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INTRODUCTION |
The plasminogen activator system is an important proteolytic
cascade that plays a role in the removal of blood clots from the
circulation and the turnover of a variety of extracellular matrix
proteins (2). The effector enzyme of this system is the powerful
protease plasmin, generated from its inactive precursor plasminogen by
the plasminogen activators, namely urokinase- or tissue type
plasminogen activator (u-PA1
and t-PA). These proteases are themselves regulated by the plasminogen activator inhibitors (PAIs), PAI-1 and PAI-2. PAI-1 effectively inhibits both t-PA and u-PA; however, PAI-2 is widely considered to
modulate u-PA activity in the extracellular compartments and plays a
less important role in regulating t-PA. Although PAI-2 is found as a
secreted glycosylated protein, a more abundant form exists within the
cytosolic compartment (3). The predominant intracellular location of
PAI-2 has fueled much speculation about additional functions for this
inhibitor. Indeed, growing evidence has indicated a role for PAI-2 in
the intracellular events associated with differentiation (4),
proliferation (5), apoptosis (6), and signal transduction (7).
The gene encoding PAI-2 has generated particular interest, not only for
its extracellular and presumed intracellular roles but also because of
its impressive regulatory profile. PAI-2 gene transcription rates are
markedly increased in response to the tumor promoter phorbol
12-myristate 13-acetate (PMA) (8) and the phosphatase inhibitor,
okadaic acid (9). The PAI-2 gene is also one of the most tumor necrosis
factor- (TNF) (10) and lipopolysaccaride (LPS) (11)-responsive genes
described. For the latter, this has been further confirmed by
serial analysis of gene expression analysis of primary human
monocytes, whereby PAI-2 mRNA levels were shown to be increased
105-fold by LPS (12), being the third most LPS-induced transcript
produced in these cells.
Notwithstanding the important contribution of transcriptional control
of PAI-2 expression (13, 14), the role of post-transcriptional regulation of the PAI-2 gene has recently been highlighted (1, 15).
These investigations stemmed from earlier results whereby treatment of
HT-1080 fibrosarcoma cells with a combination of PMA and TNF produced a
50-100-fold increase in PAI-2 gene transcription but a 1500-fold
increase in PAI-2 mRNA over a 24-h period (9). The discrepancy in
the degree of mRNA production was suggestive of inducible
stabilization of PAI-2 mRNA under these conditions. Subsequent
studies have shown that the rate of PAI-2 mRNA decay can indeed be
reduced by phorbol ester (15) and dioxin (16) or increased by
dexamethasone (17).
Functional studies on the 3'-UTR of PAI-2 mRNA led to the
identification of an AU-rich mRNA destabilizing determinant (15). This element provides a binding site for a number of cytoplasmic and
nuclear proteins including HuR (1), an mRNA-stabilizing protein
that can shuttle between the nucleus and the cytoplasm (18, 19).
Although the degree of PAI-2 mRNA instability is influenced by the
AU-rich motif in the 3'-UTR, elements within the coding region and
possibly the 5'-UTR are also likely to contribute to the control of
PAI-2 mRNA stability since the PAI-2 transcripts lacking the 3'-UTR
are still relatively unstable (1). Indeed, functional mRNA
stability determinants have been detected within the coding region of a
growing list of mRNAs including the mRNAs of c-Myc
(20-22), yeast Mat
1 (23-25), vascular endothelial growth factor (VEGF) (26), u-PAR (27) and c-Fos (28, 29).
Here, we have analyzed exons within the PAI-2 coding region for
functional mRNA instability elements. Our findings indicate that
the control of PAI-2 mRNA stability is controlled by
cis-elements located throughout the coding region, most
notably within exon 4, whereas a 28-nt region within this exon provides
a specific binding site for cytoplasmic factors. Hence PAI-2 mRNA
decay is influenced by both coding region instability elements as well as the AU-rich instability element in the 3'-UTR. Of further interest is that the region immediately adjacent to the 5' end of the exon 4-binding site bears homology to mRNA instability elements located within the coding region of five other mRNAs. This suggests that a
common coding region instability motif may be involved in mRNA turnover.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Mouse NIH3T3 cells were cultured in
Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.),
supplemented with 10% (v/v) heat-inactivated fetal calf serum
(HI-FCS), 2 mM glutamine, 50 µg/ml streptomycin, and 50 units/ml penicillin, in a humidified atmosphere at 37 °C with 5%
CO2. For mRNA decay experiments, 4 × 105 NIH3T3 cells were plated onto 10-cm2 dishes
and grown for 24 h in 10% HI-FCS DMEM, washed twice in phosphate-buffered saline solution, serum-starved in 0.5% HI-FCS DMEM
for 48 h prior to stimulation with 15% HI-FCS DMEM. Cells were
then harvested at selected intervals up to 24 h (30).
Probes and Plasmids, c-fos Promoter-driven PAI-2
Constructs--
Plasmid pfos-HGH (30) was kindly provided by Dr.
Gregory Goodall (Hanson Center, Adelaide, Australia). This vector
harbors the human growth hormone gene (HGH) placed under the control of the serum-responsive chicken c-fos promoter as well as the
neomycin resistance gene (see Fig. 1, panel A). Plasmid pfos
was generated by removing the HGH insert from pfos-HGH using the
restriction enzymes HindIII and SacI (1).
Individual or groups of PAI-2 exons were amplified by PCR using the pJ7
PAI-2 cDNA (8) as a template. KpnI and SacI
sites were engineered into the 5' and 3' sites of the PCR products, respectively, to facilitate the ligation into the c-fos HGH
vector. The sizes of the exons are indicated in Fig. 1, panel
A. The primers used for the amplification are provided in Table
I.
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Table I
Oligonucleotides synthesised to amplify individual or groups of exons
of the PAI-2 cDNA
Bold type in uppercase represents restriction sites for KpnI
and SacI. Lowercase extensions represent nucleotides added
to facilitate improved digestion with restriction enzymes.
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Generation of Plasmids for in Vitro Transcription--
DNA
templates for the in vitro transcription of labeled RNAs for
the RNA electrophoretic mobility shift assays (REMSAs, see below) were
prepared. The full-length exon 4 was amplified by PCR from the pJ7
PAI-2 cDNA template and inserted into the KpnI and
HindIII sites of pBluescript (Stratagene). The sequence of the exon 4 sense primer used for this is provided in Table I. The
antisense exon 4 primer is also indicated in Table I but had
HindIII restriction site added at the 5' end, rather than SacI. The generation of the shorter RNA probes containing
the overlapping exon 4 sequences was prepared by annealing
5'-phosphorylated oligonucleotides encompassing the sense and antisense
sequences of exon 4, regions 4A, 4B, and 4C, and then directly inserted into the KpnI and HindIII sites of pBluescript II
KS+ (Stratagene). The orientation of these inserts was
assessed by DNA sequencing. The sequence of the oligonucleotides used
for this are provided in Table II.
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Table II
Oligonucleotides synthesized to prepare overlapping exon 4 RNA probes
Bold type in uppercase represents restriction sites for KpnI
and HindIII.
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Mutagenesis--
The plasmid containing the full-length PAI-2
cDNA driven by the fos promoter (pfos-PAI-2) has been
described previously (1). Removal of exon 4 from the PAI-2 cDNA
using pfos-PAI-2 as a template was performed by site-directed
mutagenesis using the Transformer DNA kit
(CLONTECH). The mutagenic primer designed to delete
exon 4 had the following sequence: PAI-2 exon 4 deletion primer,
5'-CCTGATGCGATTTTGCAGGAATATATTCGACTCTGTC-3'. The selection primer used
to prepare pfos-PAI-2 exon 4 mutant was designed to replace the
BamHI site in pfos-PAI-2 with an EcoRI site
(underlined) as follows: selection primer (pfos-HGH),
5'-CATGTCTGAATTCCGTCGACCTCG-3'. The pfos vector harboring
the PAI-2 mutant cDNA was confirmed by sequencing.
Transfection Studies--
Stable transfection of plasmids into
NIH3T3 cells was performed by calcium phosphate precipitation procedure
(31) using 5 µg of DNA. Transfected clones were selected in medium
supplemented with 600 µg/µl of G-418 (Life Technologies, Inc.), and
resistant colonies (>200) were pooled by trypsinization.
Northern Blot Analyses--
Total RNA was purified from selected
cells as described by Chomczynski and Sacchi (32). Aliquots of 5 µg
of RNA were electrophoresed through 1% agarose gels containing 20%
formaldehyde and subsequently transferred to
Hybond-N+ membranes (Amersham Pharmacia Biotech). The
filters were hybridized with the 32P-labeled DNA probes as
described (33). Membranes were processed by standard techniques and
exposed to Kodak BioMax film (Eastman Kodak Co.) at
80 °C with two
intensifying screens. Signals were quantitated using a Fujix BAS 1000 PhosphorImager or by densitometry using a Linotype-Hell scanner.
The labeled inserts used for hybridization were obtained as follows:
the 1.8-kilobase pair EcoRI cDNA fragment of plasmid pJ7
containing the full-length PAI-2 cDNA (8); the various labeled
PAI-2 exons were prepared by PCR amplifying using plasmid pJ7 as a
template; the 679-bp BamHI/HindIII HGH cDNA
fragment of pfos-HGH containing the human growth hormone cDNA (30);
the 1.2-kilobase pair PstI cDNA fragment of mouse
-actin (34); the 972-bp BamHI/HindIII cDNA
fragment of neomycin from pCI-neo.
In Vitro Transcription and RNA Electrophoretic Mobility Shift
Assays (REMSAs)--
The pBluescript DNA templates used to transcribe
the PAI-2 exon 4 RNA probes were first linearized with
EcoRI. For in vitro transcription, 500 ng of
template was incubated for 2 h at 37 °C in the presence of 50 µCi of [
-32P]UTP (DuPont), 10 µM UTP,
0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 20 units RNase inhibitor (Promega), and 50 units of T3 RNA polymerase. Templates harboring the 29-nt AU-rich element in the 3'-UTR were linearized with XbaI, and labeled RNA was transcribed
in vitro as described above, but using 50 units of T7
polymerase. RNA probes were purified on a 6% polyacrylamide-urea
denaturing gel, eluted in 500 mM
NH4CH3COO, 1 mM EDTA solution
overnight at room temperature, ethanol-precipitated at
80 °C, and
resuspended in water (500 cps/µl).
Unlabeled RNA competitors were also prepared by in vitro
transcription, but using 3 µg of template. The relative
concentrations of the cold RNAs were estimated by ethidium bromide
staining on agarose gels. When used in the binding assays, cold
competitors were preincubated with the protein extracts for 15 min at
room temperature prior to adding the labeled probe. It was estimated that the cold competitor was used at a minimum of 50-1200-fold molar
excess over the labeled probe in the competition experiments (see
figure legends). However, it is difficult to calculate precisely the
fold excess of the cold RNA over the labeled counterpart because of the
different methodologies used during the in vitro
transcription reactions.
To prepare protein extracts for the REMSAs, confluent cells were
collected by trypsinization, washed three times with phosphate-buffered saline, and then lysed for 5 min on ice in 100 µl/106
cells of cytoplasmic extraction buffer (CEB: 10 mM HEPES,
pH 7.1, 3 mM MgCl2, 14 mM KCl,
0.2% Nonidet P-40, 1 mM dithiothreitol, 2 µg/ml
aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). The nuclei were pelleted for 1 min at 1,000 × g at 4 °C, and the supernatant containing the
cytosolic fraction was aliquoted, snap-frozen in liquid nitrogen,
and stored at
80 °C. Nuclear protein extracts were prepared from
nuclei as described previously (35). Protein concentrations were
determined by using the Bio-Rad protein dye reagent.
For the binding assays, 2-4 µg (see figure legends) of protein
extracts were preincubated with 5 µg/µl of heparin (Sigma) in a
total volume of 20 µl, for 10 min at room temperature before addition
of the RNA probe (500 cps). The probe was heated to 65 °C for 5 min
and then cooled on ice before adding to the sample. After a 30-min
incubation at room temperature, samples were treated with 1 unit of
RNase T1 (Roche Molecular Biochemicals) for 10 min at room temperature
and then subjected to electrophoresis through a 5% native
polyacrylamide gel, and protein-RNA complexes were visualized by autoradiography.
REMSA supershift experiments were performed as described (1).
Antibodies (1 µl of 1:2 diluted material) were added to the samples
immediately following the 30-min incubation of the extract with the
labeled RNA and left for 1 h on ice. For supershift experiments, the RNase T1 step was omitted.
Antisense DNA Masking Experiments--
This procedure was
performed as described by Coulis et al. (36). Four
overlapping antisense DNA oligonucleotides were prepared and annealed
to the exon 4A RNA probe that includes the first 50 bp of PAI-2 exon 4 (see Table II). Oligonucleotides 1-3 are 15 nt in length, and
oligonucleotide 4 is 16 nt in length (see Fig. 6, panel A).
The sequence of these oligomers is as follows: oligo 1, 5'-GAATGGATTTTATCT-3'; oligo 2, 5'-GAGCGGAAGGATGAA-3'; oligo 3, 5'-TGCAGAGCTGAGAGAG-3'; and oligo 4, 5'-GTCATCACAGGGTCCTGA-3'. An
unrelated DNA oligonucleotide 5'-GTCATCACAGGGTCCTGA-3' was used as a
negative control. The DNA oligonucleotides were added to give a final
concentration of either 0.1, 1.0, or 10 pmol. Following annealing of
the oligomers to the RNA template, cytoplasmic extracts and heparin
were added as described in the REMSA protocol.
UV-Cross-linking Assays--
UV-cross-linking of RNA cellular
proteins to RNA probes followed by SDS-PAGE was performed as described
(1) with slight modifications. Briefly, following the binding reaction
(REMSA protocol) using 15-20 µg of cytoplasmic extract, samples were digested with 1.0 units of RNase T1 for 10 min at room temperature and
then transferred to microtiter plate wells and placed on ice. Samples
were placed 7 cm from a UV source (Ultra LUM modeUVB-20) and
cross-linked for 15 min. RNase A (Roche Molecular Biochemicals) was
added directly to the wells (final concentration of 100 µg/ml) and
left at 37 °C for 15 min. Samples were transferred to Eppendorf tubes and denatured at 100 °C for 5 min in the presence of 6× SDS-PAGE loading buffer containing dithiothreitol before being resolved
on 10% SDS-PAGE gels under reducing conditions. Gels were dried and
labeled RNA-protein complexes detected by autoradiography. For
competition experiments, higher concentrations of unlabeled RNA were
included in these experiments compared with the REMSAs due to the
higher concentration of protein extract.
Western Blot Assays--
Western blot analysis for PAI-2 antigen
was performed as described previously (15). 50 µg of cytoplasmic
extract was subjected to 10% SDS-polyacrylamide gel electrophoresis
under reducing conditions and blotted onto a polyvinylidene difluoride
membrane. Membranes were initially blocked with TBS-T buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween
20) containing 5% nonfat dry milk for 2 h at room temperature.
Membranes were then washed and incubated with a primary anti-PAI-2
antibody (American Diagnostica) at a final dilution of 1:4000 and
incubated overnight at 4 °C. Finally, membranes were washed in TBS-T
and incubated with the appropriate horseradish peroxidase-conjugated
secondary antibody (1:10,000 dilution) for 1 h at room
temperature. Immunoreactive proteins were detected by the enhanced
chemiluminescence system (ECL reagents, PerkinElmer Life Sciences).
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RESULTS |
PAI-2 mRNA Coding Region Contains Instability Motifs--
The
pfos-HGH mRNA decay system was used to identify mRNA
instability determinants within the PAI-2 coding region. To this end, a
series of individual exons or groups of consecutive exons of the PAI-2
coding region were amplified by PCR and introduced into the 3'-UTR of
the HGH gene (see Fig. 1, panel
A). NIH3T3 cells stably transfected with these constructs were
subjected to serum treatment, and the decay rate of HGH containing
transcripts was determined by Northern blotting and quantitated by
densitometric analyses.

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Fig. 1.
Various exons within PAI-2 mRNA confer
instability to the HGH reporter mRNA. Panel A,
schematic representation of the PAI-2 cDNA showing the exons of
PAI-2 that were amplified by PCR and inserted into the 3'-UTR of the
human growth hormone (HGH) reporter gene driven by the serum-responsive
chicken c-FOS promoter (pfos-HGH). E,
exon. Panel B, mRNA decay curves of HGH-containing
transcripts in NIH3T3 cells stably transfected with pfos-HGH alone
(HGH) or containing the following PAI-2 exons inserted in to the 3'-UTR
of the HGH gene: exon 2, 2-3, 2-4, 2-5, and
2-6 (as indicated in the figure). For these experiments,
cells were treated with 15% FCS, and RNA was harvested at the
indicated time points and assessed for HGH transcripts by Northern blot
analysis using a cDNA probe specific for HGH. Signals were
quantitated by densitometric analysis, and results were expressed
relative to the maximal signal obtained. Results presented are the mean
data of 3 or 4 separate experiments. Bars represent
S.E.
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As shown in Fig. 1, panel B, serum treatment of cells
transfected with the parent pfos-HGH plasmid produced a stable HGH
transcript that displayed a half-life in excess of 3 h. Insertion
of exon 2 alone into the 3'-UTR of HGH mRNA slightly increased the
decay rate of the HGH reporter transcript (half-life 21/2 h).
However, insertion of a fragment containing exon 2 + 3 together did not alter the decay rate of the chimeric HGH mRNA. These results
suggest that exon 2 possesses instability determinants that are
counteracted by sequences present within exon 3. Interestingly, HGH
transcripts containing PAI-2 exons 2 + 3 + 4 were particularly
unstable, with the half-life of the chimeric transcript reduced to less
than 1 h. These data suggests that exon 4 possesses particularly
powerful destabilizing elements. Curiously, longer chimeric transcripts containing exons 2-5 or exons 2-6, although promoting destabilization of the reporter transcript (mRNA half-lives: 11/2 and
21/2 h, respectively), were not as effective as exons 2-4
alone. This also suggests that sequences within exon 5 or 6 contained
motifs that counteract the instability elements present in exon 4. Insertion of fragments containing exons 7 and 8 into the 3'-UTR of the
pfos-HGH plasmid conferred a destabilizing effect upon the reporter
transcript to an extent similar to that produced by exons 2-4 (data
not shown). Taken together, these results suggest that instability and
stability determinants are located throughout the PAI-2 coding region,
with powerful instability elements associated with the presence of exon
4 and also within exons 7 and 8. In this study, we focused our efforts
to assess the role of exon 4 in the control of PAI-2 mRNA
stability. The instability elements within exons 7 and 8 will be
investigated in a separate study.
To assess the stability of exon 4 in isolation, the entire exon 4 sequence was introduced into the 3'-UTR of HGH and the mRNA half-life determined. As shown in the Northern blot experiment presented in Fig. 2 (panel A),
HGH-exon 4 chimeric transcripts were induced after 1 h of serum
treatment, but decayed very rapidly with the signal barely detectable
after 2 h. Quantitation of the mRNA signals indicated that the
half-life of the exon 4 containing transcript was ~30 min
(panel B). The observation that exon 4 in isolation produced
greater instability to the reporter transcript than seen in the context
of other exons further supports the notion that mRNA stability
determinants exist within neighboring exons to counteract partially the
effects of the destabilizing elements in exon 4.

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Fig. 2.
Exon 4 of PAI-2 mRNA confers marked
instability to the HGH-reporter transcript. Panel A,
NIH3T3 cells stably transfected with c-fos-HGH alone or the
same plasmid containing the complete exon 4 sequence inserted into the
3'-UTR of the HGH gene (HGH exon 4) were subjected to serum
treatment, and HGH transcripts were assessed by Northern blot analysis.
The same filter was stripped and re-hybridized for -actin mRNA.
Panel B, mRNA decay curves of HGH-containing transcripts
in NIH3T3 cells stably transfected with pfos-HGH alone (HGH,
dotted line) or containing exon 4 inserted into the 3'-UTR
of the HGH gene (HGH-exon 4, solid line). Results
presented are the mean data of four separate experiments. Signals were
quantitated by densitometric analysis, and results were expressed
relative to the maximal signal obtained. Bars represent
S.E.
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Removal of Exon 4 from the PAI-2 cDNA Results in a Doubling of
PAI-2 mRNA Stability--
To provide more evidence that sequences
within exon 4 play a role in PAI-2 mRNA stability, the 129-bp exon
4 was deleted in-frame from the full-length PAI-2 cDNA using
plasmid pfos-PAI-2 as a template (Fig. 3,
panel A). The resulting construct (pfos-PAI-2
4) as well
as the construct containing the full-length wild-type PAI-2 cDNA
were stably transfected into NIH3T3 cells. Two independent series of
transfection experiments were performed. The collective results of two
individual serum time courses and Northern blot experiments of both
series of transfected cells indicated that the mRNA half-life of
the wild-type PAI-2 transcript to be ~1 h, which is in agreement with
previous results (1). However, the half-life of the mutant PAI-2
transcript was extended to 2 h, double that of its wild-type
counterpart. These data indicate that sequences within exon 4 promote
PAI-2 mRNA instability.

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Fig. 3.
Deletion of exon 4 from the full-length PAI-2
transcript doubles the half-life of PAI-2 mRNA. Panel
A, schematic representation of the full-length PAI-2 construct
(wild-type PAI-2) and the mutant construct containing an
in-frame deletion of exon 4 (Mutant PAI-2). The PAI-2
constructs were introduced into plasmid pfos as described under
"Experimental Procedures." E, exon. Panel
B, the decay rate of PAI-2 mRNA in NIH3T3 cells
transfected with either pfos-PAI-2 (wild-type PAI-2, solid
line) or PAI-2 exon 4 (Mutant PAI-2, dotted
line) was assessed by Northern blot analysis following a serum
time course up to 3.5 h. The intensity of the signals was
quantitated by densitometric analysis. The data shown were derived from
two independent transfection experiments with each series of
transfected cells being assessed at least two times (n = 4). Data for each experiment was expressed as a percentage of the
maximal mRNA signal obtained following serum treatment. The
half-life of the wild-type PAI-2 transcript was calculated to be 1 h, whereas the half-life of the exon 4-deleted transcript was extended
to ~2 h.
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PAI-2 Exon 4 Provides a Specific Binding Site for Cytoplasmic
Proteins--
Experiments were conducted to determine whether regions
within exon 4 provided binding sites for cellular factors. To this end,
RNA probes containing the full-length (129 nt) exon 4 sequence were
incubated with cytoplasmic extracts prepared from both NIH3T3 cells and
HT-1080 fibrosarcoma cells, and binding activity was assessed by REMSA
analysis. HT-1080 cells were utilized in these experiments because
these cells express the endogenous PAI-2 gene and are widely used to
study PAI-2 gene regulation. As shown in Fig.
4, numerous protein-RNA complexes
were formed on the exon 4 probe that displayed different migration
profiles on the native gel (lane 1). To determine the
specificity of these interactions, competition experiments were
performed whereby increasing concentrations of unlabeled RNA of
identical (lanes 2-4) or unrelated sequence (lanes
5-7) were added to the samples at the same time as the exon 4 probe. Results indicated that one of the faster migrating complexes was
indeed specific (indicated with arrow) as formation of this
complex was competed by increasing concentrations of unlabeled exon 4 RNA (lanes 2-4) but not by unrelated RNA (lanes
5-7). Similar experiments performed using NIH3T3 cells produced
essentially identical results (data not shown).

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Fig. 4.
Cytoplasmic proteins specifically recognize
sequences within exon 4 of PAI-2 mRNA. Cytoplasmic extracts
prepared from HT-1080 cells (4 µg) were incubated with RNA probes
containing the full-length 129-nt exon 4 sequence, and binding was
assessed by REMSA. Up to three complexes are produced in the absence of
competitor (0) (lane 1), but only the central migrating
complex (arrow) is specific as this was competed by addition
of increasing concentrations (~50-, 250-, and 500-fold molar excess)
of unlabeled exon 4 RNA (lanes 2-4, respectively) but not
by addition of similar concentrations of an unrelated RNA (lanes
5-7).
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The First 52 nt of Exon 4 Provide Protein-binding Sites for
Cytoplasmic Proteins--
To localize further the protein binding
region within the 129-nt exon 4 sequence, a series of REMSAs was
performed using shorter RNA probes. Three overlapping RNA probes
spanning the exon 4 sequence were generated (regions A (52 nt), B (52 nt), and C (51 nt); see Fig. 5,
panel A) and used in a REMSA assay using cytoplasmic
proteins prepared from HT-1080 cells.

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Fig. 5.
The first 52 nt of exon 4 provides binding
sites for cellular factors. Panel A, schematic
representation of the overlapping RNA probes (4A, 4B, and
4C) used to localize the protein-binding site(s) within the
129-nt PAI-2 exon 4 sequence. These sequences were introduced into
pBluescript, and RNA from linearized plasmids was transcribed in
vitro using T3 polymerase. Panel B, REMSA experiments
were performed using the RNA probe harboring the first 52 nt of exon 4 (4A probe). Incubation of cytoplasmic extracts with the 4A
probe produced two specific complexes (I and II,
indicated with arrows). The formation of these complexes was
competed with increasing concentrations (~50-, 250-, and 500-fold
molar excess) of unlabeled 4A RNA (lanes 3-5,
respectively), but not with similar concentrations of unlabeled 4B
(lanes 6-8), 4C (lanes 9-11), or an unrelated
RNA (lanes 12-14). No specific complex formation was
produced when using the 4B or 4C RNA sequences as probes (data not
shown).
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As shown in Fig. 5, panel B, the first 52 nt of exon 4 (exon
4A) produced three protein RNA complexes (lane 2).
Competition experiments confirmed that the two fastest migrating
complexes (referred to as complexes I and II) were specific as only the unlabeled 4A RNA sequence competed for binding (lanes 3-5).
Binding was not competed by cold RNA sequences harboring the 4B and 4C region of exon 4 (lanes 6-11) or by RNA of unrelated (97-nt
prothrombin 3'-UTR) sequence (lanes 12-14). No specific
binding of cytoplasmic proteins was observed to either the 4B or 4C
probes (data not shown). To confirm further that the first 52 nt of
exon 4 contained the protein-binding elements, competition REMSA
experiments were performed using the full-length exon 4 sequence as a
probe. Complexes produced using the full-length exon 4 probe were
competed by the unlabeled 4A RNA but not at all by unlabeled 4B or 4C
competitors (data not shown).
Protein-binding Sites Reside within a 28-nt Region of Exon
4--
To define more closely the binding sites for the proteins that
bind to the first 52 nt of exon 4, an antisense DNA oligonucleotide masking experiment was performed as described by Coulis et
al. (36). In this approach, antisense DNA oligomers directed
against overlapping sequences within the 4A RNA probe (see Fig.
6, panel A) were allowed to
anneal to the RNA probe before the addition of the cell extracts. As
shown in Fig. 6 (panel B), the formation of complexes I and
II was not at all inhibited by antisense oligomers complementary to the
first 15 nt (oligo 1; lanes 3-5). Antisense oligo 2 caused inhibition only at the highest concentration used (10 pmol) (lane 8), whereas antisense oligo 3 completely
inhibited complex formation (lanes 9-11), even at the
lowest concentration used (0.1 pmol). Antisense oligo 4 also
substantially inhibited complex formation (lanes 12-14) and
was essentially as effective as oligo 3 at inhibiting complex
formation. As a negative control, antisense oligomers of unrelated
sequence had no effect on binding activity (lane 15) at a
concentration of 10 pmol. Since oligonucleotides 3 and 4 correspond to
the last 28 nt of the 4A probe, these date indicate that these 28 residues contain sequences necessary for the formation of complexes I
and II. This corresponds to residues 344-371 of the PAI-2 J7 cDNA
(numbering based on the sequence published by Schleuning et
al. (8)).

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Fig. 6.
Fine map localization of the protein-binding
sites within exon 4 of PAI-2 mRNA. Panel A,
schematic representation of the relative position of the four antisense
oligonucleotides (Oligo 1-4), complementary to the
sequences within region 4A of PAI-2 exon 4, used to perform the
oligonucleotide masking experiments. Panel B, RNA probes
containing the 4A region of exon 4 were incubated with increasing
concentrations of antisense oligonucleotides (Oligo) 1-4. HT-1080
cytoplasmic extracts were added, and binding activity was assessed by
REMSA analysis. Formation of complex I and II was not inhibited by
inclusion of increasing concentrations (0.1, 1.0, or 10 pmol) of
antisense oligo 1 (lanes 3-5, respectively). Antisense
oligo 2 did not inhibit complex formation at the lower concentrations
(0.1 and 1.0 pmol; lanes 6 and 7) but did inhibit
when included at the highest concentration (10 pmol, lane
8). Antisense oligo 3 (lanes 9-11) and oligo 4 (lanes 12-14) inhibited complex formation even at the
lowest concentrations used. Addition of 10 pmol of antisense
oligonucleotide of unrelated sequence (UR) did not inhibit
complex formation (lane 15).
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Proteins of 50-52 kDa Recognize Exon 4--
To assess the
molecular weight of the proteins binding to exon 4, UV cross-linking
and SDS-PAGE studies were performed using the 52-nt 4A RNA probe.
HT-1080 cytoplasmic extracts were incubated with the labeled 4A RNA and
run on a 10% SDS-PAGE along with molecular weight markers (Life
Technologies, Inc.). As shown in Fig. 7, two distinct closely spaced proteins (indicated by arrows)
were detected that migrated with an apparent molecular mass of
~50-52 kDa (lane 2). To assess the specificity of the
complexes binding to the probe, unlabeled RNA of the identical sequence
to the labeled probe and unlabeled RNA identical to the last 51 nt of
exon 4 (i.e. exon 4C RNA) were used as competitors. As
indicated in the figure, the formation of the 50-52-kDa proteins was
competed by increasing concentrations of the unlabeled 4A sequence
(lanes 3-5) but not by the 4C sequence (lanes
6-8).

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Fig. 7.
Proteins with an apparent molecular mass of
50-52 kDa associate with exon 4 of PAI-2 mRNA. Cytoplasmic
extracts (15 µg) prepared from HT-1080 cells were incubated with an
RNA probe harboring the first 52 nt of exon 4 (probe 4A).
Samples were UV cross-linked and then subjected to SDS-PAGE, and
labeled protein-RNA complexes were visualized after exposure to x-ray
film. Protein-RNA complexes with apparent molecular masses of 50-52
kDa were produced with cytoplasmic extracts in the absence of
competitor (0, lane 2). The formation of the
cross-linked proteins was reduced in the presence of increasing
concentrations (~100-, 400-, and 1200-fold molar excess) of unlabeled
4A RNA (lanes 3-5, respectively) but not by similar
concentrations of unlabeled 4C RNA (lanes 6-8).
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Effect of PMA and TNF Treatment on the Binding Activity of Proteins
Binding to Exon 4 and the AU-rich Motif in the 3'-UTR--
PAI-2
mRNA stability in HT-1080 cells is regulated by PMA and TNF
treatment. To confirm and extend these findings, HT-1080 cells were
treated with PMA + TNF for various times up to 24 h, and changes
in PAI-2 mRNA and antigen levels were assessed by Northern and
Western blotting, respectively. As shown in Fig. 8, panel A, PAI-2 mRNA
levels were markedly increased by PMA + TNF treatment, with an increase
in levels apparent after 2 h and maximal levels after 24 h
treatment. Western blot analysis of cytoplasmic extracts prepared from
the same cells further demonstrated the marked increase in endogenous
PAI-2 antigen as a result of this treatment.

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Fig. 8.
Treatment of HT-1080 cells with PMA + TNF
causes a marked increase in PAI-2 mRNA and antigen.
Panel A, HT-1080 cells were treated with PMA + TNF for 0, 1, 2, 4, 8, or 24 h. Cells were harvested, and PAI-2 mRNA levels
were assessed by Northern blot analysis using a PAI-2 cDNA probe.
Ethidium bromide staining of the agarose gel is shown below
the image. Panel B, cytoplasmic extracts (50 µg) prepared
from HT-1080 cells treated with PMA+TNF were subjected to SDS-PAGE.
PAI-2 antigen levels were assessed by Western blot analysis using an
anti-PAI-2 monoclonal antibody.
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Experiments were then performed to determine whether the time course of
PAI-2 mRNA induction by PMA + TNF could be correlated with changes
in the binding profiles or activities of proteins associating with
PAI-2 mRNA instability sequences. To this end, REMSAs were
performed using labeled RNA probes specific for the 4A sequence and the
29-nt region within the PAI-2 3'-UTR that contains the nonameric
AU-rich instability motif and cytoplasmic extracts prepared from cells
treated with PMA + TNF for 1, 2, 4, 8 or 24 h. The AU-rich motif
in the 3'-UTR is known to promote PAI-2 mRNA instability and to
provide a binding site for cellular factors (1). As shown in Fig.
9 (panel A), PMA + TNF
treatment did not alter the intensity of any of the complexes
associating with the 4A sequence (lanes 2-7). In contrast,
a transient increase in the intensity of one of the complexes formed
with the AU-rich motif was observed following PMA + TNF treatment
(lanes 9-14). This complex was only weakly detected under
nontreated conditions but was clearly increased after 1 h and
decreased to basal levels after 8 h of treatment (indicated with
arrow). This was observed on three independent occasions
using extracts prepared from different preparations of HT-1080 cells
treated with PMA + TNF.

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Fig. 9.
Treatment of HT-1080 cells with PMA and TNF
does not alter the formation of complexes on PAI-2 exon 4 but causes a
transient increase in the binding of proteins to the AU-rich
instability motif in the 3'-UTR. Cytoplasmic extracts were
prepared from HT-1080 cells treated with PMA + TNF for 0, 1, 2, 4, 8, and 24 h (as indicated), and protein binding activity to labeled
PAI-2 exon 4 (probe 4A) and the 29-nt AU-rich mRNA instability
sequence present within the 3'-UTR of PAI-2 mRNA were assessed by
REMSA. No change in the migration pattern or intensity of complexes
associating with the exon 4 sequence was observed at any time point
(lanes 2-7). However, a transient increase in the intensity
of a complex formed with the AU-rich probe is seen following PMA + TNF
treatment (lanes 9-14). The level of intensity of this
transiently induced complex is maximal after 2 h of treatment with
PMA + TNF (lane 11). The position of the induced complex is
shown by the arrow to the left of lane 9. No Ext, no extract added. Lanes 15 and 16, extracts prepared from 24-h nontreated HT-1080 cells were used in a
REMSA supershift using an antibody directed against HuR. The position
of the HuR containing complex is indicated by the solid
arrow next to lane 16. The supershifted complexes are
visible as faint slow migrating complexes (indicated with dotted
arrows at the top of lane 16). The increase
in intensity of the complexes shown in lanes 15 and
16 is due to the omission of RNase T1 (see "Experimental
Procedures"). Ab, antibody.
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The mRNA-stabilizing protein, HuR, is known to recognize the
AU-rich instability element in the 3'-UTR. As confirmed in this figure,
HuR is associated with the fastest migrating complex formed with the
AU-rich RNA probe, as the inclusion of anti-HuR antibodies fully
displaced the fastest migrating complex (lane 16, indicated with arrow). The intensity of the HuR containing complex
did, however, appear to be slightly increased by treatment with these agents, but this was not a consistent observation. Taken together, inducible stabilization of PAI-2 mRNA by PMA + TNF is associated with a transient increase in the binding activity of cytoplasmic proteins to the AU-rich instability element in the 3'-UTR of PAI-2 mRNA.
Sequences Flanking the Exon 4-Binding Site Are Homologous to Coding
Region Instability Elements in Other mRNAs--
The protein(s)
interacting with exon 4 remain to be identified. However, it is
intriguing to point out that a significant degree of sequence homology
exists between a 10-nt A-rich region (GATAAAATCC) that partially
overlaps (3 nt) and the protein-binding site in exon 4, with sequences
within the mRNAs of yeast MAT
1 (23-25), c-Myc (20-22), uPAR
(27), VEGF (26), and TFIIIA (41) (Fig.
10). For MAT
1, c-Myc, and uPAR,
this area of homology also occurs within known or suspected binding
sites for proteins associated with mRNA instability. The region of
homology with MAT
1 is found within a 19-nt region of MAT
1
mRNA instability determinant that is postulated to provide a
protein-binding site (25) (boxed sequence in Fig. 10). The
region of similarity with c-Myc mRNA also partially overlaps a
15-nt protein-binding site for the coding region determinant binding
protein (CRD-BP) (36) (Fig. 10). Significant homology exists within the
uPAR, and part of this sequence also overlaps with the beginning of the
51-nt instability region of the uPAR transcript that provides a binding
site for a 50-kDa protein (27, 37). Although VEGF mRNA possesses
instability determinants throughout the transcript (26), no specific
protein-binding sites have been identified. Nonetheless, the region of
homology of the PAI-2 exon 4 motif with the VEGF mRNA occurs within
the splice site for exons 5 and 7 of the VEGF 164 gene (38) (indicated with arrow in Fig. 10).

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Fig. 10.
A region flanking the exon 4-binding site
bears homology with sequences present within instability determinants
of other transcripts. Alignment of sequences present within exon 4 of PAI-2 mRNA, with known or suspected instability determinants
with yeast MAT 1, c-Myc, uPAR, VEGF and TFIIIA mRNA. A 10-nt
region immediately upstream of the PAI-2 exon 4-binding site with
homology to sequences in other transcripts is shown within the
shaded area. Residues with identity to PAI-2 exon 4 are
indicated with dashes. The protein-binding site within exon
4 is indicated with the double-headed arrow above the exon 4 sequence. The tandem 19-nt near-repeat sequences within MAT 1 that
are thought to provide a binding site for proteins are
boxed. Similarly, known protein-binding sites within the
coding region instability determinants of c-Myc and uPAR are also
boxed. The arrow within the shaded area
below the VEGF sequence indicates the splice site between exons 5 and 7 of the VEGF gene.
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The area of homology found within the TFIIIA transcript (position
480-489) is found within a 528-nt sequence of the coding region
(position 342-870) that is associated with TFIIIA mRNA instability
(41). Interestingly, two additional related motifs are also found in
this region; positions 529-538 (CACAAAATCA) and 638-647 (CTGAAAATCC).
Finally, it is interesting to note that where there is sequence
divergence in some of these mRNAs to the PAI-2 sequence, the substituted residue is highly conserved. The presence of a homologous motif within the coding region of six transcripts, each of which possess coding region instability determinants, suggests that a common
coding region element plays a role in the regulation of mRNA decay.
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DISCUSSION |
PAI-2 is an impressively regulated serine protease inhibitor.
Numerous in vitro systems have demonstrated that the PAI-2
gene is increased in many cell types by inflammatory mediators
including TNF and LPS. Although many earlier studies focused on the
transcriptional mechanisms underlying the induction of the PAI-2 gene,
it is now well established that the post-transcriptional events
controlling PAI-2 expression are very important. Indeed,
post-transcriptional mechanisms account for most of the increase in
PAI-2 expression in response to TNF and PMA treatment in HT-1080 cells
(1, 15). PAI-2 mRNA is inherently unstable displaying a half-life
of 1 h, and part of this instability is attributed to an AU-rich
element located within the 3'-UTR that provides a binding site for HuR which is mainly nuclear in location.
We have extended our study on the post-transcriptional regulation of
the PAI-2 gene and evaluated the potential mRNA-destabilizing properties of sequences within the coding region of PAI-2 mRNA. Instability elements elsewhere within the PAI-2 transcript were suspected to exist since the PAI-2 transcript was still relatively unstable in the absence of the 3'-UTR (1).
By using the c-fos-HGH mRNA stability system, we have
been able to demonstrate that individual exons or groups of exons of the PAI-2 gene could confer varying degrees of instability to the HGH
reporter transcript, suggesting that multiple instability elements reside within PAI-2 mRNA, including exons 4, 7, and 8. We focused our efforts on the role of exon 4 in the regulation of PAI-2
mRNA stability as this exon conferred the most potent destabilizing
effects upon the HGH reporter transcript, reducing the half-life of the
chimeric HGH transcript to ~30 min. Instability elements within exons
7 and 8 were not pursued further in this present study.
To provide further evidence for a role of sequences within exon 4 in
the regulation of PAI-2 mRNA stability, an in-frame deletion of
exon 4 resulted in a doubling of the PAI-2 mRNA half-life. Although
this evidence is reasonably strong, we cannot exclude the possibility
that the removal of an entire exon disrupts the secondary structure of
PAI-2 mRNA in such a way as to extend the half-life of the PAI-2
transcript. Since fine mapping REMSA and DNA masking studies have
mapped the protein-binding site to a 28-nt region within exon 4 (residues 344-371 of the PAI-2 transcript), it would be interesting to
assess the decay rate of the PAI-2 transcript that contained a series
of point mutations within this particular region.
UV cross-linking and SDS-PAGE experiments revealed that protein-RNA
complexes with an apparent molecular mass of 50-52 kDa are formed when
cytoplasmic extracts are incubated with RNA probes containing the first
52 nt of exon 4. The identity of this protein(s) and their role in the
implementation of mRNA instability remain to be determined.
PAI-2 mRNA is markedly induced in HT-1080 cells treated with either
TNF or PMA. Transcriptional increase accounts for about 10-20% of
this induction suggesting that the major level of PAI-2 induction under
these conditions is post-transcriptional. However, we could find no
evidence to indicate that the binding of factors to the exon 4 instability element was altered by treatment with these agents. In
contrast, we observed a transient increase in the binding activity of
an inducible subset of cytoplasmic protein(s) that recognized the
AU-rich motif in the 3'-UTR. This provides circumstantial evidence that
these transiently induced proteins play a role in stabilizing PAI-2
mRNA during induction with PMA + TNF. The identity of these
protein(s) and a more detailed assessment of their role in inducible
regulation of PAI-2 mRNA requires further study. To date, the only
known protein to recognize the AU-rich element in the 3'-UTR is HuR.
Although HuR resides mainly in the nuclear compartment, a significant
amount of HuR is present in the cytoplasm of HT-1080 cells, yet we saw
no convincing evidence that the binding activity of HuR altered during
treatment of cells with PMA + TNF.
The literature describing the functional coding region mRNA
instability determinants in post-transcriptional regulation is rapidly
expanding. Indeed the mRNAs for
-tubulin (39, 40), TFIIIA (41),
c-Myc (20-22), yeast Mat
1 (23-25), vascular endothelial growth
factor (VEGF) (26), urokinase receptor (27, 37) and c-fos
(28, 29) all contain coding region instability sequences. Furthermore,
some of these contain binding sites for cellular factors. The mechanism
by which these coding region mRNA stability/instability determinants influence mRNA decay is obscure. However, one of the
best-studied coding region instability determinants (CRD) is found in
the c-Myc transcript (20-22, 42). In this example, a 70-kDa
CRD-binding protein (CRD-BP) binds to a region within the last 180 bp
of the c-Myc coding region (36, 42, 43) that is proposed to shield the
coding region determinant from a ribosome-associated endonuclease (44,
45). Binding of the CRD-BP to this 180 nt was inhibited by a 15-nt DNA
oligonucleotide that corresponded to positions 1763-1777 of the c-Myc
transcript. It was noted, however, that the binding region may extend
beyond these positions (23). The yeast gene MAT
1 also contains a 65-nt instability determinant within its coding region (23, 24), and
two near-identical 19-nt regions within this determinant have been
postulated to provide a recognition site(s) for an RNA-binding protein(s) (25). The urokinase receptor (uPAR) mRNA contains a
51-nt instability determinant with the coding region (27) that provides
a binding site for a 50-kDa protein (37).
We observed a significant degree of sequence homology between a 10-nt
region within exon 4 with the sequence of CRDs in five other
transcripts as follows: Mat
1, c-Myc, uPAR, VEGF, and TFIIIA (Fig.
10). This 10-nt region is also perfectly conserved in the mouse PAI-2
mRNA sequence. This suggests a common coding region motif may play
a broad role in the control of mRNA turnover. Whether this region
provides a binding site for a common factor and/or facilitates binding
of proteins to neighboring sites remains to be determined. We are
presently performing competition experiments to determine whether the
homologous sequences within the above-mentioned transcripts influence
the binding of proteins to the PAI-2 exon 4-binding site.
In summary, we describe for the first time an mRNA instability
determinant within exon 4 of the PAI-2 mRNA that provides a binding
site for cellular factors with an apparent molecular mass of 50-52
kDa. Based on these results, we suggest that both the PAI-2 exon 4 instability element and the AU-rich element in the 3'-UTR play a role
in destabilizing PAI-2 mRNA under constitutive conditions. However,
a change in the assembly of proteins binding to the AU-rich motif
occurs during treatment with PMA + TNF suggesting that these proteins
play a role during inducible PAI-2 mRNA stabilization.
Finally, the region immediately upstream and partially overlapping the
exon 4-binding site is homologous to coding region instability
determinants of five other transcripts, suggesting the presence of a
common element that may play a broad role in the post-transcriptional
regulation of mRNA.