MOLECULAR AND CELLULAR BIOLOGY, Aug. 2007, p. 5607–5618
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?
Sreerama Shetty,1* Thirunavukkarasu Velusamy,1Steven Idell,1Praveenkumar Shetty,1
Andrew P. Mazar,2Yashodhar P. Bhandary,1and 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 75708,1and 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 (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 ?-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 down-
regulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway
epithelial or 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 re-
modeling of the extracellular matrix in acute and chronic lung
injury, repair (5, 22), and neoplasia (14, 33) has become in-
Lung epithelial cells synthesize and secrete a 55-kDa proen-
zyme single-chain form of uPA, which is activated by plasmin
and other proteases (20). These cells also synthesize and ex-
press a cell surface receptor for uPA, uPAR, which is a glyco-
sylphosphatidylinositol-linked receptor (23).
Lung epithelial cell viability and uPA-mediated tissue re-
modeling may contribute to the pathogenesis of diverse lung
diseases, such as acute respiratory distress syndrome or a va-
riety of interstitial lung diseases. These diseases share a pro-
pensity for apoptosis of lung epithelial cells and the develop-
ment of progressive fibrosis that predisposes the individual to
respiratory failure (5). Recent evidence suggests a close rela-
tionship 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. Sup-
pression of p53 function due to mutation or deletion (10, 11,
31) and overexpression of uPAR occurs in tumor cells (2, 12,
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.
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
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 tran-
scription, 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 de-
scribed earlier (25).
* Corresponding author. Mailing address: The Texas Lung Injury In-
stitute, 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
?Published ahead of print on 4 June 2007.
Transfection of p53-deficient H1299 cells with p53 cDNA. p53 cDNA was
cloned into the eukaryotic expression vector pcDNA3.1. H1299 cells were trans-
fected with vector cDNA or vector DNA containing p53 cDNA by using Lipo-
fectamine, 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 uPA-
mediated uPAR mRNA synthesis was determined by run-on-transcription.
uPAR mRNA stability was assessed by transcription chase experiments as de-
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
T7or Sp6polymerase (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 pGEX-
2TK prokaryotic expression vector, and transformed into Escherichia coli BL21.
The recombinant p53 (rp53) protein was isolated using 0.5 ml of glutathione-
Sepharose. 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 sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Coomassie
blue staining and Western blotting.
Gel mobility shift assay. Binding assays were performed by incubating uni-
formly32P-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 T1and 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 autoradio-
graphed 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
32P-labeled uPAR 3?UTR transcript in the above-mentioned gel shift buffer
containing 150 mM NaCl and analyzed for uPAR mRNA 3?UTR-rp53 inter-
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
using32P-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 of32P-labeled
uPAR 3?UTR transcript in the presence of various amounts (0 to 200-fold
excess) of unlabeled uPAR sense or antisense 3?UTR mRNA at 30°C for 30 min
and then treated with RNase T1and 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 the32P-labeled uPAR mRNA and RNase T1steps.
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 of32P-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 3?UTR. 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
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 using32P-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 express-
ing 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 tran-
script, 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 3?UTR 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 sub-
confluence 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
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 V-
phosphatidylserine 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.
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
5608SHETTY ET AL.MOL. CELL. BIOL.
high uPA concentrations (?250 ng/ml), p53 expression is to-
tally suppressed in lung epithelial cells (24). In order to deter-
mine 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 with-
out uPA (Fig. 1A, panel ii). Next, membrane extracts of these
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 using32P-labeled p53 cDNA and32P-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 using32P-labeled uPAR cDNA and32P-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.
VOL. 27, 2007REGULATION OF UROKINASE RECEPTOR mRNA BY p535609
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 incre-
ments 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 concen-
tration (1 ?g/ml) induced cell surface uPAR while totally sup-
pressing 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 observa-
tion that p53 expression is regulated mainly by posttransla-
tional control under these circumstances (24).
Effect of p53 expression on uPAR protein and mRNA ex-
pression. 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 an-
tibody. Cells transfected with p53 cDNA, expressed p53 pro-
tein 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 cDNA-
transfected 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 ex-
pression, and these cells partially responded to uPA stimula-
tion 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
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 using32P-labeled uPAR cDNA and32P-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 using32P-labeled uPAR cDNA. Densitometric scanning of individual bands (mean ?
standard deviation) from four independent experiments is shown at the bottom.
5610SHETTY ET AL.MOL. CELL. BIOL.
(P ? 0.01) (Fig. 2A, panel iii). These results support our
hypothesis that p53 directly affects uPAR expression in H1299
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 uPA-
mediated 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 nonspe-
cific 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
Effects of p53 expression on the rate of uPAR mRNA syn-
thesis and degradation. p53 binds to DNA promoter se-
quences and regulates expression of p53 target genes respon-
sible 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
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.
VOL. 27, 2007REGULATION OF UROKINASE RECEPTOR mRNA BY p535611
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
5612 SHETTY ET AL.MOL. CELL. BIOL.
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 transcrip-
tion, 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 regu-
lates 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 ex-
pression. 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 en-
hanced 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 sta-
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
32P-labeled uPAR mRNA CDR but formed a specific RNA-
protein complex with the uPAR mRNA 3?UTR. To determine
the specificity of the p53-uPAR mRNA 3?UTR interaction, we
incubated various amounts (0 to 5 ?g) of rp53 protein with
32P-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 man-
ner, with maximum saturation observed at 2.5 ?g. The calcu-
lated 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 am-
plified by RT-PCR. As shown in Fig. 4A (panel vi), the max-
imal 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 p53-
uPAR mRNA interaction.
We next confirmed the specificity of the p53-uPAR mRNA
3?UTR interaction by cold competition experiments. Preincu-
bation 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
mRNA 3?UTR (Fig. 4B, panel i), whereas addition of the same
amount of unlabeled antisense transcript failed to block p53
binding to the
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 interac-
tion, whereas addition of a molar excess of homopoly(G) ri-
bonucleotide 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.
32P-labeled uPAR mRNA 3?UTR (Fig. 4B,
blotting using an anti-p53 monoclonal antibody. Panel iii, rp53 protein was incubated with32P-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 with32P-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 with32P-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
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 ?g) was incubated with32P-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 T1and analyzed
by gel mobility shift assay. Fp, probe alone. RNase T1,32P-labeled uPAR 3?UTR mRNA predigested with RNase T1before exposure to p53. The
experiments were repeated at least four or five times.
VOL. 27, 2007REGULATION OF UROKINASE RECEPTOR mRNA BY p535613
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 3?UTR
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
32P-labeled deletion transcripts were prepared by in vitro tran-
scription. 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 se-
quence 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 pre-
pared 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 chi-
meric ?-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 ?-glo-
bin–uPAR 3?UTR mRNA with uPA (1 ?g/ml) to inhibit en-
dogenous p53 expression and determined the decay of chi-
meric 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 trans-
fected 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 se-
quence 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 uPA-
mediated 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 apopto-
sis 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 intro-
duction 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).
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 transcrip-
tional activation of target genes involved in cell cycle regula-
tion and apoptosis (6, 11). In most tumor cells the p53 function
is reduced, resulting in unrestricted proliferation. p53 also reg-
ulates cellular fibrinolysis via induction of PAI-1 expression
through promoter transactivation. The PAI-1 promoter exhib-
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
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 com-
plex was analyzed by gel mobility shift assay. The arrow indicates the
uPAR mRNA-p53 protein complex. Lane Fp,
mRNA probe incubated with buffer alone. Data are representative of
those from four independent analyses.
5614 SHETTY ET AL.MOL. CELL. BIOL.
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 restora-
tion 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
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.
VOL. 27, 2007REGULATION OF UROKINASE RECEPTOR mRNA BY p535615
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 sup-
ported by the observation that inhibition of p53 increased
uPAR mRNA stability without any change in the rate of uPAR
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 exis-
tence 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
whereas poly(G) inhibited the interaction, indicating that
32P-labeled uPAR mRNA 3?UTR,
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 ?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 cDNA-
overexpressing p53?/?(Vc) control and p53 cDNA-overexpressing p53?/?(p53) cells. The error bars indicate standard deviations from the mean
5616 SHETTY ET AL.MOL. CELL. BIOL.
p53 binding requires a G-rich unique sequence. The speci-
ficity 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 T1totally abolished the p53-uPAR mRNA
p53 binds to a specific nucleotide sequence on the uPAR
mRNA 3?UTR and regulates its stability. The results of dele-
tion 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 corre-
sponds 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 tran-
script 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 se-
quence 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 in-
creased expression of uPAR occurs in the cells where expres-
sion of wild-type p53 is reduced, delineating a novel regulatory
mechanism. Introduction of p53 reverses cell surface uPAR
expression through destabilization of uPAR mRNA, confirm-
ing the newly recognized function. In all, these studies dem-
onstrate, 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 p53-
deficient lung carcinoma cells. uPA regulates epithelial cell
viability in a dose-dependent fashion, and the process inversely
relates to p53 expression, demonstrating an intricate relation-
ship between uPA-mediated p53 expression, epithelial cell vi-
ability/proliferation, and uPA-mediated uPAR expression. This is
potentially an important mechanism by which uPA can regu-
late 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 prolifera-
tion in these cells in a dose-dependent manner. This process
may play a major role in the pathogenesis of several nonma-
lignant lung diseases where induction of p53 expression, epi-
thelial cell death, and fibrin deposition are the hallmark events
(4, 8, 15, 17). This process is similarly implicated in lung car-
cinoma where cell resistance to chemotherapy depends on
levels of uPA/uPAR (2) and p53 (3).
We previously found that most tumor cells overexpress
uPAR and exhibit highly stable uPAR mRNA (21, 23, 26).
Most malignant cells exhibit enhanced proliferation and resis-
tance 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 dimen-
sion to the p53 functional repertoire and represents a new link
between viability of lung epithelial cells and pericellular pro-
teolysis. This highly coordinated pathway could influence al-
tered uPAR expression and uPAR-dependent derangements
in a range of pathophysiologic situations, including lung injury
This work was supported by National Heart, Lung, and Blood In-
stitute 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 assis-
We have no conflicting financial interests.
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