p53-/- cells (p53-/-) or stable p53-/- cells over expressing vector cDNA (pcDNA 3.1) or p53 cDNA were subjected to transcription chase experiment by treating with DRB (20 μg/ml). The level of PAI-1 mRNA was measured by Northern blotting at 0–24 h. Experiments were repeated five times with similar results. B, inhibition of p53 expression destabilizes PAI-1 mRNA. Beas2B cells transfected with control nonspecific (Nsp) siRNA or p53 siRNA were treated with PBS or uPA (50 ng/ml) for 6 h to induce maximum PAI-1 mRNA. Ongoing transcription was inhibited by treatment with DRB, and the decay of PAI-1 mRNA was analyzed for 0–12 h by Northern blotting. β-Actin mRNA expression is shown for equal loading. Representative figure of four replications is shown. C, effect of p53 on PAI-1 protein translation. H1299 cells (p53-/-) or H1299 cells transfected with vector cDNA (pcDNA3.1) or p53 cDNA were subjected to metabolic labeling using [35S]methionine and [35S]cysteine in the presence of proteosome inhibitor (MG-132) for varying time periods (0–24 h). 35S-Labeled PAI-1 proteins were immunoprecipitated from the CM using anti-PAI-1 antibody, separated on SDS-PAGE, and autoradiographed. The results represent two independent experiments.
FIGURE 4.
FIGURE 4.
p53 protein binds to PAI-1 3′-UTR mRNA. A, recombinant p53 protein (rp53) expressed in prokaryotic system was incubated with the 32P-labeled PAI-1 mRNA coding (CDR) or 3′-untranslated region (3′UTR), and the mRNA·rp53 complexes were analyzed by gel mobility shift assay on 5% nondenaturing PAGE. PAI-1 mRNA was incubated with rp53 protein (p53) or buffer alone (Fp). Arrow indicates RNA·protein complex. B, Beas2B cells treated with varying amounts (0–1 μg/ml) of uPA for 24 h were lysed, and the lysates were immunoprecipitated using anti-p53 monoclonal antibody (p53 mAb) or mouse IgG (Nsp mIgG). The p53 protein-associated PAI-1 mRNA was detected by RT-PCR using 32P-labeled dCTP and verified by nucleotide sequencing of the corresponding nonradioactive PCR product. Experiments were repeated at least three times. C, rp53 protein (0–5 μg/lane) was subjected to PAI-1 mRNA 3′-UTR binding as described in A, and following heparin digestion the rp53·PAI-1 mRNA complex was exposed to UV light (2500 μJ) for 10 min on ice, separated by 8% SDS-PAGE and autoradiographed. D, rp53·PAI-1 mRNA complex was treated with or without varying amounts (0–6 μg/lane) of anti-p53 antibody (p53 mAb) or 6 μg/lane of nonspecific mIgG (NSp mIgG). The reaction mixtures were subjected to gel mobility shift assay as described in A. E, 32P-labeled PAI-1 mRNA 3′-UTR-rp53 complexes were immunoprecipitated with either anti-p53 antibody or nonspecific mIgG. Immune complexes were then separated on SDS-PAGE and developed by autoradiography. F, identification of PAI-1 3′-UTR mRNA binding region on p53 molecule. Full-length rp53 protein (Wt.p53) obtained after thrombin digestion or GST-rp53 fusion deletion fragments corresponding to amino acid residues 1–98 (1st Qtr), 99–196 (2nd Qtr), 197–295 (3rd Qtr), and 296–393 (4th Qtr) were separated on SDS-PAGE and transferred to nitrocellulose membrane. The membrane was later subjected to Northwestern analyses using 32P-labeled PAI-1 mRNA 3′-UTR as a probe. The rp53·PAI-1 mRNA complexes were detected by autoradiography to determine the specificity of p53 protein-PAI-1 mRNA 3′-UTR interaction. Competitive inhibition of p53-PAI-1 mRNA binding using unlabeled PAI-1 mRNA sense (G) and antisense (H) transcripts is shown. rp53 proteins were incubated with 32P-labeled PAI-1 3′-UTR mRNA probe in the presence of 0–100-fold molar excess of unlabeled sense or antisense transcripts and analyzed by gel mobility shift assay. I, effects of polyribonucleotides, proteinase K (Prot.K), and SDS on p53 and PAI-1 mRNA binding. rp53 protein was incubated with a 500-fold excess of unlabeled poly(A), poly(C), poly(G), and poly(U) or a 100-fold molar excess of sense (3′-UTR S-C) or antisense (3′-UTR A-C) or p53 consensus sequence (5′prom), or proteinase K (2.5 mg/ml) and 0.1% SDS for 30 min at 30 °C. 32P-Labeled PAI-1 mRNA probe was added, and the mixture was digested with RNase T1 and analyzed by gel mobility shift assay. 32P-Labeled PAI-1 3′-UTR mRNA predigested with RNase T1 was also incubated with rp53 (RNaseT1). Fp, probe alone. Experiments are representative of four independent analyses.
FIGURE 5.
FIGURE 5.
Identification of the p53 protein-binding sequences on PAI-1 3′-UTR mRNA. A, deletion map indicating the p53 protein-binding site on PAI-1 mRNA. B, rp53 protein was incubated with 32P-labeled PAI-1 mRNA full-length coding region (1) or full-length 3′-untranslated region (2) or 3′-UTR deletion transcripts (3′-UTR-DL-3–11). The RNA·protein complex was analyzed by gel mobility shift assay. Arrow indicates the PAI-1 mRNA·p53 protein complex. Fp, 32P-labeled PAI-1 mRNA probe is incubated with buffer alone. Data are representative of four independent analyses.
FIGURE 6.
FIGURE 6.
Determining the destabilizing function of p53-binding PAI-1 3′-UTR mRNA sequence. A, structure of β-globin/PAI-1 chimeric mRNA. The p53 protein-binding 70-nt 3′-UTR sequence corresponding to nt 1958–2027 (C3) and a nonbinding control sequence of similar size corresponding to the coding region from nt 468 to 538 (C4) of PAI-1 cDNA were inserted into the 3′-UTR of β-globin cDNA. The chimeric β-globin/PAI-1 cDNAs were subcloned to pcDNA 3.1. B, nucleotide sequence of the p53 binding region nt 1958–2027 (C3) or nonbinding control sequence 468–538 (C4). C, decay of β-globin/PAI-1 chimeric mRNA. Beas2B cells were transfected with the chimeric β-globin/PAI-1 3′-UTR gene containing the 70-nt (nt 1958–2027) p53-binding sequence (β-globin-PAI-1(C3)) or nonbinding control sequence of PAI-1 CDR (nt 468–538) (β-globin-PAI-1(C4)) in pcDNA 3.1. Total RNA was isolated at different time intervals after treatment with DRB as described above (Fig. 3B) and analyzed for the level of chimeric mRNA by Northern blotting. Densitometric scanning of individual bands from four experiments is shown as a line graph. D, effect of p53 binding PAI-1 mRNA 3′-UTR sequence on PAI-1 expression. H1299 cells expressing vector cDNA (pcDNA 3.1) or p53 cDNA were untreated (None) or transfected with chimericβ-globin/PAI-1 cDNA containing a non-p53-binding control (C4, Nsp) or p53-binding (C3, p53) PAI-1 3′-UTR sequence in pcDNA 3.1, as described in Fig. 6C. PAI-1 expression in the CM was determined by Western blotting using an anti-PAI-1 antibody. The data shown are representative of the findings of four independent analyses.
FIGURE 7.
FIGURE 7.
Regulation of TSE and bleomycin-induced lung epithelial cell apoptosis by p53-mediated and PAI-1 expression. A, induction of PAI-1 and p53 expression by TSE. The CM and cell CL of Beas2B cells treated with TSE (1.5%) for 0–24 h were subjected to Western blotting using anti-PAI-1 antibody. The same CL were analyzed for expression of p53 and β-actin for equal loading using anti-p53 and anti-β-actin antibodies. B, inhibition of p53 expression inhibits TSE-induced PAI-1 expression. The CM and CL of Beas2B cells expressing control nonspecific siRNA (Nsp siRNA) or p53 siRNA treated with PBS or TSE (1.5%) were analyzed for expression of PAI-1, p53, and β-actin by Western blotting as described in Fig. 7A. C, inhibition of p53 and PAI-1 expression protects lung epithelial cells from TSE-induced apoptosis. Panel i, untransfected Beas2B cells (top row) or Beas2B cells treated with nonspecific siRNA (Nsp SI), p53 siRNA, or PAI-1 siRNA (subsequent rows) were treated with PBS or 1 and 1.5% TSE for 24 h and were then subjected to TUNEL staining to assess apoptosis. The microscopic images were photographed at ×20 magnification. Panel ii, Beas2B cells treated with nonspecific siRNA (Nsp SI) or p53 or PAI-1 siRNA were treated with PBS or 1.5% TSE for 24 h. The cells were then detached and treated with anti-annexin-V antibody and propidium iodide. The apoptotic cells were analyzed by flow cytometry. The data are representative of three independent experiments. D, inhibition of p53 and PAI-1 expression protects lung epithelial cells from bleomycin (Bleo)-induced apoptosis. Panel i, untransfected Beas2B cells (top row) or Beas2B cells treated with nonspecific siRNA (NSp SI) or p53 or PAI-1 siRNA (subsequent rows) were treated with PBS or bleomycin (40 μg/ml) for 24 h, after which they were subjected to TUNEL staining to assess the apoptotic response, as described in Fig. 7C. Microscopic images were photographed at ×20 magnification. Panel ii, Beas2B cells treated with nonspecific siRNA (Nsp SI) or p53 or PAI-1 siRNA were treated with PBS or bleomycin (40 μg/ml) for 24 h, after which they were subjected to flow cytometry as described in C, panel ii, to assess the apoptotic response.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bensaad, K., and Vousden, K. H. (2005) Nat. Med. 11 1278-1289 - PubMed
    1. Chapman, H. A. (2004) J. Clin. Invest. 113 148-157 - PMC - PubMed
    1. Shetty, S., and Idell, S. (2006) in Encyclopedia of Respiratory Medicine (Laurent, J., ed) pp. 205-210, Elsevier Publications, London
    1. Dvorak, H. F. (1986) N. Engl. J. Med. 315 1650-1659 - PubMed
    1. Dano, K., Behrendt, N., Brunner, N., Ellis, V., Ploug, M., and Pyke, C. (1994) Fibrinolysis 8 189-203

Publication types

MeSH terms

-