Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

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

doi:10.1074/jbc.m710268200

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.

Highlights

Lung epithelial cells secrete the proenzyme single chain uPA,2 which is activated by plasmin and other proteases
Panel i, Beas2B cells express minimal plasminogen activator inhibitor-1 (PAI-1), and uPA (50 ng/ml) increased PAI-1 expression. This was consistent with a similar increase in expression of both 3.2- and 2.4-kb alternate spliced variants of PAI-1 mRNA in Beas2B cells stimulated with uPA (Fig. 1B, panel ii)
We found that treatment of Beas2B cells with p53 siRNA inhibited both basal and uPA-mediated p53 expression by more than 95% compared with control nonspecific siRNA (Nsp siRNA)treated cells (Fig. 2B, panel i)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Human bronchial epithelial (Beas2B) and p53deficient human lung non-small cell carcinoma (H1299) cells obtained from ATCC were maintained in LHC-9 medium containing 1% antibiotics and in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 1% glutamine, and 1% antibiotics (Invitrogen), respectively, as described previously [8, 21]. The cells were treated with PBS or uPA, and the effect of p53 expression on basal and uPA-mediated p53, PAI-1 protein and mRNA levels was analyzed by Western or Northern blotting, respectively, as described above. Determination of p53-binding Site on PAI-1 mRNA 3Ј-UTR— PAI-1 3Ј-UTR cDNA fragments of differing size were synthesized by PCR amplification of a full-length PAI-1 cDNA template using different sense and antisense oligonucleotide primers that were designed based on 5Ј and 3Ј regions of the open reading frame These deletion fragments were cloned into pcDNA 3.1 and transcribed in vitro in the presence of [32P]UTP. H1299 cells expressing vector cDNA or p53 cDNA were transfected with chimeric ␤-globin/ PAI-1 cDNA containing the 70-nt p53 protein-binding sequence of the PAI-1 mRNA 3Ј-UTR or control non-p53 binding CDR sequences as described above for 48 h to competitively inhibit p53 binding to endogenous PAI-1 mRNA. Statistical Analysis—We tested the differences between Beas2B cells treated with p53 siRNA or PAI-1 siRNA and corresponding nonspecific control siRNA transfected cells exposed to TSE or bleomycin, respectively, by Student’s t test

RESULTS

These cells were exposed to TSE or bleomycin and analyzed for

DISCUSSION