Regulation of urokinase receptor expression by p53: novel role in stabilization of uPAR mRNA

Abstract

We found that p53-deficient (p53(-/-)) lung carcinoma (H1299) cells express robust levels of cell surface uPAR and uPAR mRNA. Expression of p53 protein in p53(-/-) 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 3' untranslated region (3'UTR), and endogenous uPAR mRNA associates with p53. The p53 binding region involves a 37-nucleotide uPAR 3'UTR sequence, and insertion of the p53 binding sequence into beta-globin mRNA destabilized beta-globin mRNA. Inhibition of p53 expression in these cells reverses decay of chimeric beta-globin-uPAR mRNA. These observations demonstrate a novel regulatory role for p53 as a uPAR mRNA binding protein that down-regulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway epithelial or lung carcinoma cells.

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-β-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-β-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 μg/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 μg/ml) for 12 h was subjected to Northern blotting using ³²P-labeled uPAR cDNA and ³²P-labeled β-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 μg/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 μg/ml of uPA were analyzed for p53 expression by Western blotting using anti-p53 antibody. The same membrane was stripped and analyzed for β-actin protein. Panel ii, total RNA isolated from Beas2B cells treated with 0, 50 ng/ml, and 1 μg/ml uPA for 12 h was analyzed for uPAR, p53, and β-actin mRNA as described for panels ii in panels A and B. Experiments were replicated twice with identical results.

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 μg/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 μg/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 μg/ml) for 12 h was subjected to Northern blotting using ³²P-labeled uPAR cDNA and ³²P-labeled β-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-β-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 μg/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 μg/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.

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, naïve 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. β-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, naïve p53^−/− cells or stable p53^−/− cells overexpressing vector cDNA or p53 cDNA were subjected to transcription chase by treatment with DRB (20 μg/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 μg/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. β-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.

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 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 μg 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 μg) 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 μg/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 ³²P-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 μg) 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.

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.

FIG. 6.

Determining the destabilizing function of the p53 binding uPAR 3′UTR mRNA sequence. (i) Structure of β-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 β-globin cDNA. The chimeric β-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 β-globin-uPAR 3′UTR gene containing the 37-nt p53 binding sequence (nt 1051 to 1088) [β-Globin-uPAR(C3)] or nonbinding control sequence (nt 1094 to 1131) [β-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 β-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 β-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.

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 siRNA-treated 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 μCi/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 cDNA-overexpressing p53^−/− (Vc) control and p53 cDNA-overexpressing p53^−/− (p53) cells. The error bars indicate standard deviations from the mean values.