MOLECULAR AND CELLULAR BIOLOGY, Aug. 2007, p. 5607–5618 0270-7306/07/$08.00⫹0 doi:10.1128/MCB.00080-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 16
Regulation of Urokinase Receptor Expression by p53: Novel Role in Stabilization of uPAR mRNA䌤 Sreerama Shetty,1* Thirunavukkarasu Velusamy,1 Steven Idell,1 Praveenkumar Shetty,1 Andrew P. Mazar,2 Yashodhar P. Bhandary,1 and Rashmi S. Shetty1 Texas Lung Injury Institute, Department of Specialty Care Services, The University of Texas Health Center at Tyler, 11937 U.S. Highway 271, Tyler, Texas 75708,1 and Attenuon LLC, 11535 Sorrento Valley Rd., San Diego, California 921212 Received 15 January 2007/Returned for modification 13 February 2007/Accepted 10 April 2007
We found that p53-deficient (p53ⴚ/ⴚ) lung carcinoma (H1299) cells express robust levels of cell surface uPAR and uPAR mRNA. Expression of p53 protein in p53ⴚ/ⴚ cells suppressed basal and urokinase (uPA)induced cell surface uPAR protein and increased uPAR mRNA degradation. Inhibition of p53 by RNA silencing in Beas2B human airway epithelial cells conversely increased basal as well as uPA-mediated uPAR expression and stabilized uPAR mRNA. Purified p53 protein specifically binds to the uPAR mRNA 3ⴕ untranslated region (3ⴕUTR), and endogenous uPAR mRNA associates with p53. The p53 binding region involves a 37-nucleotide uPAR 3ⴕUTR sequence, and insertion of the p53 binding sequence into -globin mRNA destabilized -globin mRNA. Inhibition of p53 expression in these cells reverses decay of chimeric -globin–uPAR mRNA. These observations demonstrate a novel regulatory role for p53 as a uPAR mRNA binding protein that downregulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway epithelial or lung carcinoma cells. 21), and uPA induces uPAR and proliferative responses in lung epithelial cells (23). These observations prompted us to test the possibility that uPA-p53 cross talk could regulate uPAR expression and downstream uPAR-mediated responses in lung epithelial cells. We now describe a newly recognized uPA-p53 cross talk that involves p53 as a sequence-specific uPAR mRNA binding protein that regulates uPAR mRNA stability and uPA-mediated control of the viability of human airway epithelial and lung carcinoma cells.
Urokinase (uPA)-mediated plasmin generation contributes to extravascular proteolysis and tissue remodeling (22). During the last decade, evidence for the involvement of uPA in remodeling of the extracellular matrix in acute and chronic lung injury, repair (5, 22), and neoplasia (14, 33) has become increasingly clear. Lung epithelial cells synthesize and secrete a 55-kDa proenzyme single-chain form of uPA, which is activated by plasmin and other proteases (20). These cells also synthesize and express a cell surface receptor for uPA, uPAR, which is a glycosylphosphatidylinositol-linked receptor (23). Lung epithelial cell viability and uPA-mediated tissue remodeling may contribute to the pathogenesis of diverse lung diseases, such as acute respiratory distress syndrome or a variety of interstitial lung diseases. These diseases share a propensity for apoptosis of lung epithelial cells and the development of progressive fibrosis that predisposes the individual to respiratory failure (5). Recent evidence suggests a close relationship between uPA-uPAR-mediated matrix remodeling, cell sensitivity to proliferation, and programmed cell death (1, 13, 24). However, there is a paucity of evidence that directly links the two physiological processes of cell proliferation and programmed cell death. We recently found that uPA regulates lung epithelial cell apoptosis/growth through elaboration of p53 (24), demonstrating the first direct link between epithelial cell survival/cell cycle regulation and alveolar fibrinolysis. Suppression of p53 function due to mutation or deletion (10, 11, 31) and overexpression of uPAR occurs in tumor cells (2, 12,
MATERIALS AND METHODS Cell culture. Human bronchial epithelial cells (Beas2B) and p53-deficient human lung non-small-cell carcinoma cells (H1299) were obtained from the ATCC and maintained as previously described (24, 26). Total cellular membrane extraction and Western blotting. Beas2B cells or H1299 cells grown to confluence were treated with phosphate-buffered saline (PBS) or uPA in serum-free medium. uPAR expression was analyzed by Western blotting of isolated membrane proteins as previously described (23). Plasmid construction. Plasmid uPAR and p53 cDNAs were subcloned in the HindIII and XbaI sites of pcDNA3.1 (Invitrogen, CA), and the sequences of the clones were confirmed by sequencing. Random priming of uPAR or p53 cDNA. The full-length template of uPAR or p53 cDNA was released with HindIII or XbaI, purified on a 1% agarose gel, and labeled with [32P]dCTP using a rediPrime labeling kit (Amersham Biosciences, NJ). Northern blotting of uPAR or p53 mRNA. A Northern blotting assay was used to assess the level of uPAR or p53 mRNA. Beas2B or H1299 cells were treated with PBS or uPA for 12 h in RPMI medium. Total RNA was analyzed for expression of uPAR and p53 mRNAs by Northern blotting as described earlier (23, 24). uPAR mRNA stability was assessed by transcription chase experiments in which cells stimulated with PBS or uPA for 12 h were then treated with 5,6-dichloro-1--D-ribofluranosyl benzimidazole (DRB) to inhibit ongoing transcription, after which total RNA was isolated at specific time points. uPAR mRNA was measured by Northern blotting as described above. Nuclear run-on transcription activation assay. Confluent cells grown in two T182 flasks were serum starved overnight in RPMI medium. The cells were later analyzed for uPAR mRNA synthesis by transcription activation assay as described earlier (25).
* Corresponding author. Mailing address: The Texas Lung Injury Institute, Department of Specialty Care Services, The University of Texas Health Center at Tyler, 11937 U.S. Highway 271, Lab C-6, Tyler, TX 75708. Phone: (903) 877-7668. Fax: (903) 877-5627. E-mail: sreerama
[email protected]. 䌤 Published ahead of print on 4 June 2007. 5607
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Transfection of p53-deficient H1299 cells with p53 cDNA. p53 cDNA was cloned into the eukaryotic expression vector pcDNA3.1. H1299 cells were transfected with vector cDNA or vector DNA containing p53 cDNA by using Lipofectamine, and stable cell lines were created as described earlier (23). The cells were treated with PBS or uPA, and the effect of p53 expression on basal and uPA-mediated p53, uPAR protein, and mRNA expression was analyzed by Western or Northern blotting. The effect of p53 expression on basal and uPAmediated uPAR mRNA synthesis was determined by run-on-transcription. uPAR mRNA stability was assessed by transcription chase experiments as described above. Inhibition of p53 expression by RNA silencing in Beas2B cells. Beas2B cells grown to 70% confluence were treated with nonspecific small interfering RNA (siRNA) or p53-specific siRNA (Santa Cruz Biotechnologies, CA) for 36 h, after which the cells were treated with PBS or uPA and analyzed for the expression of p53 or uPAR protein and mRNA by Western or Northern blotting. The effect of p53 inhibition on basal and uPA-mediated uPAR mRNA synthesis or decay was determined as described above. In vitro transcription. Linearized plasmids containing the human uPAR mRNA transcriptional templates of uPAR cDNA were transcribed in vitro with T7 or Sp6 polymerase (Ambion, TX) in the presence of 50 Ci of [32P]UTP as described earlier (26). Molecular cloning, expression, and purification of p53. The coding sequence of p53 was PCR amplified using a previously cloned full-length cDNA packaged in pcDNA 3.1 vector, in conjunction with sense (CGC GGA TCC ATG GAG GAG CCG CAG TCA GAT CCT AGC; underlining indicates the BamHI restriction site) and antisense (CGC GGA TCC TCA GTC TGA GTC AGG CCC TTC TGT CTT GAA) oligonucleotide primers designed based on the 5⬘ and 3⬘ regions of the open reading frame. The purified PCR product was restricted with BamHI enzyme, subcloned into the BamHI site of the pGEX2TK prokaryotic expression vector, and transformed into Escherichia coli BL21. The recombinant p53 (rp53) protein was isolated using 0.5 ml of glutathioneSepharose. The bound glutathione S-transferase fusion protein was treated with 2 ml of a thrombin digestion buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 2.5 mM CaCl2) containing 5 units/ml of bovine thrombin as described before (27). The isolated rp53 proteins were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Coomassie blue staining and Western blotting. Gel mobility shift assay. Binding assays were performed by incubating uniformly 32P-labeled transcripts (20,000 cpm) corresponding to the uPAR coding region (CDR) or 3⬘ untranslated region (3⬘UTR) with rp53 protein (2 g) in a buffer containing 150 mM NaCl that was mixed with an equal volume of gel mobility shift buffer (15 mM KCl, 5 mM MgCl2, 0.25 mM EDTA, 0.25 mM dithiothreitol, 12 mM HEPES [pH 7.9], 10% glycerol) and E. coli tRNA (200 ng/l) in a total volume of 20 l at 30°C for 30 min. Reaction mixtures were treated with 50 units of RNase T1 and incubated for an additional 30 min at 37°C. To avoid nonspecific protein binding, 5 mg/ml heparin was added and the mixture was incubated at room temperature for an additional 10 min. Samples were then separated by electrophoresis on 5% native polyacrylamide gels with 0.25⫻ Tris-borate-EDTA running buffer. The gels were dried and autoradiographed at ⫺70°C using Kodak X-ray film. In order to determine the specificity of the interaction, various amounts (0 to 5 g) of rp53 were incubated with 32 P-labeled uPAR 3⬘UTR transcript in the above-mentioned gel shift buffer containing 150 mM NaCl and analyzed for uPAR mRNA 3⬘UTR-rp53 interaction. Whole-cell extraction and immunoprecipitation of RNA-protein complex. In order to confirm the direct interaction of p53 protein with uPAR mRNA in vivo, we cross-linked Beas2B cells treated with PBS and uPA (50 ng/ml or 1 g/ml) with formalin as described before (16). The cytosolic extracts were prepared by breaking the cells in lysis buffer (25 mM Tris-HCl [pH 7.9], 0.5 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride). These extracts were immunoprecipitated using nonspecific mouse immunoglobulin G followed by monoclonal antibody against human p53 protein in the presence of RNase inhibitor and rRNA for 1 h at room temperature. The immune complexes were precipitated using protein A/G-agarose, and the agarose beads were washed three times with lysis buffer. Total RNA was isolated from the immune complex using TRI reagent, and associated uPAR mRNA was amplified by reverse transcription-PCR (RT-PCR) using specific primers. The RNA associated with the p53 immune complex was amplified for -actin mRNA as a negative control. uPAR cDNA was used as the positive control. The PCR products were later identified by Southern blotting using 32P-labeled uPAR cDNA or nucleotide sequencing (27). Competitive inhibition by sense and antisense mRNAs or polyribonucleotides. rp53 protein (2 g) was incubated with approximately 0.042 ng of 32P-labeled uPAR 3⬘UTR transcript in the presence of various amounts (0 to 200-fold
MOL. CELL. BIOL. excess) of unlabeled uPAR sense or antisense 3⬘UTR mRNA at 30°C for 30 min and then treated with RNase T1 and heparin as described above, and the reaction mixtures were run on 5% native gels, dried, and autoradiographed. To determine the specificity of the RNA-protein complex, rp53 protein was pretreated with a molar excess of poly(A), poly(C), poly(G), or poly(U) ribonucleotide for 30 min at 30°C prior to the 32P-labeled uPAR mRNA and RNase T1 steps. Effects of SDS and proteinase K. rp53 protein was treated with SDS (0.1%) or proteinase K (2.5 mg/ml) for 30 min at 37°C prior to addition of 32P-labeled uPAR mRNA. The reaction mixtures were subjected to gel mobility shift assay as described above. In separate experiments, 32P-labeled uPAR 3⬘UTR mRNA was predigested with RNase T1 (50 units) for 30 min at 37°C before gel mobility shift analyses using rp53 protein. Determination of p53 binding site on uPAR mRNA 3ⴕUTR. uPAR 3⬘UTR cDNA fragments of different sizes were synthesized by PCR amplification of full-length uPAR cDNA template. Deletion fragments were cloned into pcDNA 3.1 and transcribed in vitro in the presence of [32P]UTP. 32P-labeled deletion transcripts were subsequently used as probes for gel mobility shift and UV cross-linking studies to localize the rp53 protein binding sequence on uPAR 3⬘UTR mRNA. Construction of -globin–uPAR chimeric message. Two 37-base-pair DNA fragments, one corresponding to the rp53 binding sequence (C3) and the other corresponding to the control nonbinding sequence (C4), were prepared from uPAR 3⬘UTR cDNA. Each of these cDNA fragments was inserted into the 3⬘UTR of complete human -globin cDNA. The clones were then inserted into a eukaryotic expression vector, pcDNA3.1. Beas2B cells were transfected with the prepared chimeric plasmid constructs by lipofection using Lipofectamine, and transient transfectants were grown in culture flasks. The decay of chimeric -globin–uPAR mRNA was then measured after the inhibition of transcription with DRB by Northern blotting using 32P-labeled cDNA at various time points. The half-life of the mRNA at each interval was determined by densitometry, normalized to the -actin control mRNA of the samples, and subsequently compared with the densitometric values for samples determined at the 0-h baseline of each experiment. In a separate experiment, we treated cells expressing chimeric -globin–uPAR 3⬘UTR mRNA containing p53 binding sequence with uPA (1 g/ml) to inhibit the basal p53 protein expression. In order to determine the direct involvement of p53 in the degradation of chimeric transcript, these cells were treated with DRB and the half-life of chimeric -globin– uPAR 3⬘UTR mRNA was determined as described above. Effect of p53 binding uPAR mRNA 3ⴕUTR sequence on uPAR expression. H1299 cells expressing vector cDNA or p53 cDNA were treated with p53 protein binding uPAR mRNA 3⬘UTR or control non-p53 binding sequence for 48 h. uPAR expression was determined by Western blotting of membrane proteins. Effect of p53 on uPA-mediated DNA synthesis. Naı¨ve H1299 cells and cell lines transfected with p53 cDNA in pcDNA3.1 or vector alone were grown to subconfluence in 24-well plates. Cells were treated with various amounts of uPA (0 to 2,000 ng/ml) in serum-free RPMI medium for 40 h. [3H]thymidine (1 Ci/ml; 20.3 mmol Ci) was later added to the same medium and incubated for an additional 8 h. The cells were analyzed for rate of DNA synthesis by measuring the incorporated [3H]thymidine as described previously (24). Beas2B cells treated with p53-specific siRNA or control nonspecific siRNA were treated with 0 to 2,000 ng/ml of uPA, and the rate of DNA synthesis was determined as described above. Effect of p53 on uPA-mediated lung epithelial cell apoptosis. Naı¨ve H1299 cells or H1299 cells transfected with vector cDNA or p53 cDNA were treated with various amount of uPA as described above. The cells were subjected to flow cytometry to determine programmed cell death by measuring the annexin Vphosphatidylserine interaction using the BD ApoAlert kit (BD Biosciences). In a separate experiment, nonmalignant Beas2B cells treated with p53-specific siRNA or control nonspecific siRNA in the presence of various amounts of uPA were subjected to flow cytometric analysis to assess the apoptotic response. Statistical analysis. We tested the differences between Beas2B or H1299 cells treated with p53 siRNA or p53 cDNA and corresponding control siRNA or vector cDNA-transfected control cells, respectively, by Student’s t test.
RESULTS Expression of uPAR protein and mRNA by p53-deficient lung carcinoma cells and nonmalignant lung epithelial cells. We recently reported that high concentrations of uPA (250 to 1,000 ng/ml; 5 to 20 nM) induce expression of uPAR (23) through posttranscriptional stabilization of its mRNA (28). At
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FIG. 1. p53 and uPAR expression by lung epithelial cells. (A) Expression of p53 protein and mRNA by bronchial epithelial cells (Beas2B) and p53-deficient H1299 lung non-small-cell carcinoma (p53⫺⫺) cells. Panel i, lysates of Beas2B and p53⫺/⫺ 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 anti--actin antibody for equal loading. Panel ii, the RNA isolated from Beas2B or p53⫺/⫺ cells treated with PBS or uPA (50 ng/ml) for 12 h was subjected to Northern blotting using 32P-labeled p53 cDNA and 32P-labeled anti--actin cDNA for equal loading. The bottom numbers represent densitometric scanning (mean ⫾ standard deviation) from at least four experiments. (B) Expression of uPAR protein and mRNA by Beas2B and H1299 p53⫺/⫺ cells. Panel i, membrane proteins of Beas2B and p53⫺/⫺ cells treated with PBS or uPA (1 g/ml) for 24 h were subjected to Western blotting using an anti-uPAR antibody. Panel ii, RNA isolated from Beas2B or p53⫺/⫺ cells treated with PBS or uPA (1 g/ml) for 12 h was subjected to Northern blotting using 32P-labeled uPAR cDNA and 32P-labeled -actin cDNA. These experiments were repeated at least four times, and densitometric scanning of individual bands (mean ⫾ standard deviation) is shown at the bottom. (C) Effect of uPA concentration on uPAR and p53 expression by Beas2B cells. Panel i, Beas2B cells were treated with 0, 50 ng/ml, or 1 g/ml of uPA for 24 h, and the membrane proteins were analyzed for uPAR expression by Western blotting using anti-uPAR antibody. Total cell lysates isolated from Beas2B cells treated with 0 to 1 g/ml of uPA were analyzed for p53 expression by Western blotting using anti-p53 antibody. The same membrane was stripped and analyzed for -actin protein. Panel ii, total RNA isolated from Beas2B cells treated with 0, 50 ng/ml, and 1 g/ml uPA for 12 h was analyzed for uPAR, p53, and -actin mRNA as described for panels ii in panels A and B. Experiments were replicated twice with identical results.
high uPA concentrations (⬎250 ng/ml), p53 expression is totally suppressed in lung epithelial cells (24). In order to determine if p53 influences uPAR expression, we initially compared p53 expression in nonmalignant Beas2B cells and malignant H1299 large cell lung carcinoma cells by Western blotting. As shown in Fig. 1A (panel i), Beas2B cells express small basal amounts of p53 protein (P ⬍ 0.01), and uPA regulates p53 expression in Beas2B cells with maximal induction at a low
uPA concentration (50 ng/ml). We previously showed that exposure to ⬎250 ng/ml uPA causes total suppression of p53 (24). We therefore treated Beas2B and H1299 cells with 50 ng/ml uPA. H1299 cells neither express p53 nor respond to uPA stimulation. Northern blotting indicated that uPA failed to alter expression of p53 mRNA in Beas2B cells, and p53 mRNA was undetectable in H1299 cells treated with or without uPA (Fig. 1A, panel ii). Next, membrane extracts of these
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FIG. 2. Regulation of uPAR expression by p53 in Beas2B and H1299 p53⫺/⫺ cells. (A) Reintroduction of p53 in p53⫺/⫺ cells inhibits uPAR protein and mRNA expression. Panel i, cells lacking p53 (p53⫺/⫺) 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 1 g/ml of uPA for 24 h. Cell lysates were analyzed for p53 expression by Western blotting as described in the legend to Fig. 1. Panel ii, p53⫺/⫺ cells transfected with vector pcDNA3.1 or wild-type p53 cDNA in pcDNA3.1 were treated with PBS or uPA (1 g/ml). Cell membranes were subjected to Western blotting using an anti-uPAR antibody. Panel iii, RNA isolated from p53⫺/⫺ cells transfected with vector cDNA or p53 cDNA and treated with PBS or uPA (1 g/ml) for 12 h was subjected to Northern blotting using 32P-labeled uPAR cDNA and 32P-labeled -actin cDNA. (B) Inhibition of p53 by siRNA induces uPAR protein and mRNA expression in Beas2B cells. Panel i, Beas2B cells transfected with control nonspecific siRNA (NspSiRNA) 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 antibody. The same membrane was stripped and developed with anti--actin antibody to assess equal loading. Panel ii, Beas2B cells treated with control siRNA or p53 siRNA as described for panel i were treated with PBS or uPA (1 g/ml) for 24 h. The membrane proteins were isolated and subjected to Western blotting using anti-uPAR antibody. Panel iii, Beas2B cells transfected with control siRNA or p53 siRNA were treated with PBS or uPA (1 g/ml) for 12 h. The total RNA isolated from these cells was subjected to Northern blotting using 32P-labeled uPAR cDNA. Densitometric scanning of individual bands (mean ⫾ standard deviation) from four independent experiments is shown at the bottom.
two cell types were subjected to Western blotting to determine if uPAR is differentially expressed due to differences in basal levels of p53. As shown in Fig. 1B (panel i), Beas2B and p53-deficient large cell carcinoma cells express uPAR, and uPA (1 g/ml) increased uPAR expression in both cell types. However, p53-deficient cells expressed at least 50-fold more basal uPAR, which was further induced severalfold by uPA compared to that in nonmalignant Beas2B cells. Similar increments of uPAR mRNA were observed in p53-deficient cells (Fig. 1B, panel ii). These observation led us to speculate that p53 is involved in uPA-mediated regulation of cell surface uPAR expression. In order to confirm the uPA effect on p53 and uPAR expression, we treated Beas2B cells with 50 ng/ml or 1 g/ml of uPA and tested the expression of cell surface uPAR and cellular p53 protein. As shown in Fig. 1C (panel i), low concentrations of uPA induced p53 expression but failed to induce uPAR expression. In contrast, the higher uPA concentration (1 g/ml) induced cell surface uPAR while totally suppressing the expression of p53 protein. uPA likewise induced uPAR mRNA expression at 1 g/ml (Fig. 1C, panel ii). uPA had no effect on p53 mRNA, confirming our earlier observation that p53 expression is regulated mainly by posttranslational control under these circumstances (24).
Effect of p53 expression on uPAR protein and mRNA expression. To confirm that p53 is involved in lung epithelial cell uPAR expression, H1299 cells were transfected with either vector (pcDNA 3.1) cDNA alone or p53 cDNA cloned in pcDNA3.1. Stable cell lines were created and analyzed for p53 expression by Western blotting using anti-p53 monoclonal antibody. Cells transfected with p53 cDNA, expressed p53 protein in p53-deficient H1299 cells, whereas empty pcDNA 3.1 vector cDNA-transfected cells failed to express p53 protein (Fig. 2A, panel i). Treatment of p53 cDNA-overexpressing cells with 1 g/ml of uPA reduced (P ⬍ 0.01) p53 expression (Fig. 2A, panel i), confirming its involvement in p53 translation (24). Next, these cells were analyzed for cell surface uPAR expression. As shown in Fig. 2A (panel ii), pcDNA 3.1 cDNAtransfected cells expressed elevated cell surface uPAR, and uPA stimulation further increased the expression of uPAR in these cell types. However, reintroduction of p53 through cDNA transfection in these cells suppressed basal uPAR expression, and these cells partially responded to uPA stimulation compared to vector cDNA-transfected cells (P ⬍ 0.01). We then analyzed the effects of p53 restoration by Northern blotting and found that p53 expression inhibited uPAR mRNA expression compared to that in vector cDNA-transfected cells
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FIG. 3. Regulation of uPAR mRNA expression by p53 in Beas2B and p53⫺/⫺ cells. (A) Effect of p53 overexpression on rate of uPAR mRNA synthesis and decay in p53⫺/⫺ cells. Panel i, naı¨ve H1299 cells (p53⫺/⫺) or H1299 cells transfected with vector cDNA (pcDNA3.1) or p53 cDNA were switched to serum-free medium for 12 h. Nuclei isolated from H1299 cells were subjected to the transcription reaction in the presence of [32P]UTP at 30°C for 30 min. 32P-labeled nuclear RNA was hybridized with uPAR cDNA immobilized on nitrocellulose. -Actin and pUC18 cDNAs were used as positive and negative loading controls, respectively. Densitometric scanning of individual bands (mean ⫾ standard deviation) from two independent experiments are shown at the bottom. Panel ii, naı¨ve p53⫺/⫺ cells or stable p53⫺/⫺ cells overexpressing vector cDNA or p53 cDNA were subjected to transcription chase by treatment with DRB (20 g/ml). The level of uPAR mRNA was measured by Northern blotting at 0 to 24 h. These experiments were repeated at least three times, and densitometric scanning of individual bands (mean ⫾ standard deviation) is shown as a graph. (B) Inhibition of p53 expression enhances both basal and uPA-mediated stabilization of uPAR mRNA. Beas2B cells transfected with control nonspecific (Nsp) siRNA or p53 siRNA were treated with PBS or uPA (1 g/ml) for 12 h. Ongoing transcription was inhibited by treatment with DRB, and the decay of uPAR mRNA was analyzed for 0 to 12 h by Northern blotting. -Actin mRNA expression is shown for equal loading. Densitometric scanning of individual bands (mean ⫾ standard deviation) is shown as a graph. The experiments were repeated at least four times.
(P ⬍ 0.01) (Fig. 2A, panel iii). These results support our hypothesis that p53 directly affects uPAR expression in H1299 cells. To extend these studies, we next inhibited p53 expression by RNA silencing in nonmalignant Beas2B airway epithelial cells and analyzed p53 expression by Western blotting. Treatment of Beas2B cells with p53 siRNA inhibited basal and uPAmediated p53 expression by more than 95% compared to that in control nonspecific siRNA-treated cells (Fig. 2B, panel i). We next analyzed basal and uPA-mediated cell surface uPAR expression in siRNA-treated cells by Western blotting. As shown in Fig. 2B (panel ii), Beas2B cells treated with nonspecific siRNA expressed minimal basal levels of uPAR, and uPA stimulation enhanced uPAR expression severalfold. However, inhibition of p53 expression by p53 siRNA treatment increased basal uPAR expression, and uPA augmented the effect in these cells compared to control siRNA-treated cells (P ⬍ 0.01).
Inhibition of p53 induced basal uPAR mRNA expression, and uPA further increased uPAR mRNA compared to that in control siRNA-treated Beas2B cells (P ⬍ 0.01) (Fig. 2B, panel iii). These results demonstrate that p53 protein regulates uPAR expression. Effects of p53 expression on the rate of uPAR mRNA synthesis and degradation. p53 binds to DNA promoter sequences and regulates expression of p53 target genes responsible for cell growth arrest (11) as well as uPA and PAI-1 transcription (9). We therefore tested the inference that p53 expression affects the rate of uPAR mRNA transcription in p53-deficient H1299 cells. Nuclei from p53-deficient cells transfected with or without vector or p53 cDNA in pcDNA 3.1 were isolated, and the nuclear extracts were subjected to run-on transcription to determine the rate of uPAR mRNA synthesis. As shown in Fig. 3A (panel i), p53 expression failed to affect uPAR mRNA synthesis. Because uPA induces uPAR
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FIG. 4. p53 protein binds to uPAR 3⬘UTR mRNA. (A) Expression of rp53 protein and testing of rp53 and p53 isolated from Beas2B cells for uPAR mRNA binding. Panel i, rp53 protein expressed in a prokaryotic system was analyzed by SDS-PAGE and staining with Coomassie blue. Panel ii, rp53 and Beas2B cell lysates separated by SDS-PAGE were transferred to a nitrocellulose membrane and analyzed for p53 by Western
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expression through posttranscriptional stabilization of uPAR mRNA (28), we suspected that p53 controls uPAR expression via uPAR mRNA degradation. We therefore treated naı¨ve p53-deficient cells or p53-deficient cells transfected with vector cDNA or p53 cDNA with DRB to inhibit ongoing transcription, and the decay of uPAR mRNA was analyzed by Northern blotting. H1299 cells which expressed p53 produced unstable mRNA (half-life [t1/2, ⬍3 h) compared to untransfected (t1/2, ⬃12 h) or vector cDNA-expressing (t1/2, ⬃12 h) p53-deficient cells (Fig. 3A, panel ii). These results indicate that p53 regulates uPAR mRNA stability. To confirm that p53 similarly affects uPAR mRNA turnover in nonmalignant lung epithelial cells, Beas2B cells were treated with control siRNA or p53 siRNA to suppress basal p53 expression. Beas2B cells express low basal levels of uPAR and respond to uPA stimulation (Fig. 1B), so control and p53 siRNA-transfected cells were treated with PBS or uPA for 12 h to induce maximum levels of uPAR mRNA (23). Ongoing transcription was inhibited, and the decay of uPAR mRNA was determined by Northern blotting. Our results show that uPAR mRNA is unstable (t1/2, ⬍3 h) in control siRNA-treated Beas2B cells and that uPA treatment stabilized uPAR mRNA in these cells. However, inhibition of p53 by p53 siRNA enhanced basal (t1/2, ⬎6 h) and further increased uPA-mediated (t1/2, ⬎6 h) stabilization of uPAR mRNA (Fig. 3B). These results confirm that p53 directly regulates uPAR mRNA stability. Identification of p53 as a uPAR mRNA binding protein. Posttranscriptional regulation of mRNA in general and uPAR mRNA in particular involves specific mRNA binding proteins. We therefore hypothesized that p53 binds to uPAR mRNA and thereby regulates its degradation. To test this possibility, we expressed an rp53–glutathione S-transferase fusion protein (rp53) in a prokaryotic system (Fig. 4A, panel i) and affinity purified the rp53 using a glutathione-Sepharose column. The purification (⬎95%) of rp53 protein was confirmed by Western blotting using an anti-p53 monoclonal antibody (Fig. 4A, panel ii). Next, we tested the ability of rp53 protein to interact with the uPAR mRNA CDR and 3⬘UTR by gel mobility shift assay. As shown in Fig. 4A (panel iii), rp53 protein failed to bind 32 P-labeled uPAR mRNA CDR but formed a specific RNAprotein complex with the uPAR mRNA 3⬘UTR. To determine
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the specificity of the p53-uPAR mRNA 3⬘UTR interaction, we incubated various amounts (0 to 5 g) of rp53 protein with 32 P-labeled uPAR mRNA 3⬘UTR and analyzed the binding by gel mobility shift assay. As shown in Fig. 4A (panel iv), the binding was apparent with 250 ng of p53 protein and the p53 binding to uPAR mRNA increased in a dose-dependent manner, with maximum saturation observed at 2.5 g. The calculated equilibrium dissociation constant was 444 ⫾ 69 nM. Since the buffer that we used for RNA-protein interaction studies contained a low salt concentration (15 mM KCl), we further assessed the p53-uPAR mRNA binding reaction in a gel shift buffer containing 150 mM NaCl and found that the use of higher, more physiologically relevant salt concentrations failed to alter the p53 binding interaction (Fig. 4A, panel v). To confirm that p53 also binds uPAR mRNA in vitro in cultured airway epithelial cells, we immunoprecipitated lysates of Beas2B cells treated with PBS or uPA (50 ng or 1 g/ml) with anti-p53 antibody, and p53-associated uPAR mRNA was amplified by RT-PCR. As shown in Fig. 4A (panel vi), the maximal p53-uPAR mRNA interaction was found in cells treated with 50 ng/ml uPA, indicating that the effect is concurrent with induction of p53 mediated by uPA at this concentration. At 1 g/ml uPA, the p53-uPAR mRNA association is absent, likely due to suppression of p53 (24). PCR of total RNA isolated from the p53 immunoprecipitates failed to amplify -actin mRNA (data not shown), indicating the specificity of the p53uPAR mRNA interaction. We next confirmed the specificity of the p53-uPAR mRNA 3⬘UTR interaction by cold competition experiments. Preincubation of rp53 (2 g) with a molar excess of unlabeled uPAR 3⬘UTR sense transcripts resulted in dose-dependent inhibition of the interaction between p53 and the 32P-labeled uPAR mRNA 3⬘UTR (Fig. 4B, panel i), whereas addition of the same amount of unlabeled antisense transcript failed to block p53 binding to the 32P-labeled uPAR mRNA 3⬘UTR (Fig. 4B, panel ii). Pretreatment of p53 with a 200-fold molar excess of homopoly(A), homopoly(C), or homopoly(U) ribonucleotides likewise failed to affect the p53-uPAR 3⬘UTR mRNA interaction, whereas addition of a molar excess of homopoly(G) ribonucleotide inhibited the binding of p53 protein to uPAR mRNA (Fig. 4B, panel iii), indicating that p53 binds to specific G-rich nucleotide sequences on the uPAR mRNA 3⬘UTR.
blotting using an anti-p53 monoclonal antibody. Panel iii, rp53 protein was incubated with 32P-labeled uPAR mRNA CDR or 3⬘UTR, and the mRNA-rp53 complexes were analyzed by gel mobility shift assay on 5% nondenaturing PAGE. Fp, buffer alone. The arrow indicates the RNA-protein complex. Panel iv, 0 to 5.0 g of rp53 protein was incubated with 32P-labeled uPAR mRNA 3⬘UTR and analyzed for p53-uPAR mRNA interaction by gel mobility shift assay. Panel v, rp53 protein (1 g) was incubated with 32P-labeled uPAR mRNA 3⬘UTR probe in a buffer containing 15 mM NaCl or 150 mM NaCl, and the RNA-p53 interaction was analyzed by gel mobility shift assay. Fp, probe alone. Panel vi, Beas2B cells treated with various amounts (0 to 1 g/ml) of uPA for 24 h were lysed, and the lysates were immunoprecipitated using anti-p53 monoclonal antibody (p53 mAb) or mouse immunoglobulin G (NSp mIgG). The p53 protein-associated uPAR mRNA was detected by RT-PCR using 32 P-labeled dCTP and verified by nucleotide sequencing of the corresponding nonradioactive PCR product. uPAR cDNA was used as a positive control (⫹ve), and uPAR cDNA was replaced by buffer alone for a negative control (⫺ve). (B) Determination of the specificity of the p53 protein-uPAR mRNA 3⬘UTR interaction. Competitive inhibition of p53-uPAR mRNA interaction using unlabeled uPAR mRNA sense and antisense transcripts is shown. rp53 protein (2 g) was incubated with 32P-labeled uPAR 3⬘UTR mRNA (0.042 ng) in the presence of a 0- to 200-fold molar excess of unlabeled sense (panel i) or antisense (panel ii) transcript over labeled analog. Fp, free probe. Panel iii, effects of polyribonucleotides, proteinase K, and SDS on uPAR mRNA interaction with p53. rp53 protein was incubated with a 200-fold excess of unlabeled sense (3⬘UTR S-C), antisense (3⬘UTR A-C), or p53 consensus (5⬘ prom) sequence or with poly(A), poly(C), poly(G), poly(U), proteinase K (2.5 mg/ml), or 0.1% SDS for 30 min at 30°C. 32P-labeled uPAR mRNA probe was added, and the mixture was digested with RNase T1 and analyzed by gel mobility shift assay. Fp, probe alone. RNase T1, 32P-labeled uPAR 3⬘UTR mRNA predigested with RNase T1 before exposure to p53. The experiments were repeated at least four or five times.
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FIG. 5. Identification of the p53 protein binding sequence on uPAR 3⬘UTR mRNA. (i) Deletion map indicating the p53 protein binding site on uPAR mRNA. (ii) rp53 protein expressed in E. coli was affinity purified. The rp53 was incubated with 32P-labeled uPAR mRNA full-length CDR (lane 1), full-length 3⬘UTR (lane 2), or 3⬘UTR deletion transcripts (lanes 3 to 8), and the RNA-protein complex was analyzed by gel mobility shift assay. The arrow indicates the uPAR mRNA-p53 protein complex. Lane Fp, 32P-labeled uPAR mRNA probe incubated with buffer alone. Data are representative of those from four independent analyses.
Treatment of p53 protein with proteinase K or SDS abolished its interaction with the uPAR mRNA 3⬘UTR. Lastly, a molar excess of unlabeled p53 promoter binding DNA consensus sequence failed to abolish binding of p53 protein to the uPAR mRNA 3⬘UTR, indicating that the p53 transactivation domain is independent of the uPAR mRNA binding region. Identification of the p53 binding sequence on uPAR 3ⴕUTR mRNA and its functionality. In order to identify the specific p53 protein binding sequences on the uPAR mRNA 3⬘UTR, we made PCR-based overlapping deletions of uPAR cDNA. These cDNA fragments were subcloned into pcDNA 3.1, and 32 P-labeled deletion transcripts were prepared by in vitro transcription. These transcripts were then individually tested for p53 binding by gel shift assay. As shown in Fig. 5, p53 protein bound specifically to a 37-nucleotide (nt) sequence (nt 1051 to 1088) present on the uPAR mRNA 3⬘UTR. To determine if the p53 binding 37-nt uPAR 3⬘UTR sequence contains information for message stability, we inserted the 37-nt sequence into the -globin cDNA (Fig. 6, panel i), and the chimeric -globin–uPAR 3⬘UTR cDNA containing the 37-nt p53 binding sequence (Fig. 6, panel ii, C3) was subcloned into the eukaryotic expression vector pcDNA3.1. We also prepared chimeric -globin–uPAR 3⬘UTR cDNA containing a control 37-nt non-p53 binding sequence (nt 1094 to 1131) (Fig. 6, panel ii, C4). Beas2B cells were then transfected with chimeric -globin–uPAR 3⬘UTR cDNA constructs as well as -globin cDNA alone. The decay of chimeric -globin–uPAR 3⬘UTR mRNA was determined by Northern blotting after inhibiting ongoing transcription. Figure 6 (panel iii) shows that insertion of the p53 binding 37-nt uPAR 3⬘UTR sequence (C3) destabilized -globin mRNA. However, insertion of a similarly sized control non-p53 binding sequence (C4) failed to alter the stability of -globin mRNA (Fig. 6, panel iii), and -globin mRNA was likewise quite stable (data not shown), indicating
that the p53 binding 37-nt uPAR 3⬘UTR sequence contains regulatory information that controls uPAR mRNA stability. We next treated Beas2B cells overexpressing chimeric -globin–uPAR 3⬘UTR mRNA with uPA (1 g/ml) to inhibit endogenous p53 expression and determined the decay of chimeric mRNA by transcription chase experiments. We found that inhibition of p53 expression reversed decay of chimeric -globin–uPAR mRNA (Fig. 6, panel iv), demonstrating the involvement of p53 in the regulation. We next tested the effect of the p53 binding 37-nt uPAR mRNA 3⬘UTR sequence (C3) on uPAR expression. H1299 cells transfected with vector cDNA or p53 cDNA were transfected with 37-nt p53 binding (C3) RNA or control 37-nt (C4) RNA for 48 h, and membrane proteins were analyzed for uPAR expression. As shown in Fig. 6 (panel v), treatment of p53 cDNA-transfected H1299 cells with the 37-nt p53 binding RNA but not the 37-nt control RNA reversed (P ⬍ 0.01) the inhibitory effect of p53. However, the 37-nt p53 binding sequence had no effect on vector cDNA-transfected H1299 cells. These competition studies indicate that p53 interaction with the 37-nt uPAR mRNA (C3) 3⬘UTR sequence regulates cell surface uPAR expression. Determining the role of p53 expression in uPA-mediated cell proliferation and survival. The ability of p53 to regulate uPAmediated alterations in viability of lung epithelial cells was next analyzed. Inhibition of p53 by siRNA significantly (P ⬍ 0.01) inhibited uPA-induced epithelial cell apoptosis compared to that in nonspecific siRNA-treated cells (Fig. 7A, panel i). We next found that p53 expression significantly (P ⬍ 0.01) induced uPA-mediated apoptosis of p53-deficient cells compared to that in untreated or vector cDNA-treated cells (Fig. 7A, panel ii). However, addition of uPA (⬎250 ng/ml) inhibited apoptosis of p53⫺/⫺ cells transfected with p53 cDNA. Conversely, inhibition of expression of p53 by p53 siRNA also significantly (P ⬍ 0.01 or P ⬍ 0.05) induced basal as well as uPA-induced [3H]thymidine incorporation (Fig. 7B, panel i), whereas introduction of p53 in p53-deficient cells significantly (P ⬍ 0.05) inhibited basal and uPA-induced DNA synthesis at a lower uPA concentration than for untransfected or vector cDNA transfected cells (Fig. 7B, panel ii). DISCUSSION uPAR is integrally involved in uPA-dependent pericellular proteolysis and is localized at the protruding edge of migrating cells (12, 32). uPA is also involved in several crucial cellular nonproteolytic functions, including cellular proliferation and survival (4, 21, 23, 24). These functions of uPA depend on its association with uPAR and may contribute to remodeling of the lung, as occurs in acute respiratory distress syndrome or the interstitial lung diseases and in lung cancer (12, 29, 30). Regulation of uPAR expression is therefore critical to the control of both the proteolytic and nonproteolytic functions of uPA in lung inflammation as well as neoplasia. p53 exhibits sequence-specific DNA binding and transcriptional activation of target genes involved in cell cycle regulation and apoptosis (6, 11). In most tumor cells the p53 function is reduced, resulting in unrestricted proliferation. p53 also regulates cellular fibrinolysis via induction of PAI-1 expression through promoter transactivation. The PAI-1 promoter exhib-
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FIG. 6. Determining the destabilizing function of the p53 binding uPAR 3⬘UTR mRNA sequence. (i) Structure of -globin–uPAR 3⬘UTR chimeric mRNA. The p53 protein binding 37-nt sequence corresponding to nt 1051 to 1088 (C3) and a control sequence of similar size corresponding to the nonbinding region from nt 1094 to 1131 (C4) of uPAR 3⬘UTR cDNA were inserted into the 3⬘UTR of -globin cDNA. The chimeric -globin–uPAR 3⬘UTR cDNA was subcloned into pcDNA 3.1. (ii) Nucleotide sequence of p53 binding region nt 1051 to 1088 (C3) or nonbinding control sequence 1094 to 1131 (C4). (iii) Beas2B cells were transfected with the chimeric -globin–uPAR 3⬘UTR gene containing the 37-nt p53 binding sequence (nt 1051 to 1088) [-Globin–uPAR(C3)] or nonbinding control sequence (nt 1094 to 1131) [-Globin–uPAR(C4)] of the uPAR 3⬘UTR in pcDNA3.1. Total RNA was isolated at different time intervals after treatment with DRB as described for Fig. 3A (panel ii), and the level of chimeric mRNA was analyzed by Northern blotting. Densitometric scanning of individual bands (mean ⫾ standard deviation) from four experiments is shown as a graph. (iv) Beas2B cells overexpressing the chimeric -globin–uPAR 3⬘UTR transcript containing the p53 binding sequence were treated with PBS or uPA for 12 h to inhibit endogenous p53 expression, and decay of chimeric mRNA was determined after inhibiting ongoing transcription by treatment with DRB as described for panel iii. Densitometric scanning of individual bands (mean ⫾ standard deviation) from two independent experiments after normalizing with a -actin loading control is shown as a graph. (v) Stable H1299 cells overexpressing vector cDNA (pcDNA 3.1) or p53 cDNA were untreated (None) or transfected with RNA containing the 37-nt control (C4) (Nsp) or p53 binding (C3) (p53) uPAR mRNA 3⬘UTR sequence. The membrane proteins were isolated, and uPAR expression was determined by Western blotting using an anti-uPAR antibody. Data are representative of at least four independent analyses.
its a p53 binding sequence (9). p53 inhibits uPA expression through suppression of the promoter and enhancer activity of the uPA gene. Various observations also support the potential for cross talk between p53 and uPAR expression (19, 24). We therefore sought to unravel the mechanism by which uPAR and p53 interact and to determine how p53 and uPAR are expressed and regulated by uPA. Here we show, for the first time, that expression of the uPAR gene is regulated by p53. We recently reported that uPA induces cell surface uPAR expression through attenuation of posttranscriptional uPAR mRNA decay (28) and that maximum uPAR expression occurs when lung epithelial cells are treated with uPA at a concen-
tration of greater than 10 nM (500 ng/ml). We also found that uPA regulates p53 expression in a concentration-dependent manner. However, uPA induces uPAR at a concentration at which p53 expression is totally suppressed. Our results now indicate that cells lacking p53 express elevated levels of uPAR and that uPA further increases uPAR expression. The role of p53 in uPAR expression is further supported in that restoration of p53 in p53⫺/⫺ cells suppressed basal uPAR expression. Further, induction of basal uPAR as well as the added effect of uPA in p53-inhibited (siRNA-treated) cells shows that p53 regulates cell surface uPAR expression. Our results show that expression of p53 in p53⫺/⫺ cells failed
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FIG. 7. Cellular consequences of modulation of p53 expression in Beas2B and p53⫺/⫺ cells. (A) Effect of p53 expression on Beas2B and p53⫺/⫺ cell apoptosis. Panel i, Beas2B cells transfected with control nonspecific siRNA (Vc) or p53 siRNA were treated with various amounts of uPA (0 to 2,000 ng/ml) for 48 h at 37°C in basal medium. The cells were later detached and treated with anti-annexin V antibody and propidium iodide. The apoptotic cells were analyzed by flow cytometry. Panel ii, H1299 cells (p53⫺/⫺) transfected with or without vector cDNA (Vc) or p53 cDNA (p53) were treated with various amounts of uPA for 48 h. The apoptotic cells were analyzed by flow cytometry as described for panel i. The data shown are representative of three separate experiments, and P values represent differences between nonspecific siRNA (Vc)- and p53 siRNAtreated and/or untransfected p53⫺/⫺ cells (p53⫺/⫺) or between vector cDNA-overexpressing control p53⫺/⫺ (Vc) and p53 cDNA-overexpressing p53⫺/⫺ (p53) cells. (B) Effect of p53 on uPA-mediated proliferation of Beas2B and p53⫺/⫺ cells. Panel i, Beas2B cells transfected with control nonspecific siRNA (Vc) or p53 siRNA were treated with various amounts of uPA (0 to 2,000 ng/ml) for 48 h at 37°C in basal medium, and the DNA synthesis was measured by pulse-labeling cells with 1 Ci/ml [3H]thymidine for the last 8 h of the treatment. The cells were washed and extracted with trichloroacetic acid, and the incorporated radioactivity was then measured using a scintillation counter. Panel ii, subconfluent H1299 (p53⫺/⫺) cells untransfected or transfected with vector cDNA (Vc) or p53 cDNA (p53) were treated with various amounts of uPA, and the DNA synthesis was measured as described above for panel i. The values are means from at least four independent analyses, and the P values represent differences between nonspecific siRNA (Vc)- and p53 siRNA-treated and/or untransfected p53⫺/⫺ (p53⫺/⫺) cells or between vector cDNAoverexpressing p53⫺/⫺ (Vc) control and p53 cDNA-overexpressing p53⫺/⫺ (p53) cells. The error bars indicate standard deviations from the mean values.
to alter uPAR mRNA synthesis and that the uPAR promoter lacks a p53 binding sequence, whereas p53 represses uPA mRNA synthesis. However, expression of p53 destabilized uPAR mRNA in H1299 cells, indicating that p53 is involved in the stabilization of uPAR mRNA. The involvement of p53 in posttranscriptional regulation of uPAR mRNA is also supported by the observation that inhibition of p53 increased uPAR mRNA stability without any change in the rate of uPAR mRNA synthesis. We therefore hypothesized that p53 directly interacts with uPAR mRNA to regulate its stability. We confirmed this as-
sumption by showing that rp53 interacts with the uPAR mRNA 3⬘UTR by gel shift assay. Coprecipitation of uPAR mRNA with p53 from Beas2B cell lysates confirms the existence of such an interaction in vivo. The specificity of p53 interaction with the uPAR mRNA 3⬘UTR was shown by competition experiments using an unlabeled sense analog. An unlabeled antisense probe failed to compete for specific binding. Furthermore, addition of a molar excess of poly(A), poly(C), or poly(U) homopolyribonucleotide had no effect on p53 binding to the 32P-labeled uPAR mRNA 3⬘UTR, whereas poly(G) inhibited the interaction, indicating that
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p53 binding requires a G-rich unique sequence. The specificity of p53 binding to uPAR 3⬘UTR mRNA is buttressed by the findings that pretreatment of p53 with proteinase K or SDS or predigestion of uPAR mRNA probe with RNase A and RNase T1 totally abolished the p53-uPAR mRNA 3⬘UTR complex. p53 binds to a specific nucleotide sequence on the uPAR mRNA 3⬘UTR and regulates its stability. The results of deletion experiments show that rp53 protein specifically binds to 37-nt uPAR mRNA 3⬘UTR sequences and that the binding region is located downstream of the stop codon. Our studies indicate that there is no p53 binding sequence on the uPAR CDR or 3⬘UTR other than this 37-nt region, which corresponds to the uPAR cDNA sequence from nt 1051 to 1088 (18). We found that insertion of a uPAR 3⬘UTR sequence containing the 37-nt p53 binding sequence into -globin mRNA destabilized the chimeric transcript in Beas2B cells, indicating that the p53 binding sequence contains information for mRNA destabilization. Stabilization of the chimeric transcript containing the p53 binding 37-nt uPAR mRNA 3⬘ UTR sequence by inhibition of p53 expression in Beas2B cells by uPA treatment further supports the role of p53 in uPAR mRNA expression. Treatment with the 37-nt p53 binding sequence of the uPAR mRNA 3⬘UTR also induced uPAR in p53-expressing H1299 cells, confirming that the p53 interaction with the 37-nt uPAR mRNA 3⬘UTR sequence destabilizes uPAR mRNA. Our observations reveal that p53 interacts with the 37-nt uPAR mRNA-destabilizing element and that increased expression of uPAR occurs in the cells where expression of wild-type p53 is reduced, delineating a novel regulatory mechanism. Introduction of p53 reverses cell surface uPAR expression through destabilization of uPAR mRNA, confirming the newly recognized function. In all, these studies demonstrate, for the first time, a novel function for p53 as an mRNA binding protein that regulates the expression of uPAR mRNA by altering its stability. Expression of p53 also reduced uPA-mediated proliferation and increased apoptosis of p53deficient lung carcinoma cells. uPA regulates epithelial cell viability in a dose-dependent fashion, and the process inversely relates to p53 expression, demonstrating an intricate relationship between uPA-mediated p53 expression, epithelial cell viability/proliferation, and uPA-mediated uPAR expression. This is potentially an important mechanism by which uPA can regulate alveolar fibrinolysis and remodeling through uPA- and p53-mediated control of uPAR. p53 expression is also dependent on uPA’s association with uPAR (24). Inhibition of p53 in nonmalignant lung epithelial cells by RNA inhibition causes basal as well as uPA-mediated proliferation, and these cells fail to undergo apoptosis induced by lower concentrations of uPA. In contrast, reintroduction of p53 in p53-deficient cells enhances apoptosis of p53-deficient cells. However, uPA inhibits apoptosis and induces proliferation in these cells in a dose-dependent manner. This process may play a major role in the pathogenesis of several nonmalignant lung diseases where induction of p53 expression, epithelial cell death, and fibrin deposition are the hallmark events (4, 8, 15, 17). This process is similarly implicated in lung carcinoma where cell resistance to chemotherapy depends on levels of uPA/uPAR (2) and p53 (3). We previously found that most tumor cells overexpress
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uPAR and exhibit highly stable uPAR mRNA (21, 23, 26). Most malignant cells exhibit enhanced proliferation and resistance to apoptosis due to decreased p53 expression (7, 10). We now show that posttranscriptional regulation of uPAR mRNA is p53 dependent. In summary, we confirmed that the p53-uPAR mRNA 3⬘UTR interaction regulates uPAR mRNA stability in lung epithelial cells. This is first description of the ability of p53 to interact with uPAR mRNA and control its uPAR expression at the posttranscriptional level. This pathway adds a new dimension to the p53 functional repertoire and represents a new link between viability of lung epithelial cells and pericellular proteolysis. This highly coordinated pathway could influence altered uPAR expression and uPAR-dependent derangements in a range of pathophysiologic situations, including lung injury or neoplasia. ACKNOWLEDGMENTS This work was supported by National Heart, Lung, and Blood Institute grants R01-HL071147 and P01HL-62453. We are grateful to Ming Cheh Liu for his help in expressing rp53 protein and to Brad Low and M. B. Harish for their technical assistance. We have no conflicting financial interests. REFERENCES 1. Alfano, D., P. Franco, I. Vocca, N. Gambi, V. Pisa, A. Mancini, M. Caputi, M. V. Carriero, I. Iaccarino, and M. Stoppelli. 2005. The urokinase plasminogen activator and its receptor; role in cell growth and apoptosis. Throm. Haemost. 93:205–211. 2. Andreasen, P. A., R. Egelund, and H. H. Petersen. 2000. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol. Life Sci. 57:25–40. 3. Bensaad, K., and K. H. Vousden. 2005. Savior and slayer: the two faces of p53. Nat. Med. 11:1278–1289. 4. Chapman, H. A. 1997. Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Curr. Opin. Cell Biol. 9:714–724. 5. Chapman, H. A. 2004. Disorders of lung matrix remodeling. J. Clin. Investig. 113:148–157. 6. He, G., Z. H. Siddik, Z. Huang, R. Wang, J. Koomen, R. Kobayashi, A. R. Khokhar, and J. Kuang. 2005. Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities. Oncogene 24:2929–2943. 7. Iskander, K., A. Gaikwad, M. Paquet, D. J. Long II, C. Brayton, R. Barrios, and A. K. Jaiswal. 2005. Lower induction of p53 and decreased apoptosis in NQO1-null mice lead to increased sensitivity to chemical-induced skin carcinogenesis. Cancer Res. 65:2054–2058. 8. Jeffery, P. K. 2001. Remodeling in asthma and chronic obstructive diseases. Am. J. Respir. Crit. Care Med. 164:S28–S38. 9. Kunz, C., S. Pebler, J. Otte, and D. Von Der Ahe. 1995. Differential regulation of plasminogen activator and inhibitor gene transcription by the tumor suppressor p53. Nucleic Acids Res. 23:3710–3717. 10. Lane, D. 2004. Curing cancer with p53. N. Engl. J. Med. 350:2711–2712. 11. Levine, A. J. 1997. p53, the cellular gatekeeper for growth and division. Cell 88:323–331. 12. Mazar, A. P., J. Henkin, and R. H. Goldfarb. 1999. The urokinase plasminogen activator system in cancer: implications for tumor angiogenesis and metastasis. Angiogenesis 3:15–32. 13. Mazzieri, R., and F. Blasi. 2005. The urokinase receptor and the regulation of cell proliferation. Thromb. Haemostat. 93:641–646. 14. Mignatti, P., and D. B. Rifkin. 2000. Nonenzymatic interactions between proteinases and the cell surface: novel roles in normal and malignant cell physiology. Adv. Cancer Res. 78:103–157. 15. Mishra, A., N. A. Doyle, and W. J. Martin II. 2000. Bleomycin-mediated pulmonary toxicity: evidence for a p53-mediated response. Am. J. Respir. Cell Mol. Biol. 22:543–549. 16. Niranjanakumari, S., E. Lasda, R. Brazas, and M. A. Garcia-Blanco. 2002. Reversible cross-linking combined with immunoprecipitation to study RNAprotein interaction in vivo. Methods 26:182–190. 17. Plataki, M., A. V. Koutsopoulos, K. Darivianaki, G. Delides, N. M. Siafakas, and D. D. Bouros. 2005. Expression of apoptotic and antiapoptotic markers in epithelial cells in idiopathic pulmonary fibrosis. Chest 127:266–274. 18. Roldan, A. L., M. V. Cubellis, M. T. Masucci, N. Behrendt, L. R. Lund, K. Dano, E. Appella, and F. Blasi. 1990. Cloning and expression of the receptor
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19.
20.
21.
22.
23. 24. 25.
26.
SHETTY ET AL.
for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis. EMBO J. 9:467–474. Shetty, S., A. Kumar, A. R. Johnson, and S. Idell. 1995. Regulation of mesothelial cell mitogenesis by antisense oligonucleotides for the urokinase receptor. Antisense Res. Dev. 5:307–314. Shetty, S., and S. Idell. 2000. Post-transcriptional regulation of urokinase mRNA: identification of a novel urokinase mRNA-binding protein in human lung epithelial cells in vitro. J. Biol. Chem. 275:13771–13779. Shetty, S., and S. Idell. 1999. Pottranscriptional regulation of urokinase receptor gene expression in lung carcinoma and malignant mesothelioma cells in vitro. Mol. Cell. Biohem. 199:189–200. Shetty, S., and S. Idell. 2006. Fibrinolysis/plasminogen activator and plasmin, p. 205–210. In J. Laurent (ed.), Encyclopedia of respiratory medicine. Elsevier Publications, London, United Kingdom. Shetty, S., and S. Idell. 2001. Urokinase induces expression of its own receptor in Beas2B lung epithelial cells. J. Biol. Chem. 276:24549–24556. Shetty, S., M. Gyetko, and A. Mazar. 2005. Induction of p53 by urokinase in lung epithelial cells. J. Biol. Chem. 280:28133–28141. Shetty, S., B. Khalil, D. B. Cines, and S. Idell. 2003. Induction of plasminogen activator inhibitor-1 by urokinase in lung epithelial cells. J. Biol. Chem. 278:18124–18131. Shetty, S., A. Kumar, and S. Idell. 1997. Posttranscriptional regulation of urokinase receptor mRNA: identification of a novel urokinase receptor
MOL. CELL. BIOL.
27. 28. 29. 30.
31. 32. 33.
mRNA binding protein in human mesothelioma cells. Mol. Cell. Biol. 17: 1075–1083. Shetty, S., H. B. Muniappa, P. K. S. Halady, and S. Idell. 2004. Regulation of urokinase receptor expression by phosphoglycerate kinase. Am. J. Respir. Cell Mol. Biol. 31:100–106. Shetty, S., T. Velusamy, S. Idell, H. Tang, and P. K. Shetty. 2007. Regulation of urokinase receptor expression by protein tyrosine phosphatases. Am. J. Physiol. Lung Cell Mol. Physiol. 292:L414–L421. Sisson, T. H., N. Hattori, Y. Xu, and R. H. Simon. 1999. Treatment of bleomycin-induced pulmonary fibrosis by transfer of urokinase-type plasminogen activator genes. Hum. Gene Ther. 10:2315–2323. Swaisgood, C. M., E. L. French, C. Noga, R. H. Simon, and V. A. Ploplis. 2000. The development of bleomycin-induced pulmonary fibrosis in mice deficient for components of the fibrinolytic system. Am. J. Pathol. 157:177– 187. Vogelstein, B., D. Lane, and A. J. Levine. 2000. Surfing the p53 network. Nature 408:307–310. Wei, Y., J. A. Eble, Z. Wang, J. A. Kreidberg, and H. A. Chapman. 2001. Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1. Mol. Biol. Cell 12:2975–2986. Yanamandra, N., S. D. Konduri, S. Mohanam, D. H. Dinh, W. C. Olivero, M. Gujrati, G. L. Nicolson, M. Obeyeseke, and J. S. Rao. 2005. Downregulation of urokinase-type plasminogen activator receptor (uPAR) induces caspase-mediated cell death in human glioblastoma cells. Clin. Exp. Metast. 18:611–615.
