Transactivation of the Urokinase-type Plasminogen Activator
Receptor Gene through a Novel Promoter Motif Bound with an
Activator Protein-2
-related Factor*
Heike
Allgayer,
Heng
Wang,
Yao
Wang
,
Markus M.
Heiss§,
Reinhard
Bauer¶,
Okot
Nyormoi, and
Douglas
Boyd
From the Department of Cancer Biology and ¶ Department of
Biochemistry, M.D. Anderson Cancer Center, Houston, Texas 77030, the
Department of Renal Medicine, Westmead Hospital,
Westmead, University of Sydney, Westmead, New South Wales, Australia,
and the § Department of Surgery, Klinikum Grosshadern,
Ludwig-Maximilians University, D-81377 Munich, Germany
 |
ABSTRACT |
The urokinase receptor overexpressed in invasive
cancers promotes laminin degradation. The current study was undertaken
to identify cis elements and trans-acting
factors activating urokinase receptor expression through a footprinted
(
148/
124) region of the promoter containing putative activator
protein-2- and Sp1-binding motifs. Mobility shifting experiments using
nuclear extract from a high urokinase receptor-expressing cell line
(RKO) indicated that Sp1, Sp3, and a factor similar to, but distinct
from, activator protein-2
bound to this region. Mutations preventing
the binding of the activator protein 2
-related factor diminished
urokinase receptor promoter activity. In RKO cells, the expression of a negative regulator of activator protein-2 function diminished urokinase
receptor promoter activity, protein, and laminin degradation. Conversely, urokinase receptor promoter activity in low urokinase receptor-expressing GEO cells was increased by activator protein-2
A expression. Although using GEO nuclear extract, little activator protein-2
-related factor bound to the footprinted region, phorbol 12-myristate 13-acetate treatment, which induces urokinase receptor expression, increased complex formation. Mutations preventing the
activator protein-2
-related factor and Sp1/Sp3 binding reduced urokinase receptor promoter stimulation by this agent. Thus, the constitutive and phorbol 12-myristate 13-acetate-inducible expression of the urokinase receptor is mediated partly through
trans-activation of the promoter via a sequence
(
152/
135) bound with an activator protein-2
-related factor.
 |
INTRODUCTION |
The urokinase-type plasminogen activator (urokinase) is a serine
protease that converts the inert zymogen plasminogen into plasmin, a
protease with broad substrate specificity leading to extracellular
matrix degradation and tumor invasion (1-3). Urokinase can bind
specifically and with high affinity (KD ~0.5 nM) to a 45-60-kDa heavily glycosylated cell surface
receptor (u-PAR)1 (4, 5)
composed of three similar repeats approximately 90 residues each (6,
7). The amino-terminal domain binds the plasminogen activator with the
carboxyl terminus domain serving to anchor the binding protein to the
cell surface via a glycosyl-phosphatidylinositol chain (6, 7).
The u-PAR has multiple functions. First, urokinase bound to the u-PAR
activates plasminogen at a much faster rate than fluid phase
plasminogen activator (8, 9), and this contributes to type IV
collagenase activation (10). Second, the binding site clears
urokinase-inhibitor complexes from the extracellular space (11) via a
2-macroglobulin receptor-dependent mechanism (12). Third, the u-PAR interacts with the extracellular domain of
integrins to connect to the cytoskeleton, thereby mediating cell
adhesion and migration (13-15). Fourth, the u-PAR is chemotactic for
human monocytes and mast cells, and this may require the cleavage of
the binding site between domains 1 and 2 (16, 17).
The u-PAR gene is 7 exons long and is located on chromosome 19q13 (18,
19). Transcription of the u-PAR gene yields a 1.4-kilobase mRNA or
an alternatively spliced variant lacking the membrane attachment
peptide sequence (20, 21). The amounts of u-PAR are controlled mainly
at the transcriptional level, but altered message stability and
receptor recycling may represent other means of controlling the amount
of this gene product at the cell surface (22-25).
The transcriptional regulation of the u-PAR gene is still poorly
understood. Soravia et al. (26) reported that the basal expression of the gene was regulated via Sp1 motifs proximal and upstream of the transcriptional start site. Our laboratory showed that
both the constitutive and PMA-inducible expression of the gene required
a footprinted region (
190/
171) of the promoter containing an AP-1
motif (22). We also observed a second footprinted region of the
promoter (
148/
124), and deletion of this region caused a dramatic
reduction in the constitutive u-PAR promoter activity in a colon cancer
cell line characterized by its high expression of this gene.
Interestingly, this region of the promoter contained noncanonical AP-2
(
142/
134) and Sp1 (
147/
138) motifs overlapping with each other
as well as nonconsensus polyomavirus activator 3 (
133/
127) motifs.
The u-PAR has been implicated in a number of physiological and
pathological processes involving tissue remodeling, although it is not
critical for mouse development (27). In cancer, several experimental
and clinical findings support the view that the u-PAR plays a prominent
role in tumor cell invasion and metastasis. For example, the u-PAR
mRNA is expressed in the tumor cells of invasive colon cancers (28,
29), and a high u-PAR protein level is predictive of short survival
times for patients with this disease (30). Further, earlier studies
have shown that the overexpression of a human u-PAR cDNA increased
the ability of human osteosarcoma cells to invade into an extracellular
matrix-coated porous filter (31). Conversely, down-regulating u-PAR
levels using antisense expression constructs, oligonucleotides, or
synthetic compounds reduced the ability of divergent invasive cancers
to invade in vitro and in vivo (32-36). Since
the u-PAR is a key factor in promoting tumor-associated proteolysis,
down-regulation of its expression could be a promising strategy for
inhibiting cancer invasion and metastasis. We therefore undertook a
study with two objectives: (a) to identify
cis-elements and trans-acting factors regulating
constitutive and PMA-inducible u-PAR gene expression via the
footprinted region spanning nucleotides
148/
124 and (b)
to determine the effect of interfering with transcription factors
binding to this region on u-PAR-directed laminin degradation.
 |
EXPERIMENTAL PROCEDURES |
Vectors and Antibodies--
The u-PAR CAT reporter consisted of
449 base pairs of sequence (37) stretching from
398 to +51 (relative
to the transcription start site) cloned into the XbaI site
of the pCAT-Basic vector (Promega, Madison, WI). Reporter constructs
regulated by truncated u-PAR promoter fragments were as described
previously (22). The urokinase CAT reporter consisted of 2345 base
pairs of 5'-flanking region fused directly to the reporter (38).
Antibodies to Sp1, Sp2, Sp3, and AP-2 isoforms were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Jun-D expression
construct was described elsewhere (22). Oligonucleotides were purchased
from Genosys Biotechnologies (The Woodlands, TX). Recombinant AP-2
A
and Sp1 (full-length human proteins) were obtained from Promega
(Madison, WI). Expression vectors for AP-2
A, AP-2
B, and AP-2
antisense (39) consisted of the cloned sequences inserted into the
EcoRI site of pSG5 (Stratagene, La Jolla, CA) and were
kindly provided by Dr. Michael Tainsky. The AP-2 pBLCAT2 reporter
construct contained three consensus AP-2 motifs 5' of the pBLCAT2
reporter (40). For the generation of the R2 CAT reporter construct, an
oligonucleotide spanning nucleotides
154/
128 was cloned into the
XbaI site of pCATbasic (Promega).
Preparation of Nuclear Extracts and EMSA--
Nuclear extracts
and EMSA were carried out as described elsewhere (22). EMSA was carried
out using nuclear extract (8 µg), 0.6 µg of poly(dI/dC), and 2 × 104 cpm of a T4 polynucleotide kinase-labeled
[
-32P]ATP oligonucleotide. The sequences of the AP-2
and Sp1 consensus oligonucleotides were: 5'-GAT CGA ACT GAC CGC CCG CGG
CCC GT-3' (Santa Cruz Biotechnology catalog no. sc-2513) and 5'-ATT CGA TCG GGG CGG GGC GAG C-3' (Santa Cruz Biotechnology catalog no. sc-2502), respectively. The sequence of the mutated (underlined nucleotides) AP-2 consensus-containing oligonucleotide was 5'-GAT CGA
ACT GAC CGC TTG CGG CCC GT-3' (Santa Cruz Biotechnology
catalog no. sc-2516).
Site-directed Mutagenesis--
This was performed according to
the protocol of the Site-Directed Mutagenesis Kit (5Z701) of
(Palo Alto, CA). For generation of the
Sp1/Sp3mt u-PAR CAT, pCATbasic (Promega) regulated by 398 base pairs of
the u-PAR promoter (37) served as a template. A mutation primer
substituting T for G at positions
148,
147,
144, and
142 and
its corresponding selection primer (5'-CTTATCATGTCTGGTACCCCCGGAATTC-3') converting the BamHI site of pCATbasic to a KpnI
site were annealed to the denatured template plasmid, and the plasmid
was amplified according to the protocol. Remaining wild-type plasmid
was eliminated by two BamHI digestions for 4 h, each of
them followed by transformation of nondigested DNA into BMH 71-18
mutS cells (, Palo Alto, CA).
DNA of selected clones was isolated and sequenced using the Amersham
Pharmacia Biotech T7-Sequenase 2.0 Kit.
For generation of the AP-2/Sp1/Sp3mt u-PAR CAT construct, the Sp1/Sp3mt
u-PAR CAT plasmid served as a template. The second mutation primer
substituted A for C at positions
146,
145,
142, and
141 of the
u-PAR promoter, and the selection primer
(5'-CTTATCATGTCTGGATCCCCCGGAATTC-3') changed the KpnI site
generated above back to BamHI. Selection for
AP-2/Sp1/3-mutated plasmids was done by KpnI digestion. The procedure was continued as described above.
CAT Assays--
Cells were transfected at 60% confluency using
poly-L-ornithine as described previously (41). All
transient transfections were performed in the presence of a luciferase
expression vector (4 µg), and transfection efficiencies were
determined by assaying for luciferase activity. CAT activity was
measured as described previously (22). The amount of acetylated
[14C]chloramphenicol was determined using a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant
software. Student's t test analysis was performed two-sided
using the SPSS for Windows statistical software (release 6.1.3) (SPSS
Inc., Chicago, IL). Statistical significance was defined as
p
0.05.
Magnetic Separation of Transfected and Nontransfected
Cells--
Transfected cells were enriched by the MACS-Select method
of Miltenyi Biotech (Auburn, CA). RKO cells were co-transfected with
the AP-2
B expression construct and a plasmid encoding a mutated CD4
molecule (pMACS 4) in a 3:1 ratio. Cells were harvested after 42 h
in 320 µl of PBE buffer (phosphate-buffered saline, 0.5% bovine
serum albumin, 5 mM EDTA) and incubated for 15 min with 80 µl of a magnetic bead-conjugated antibody directed against the
mutated CD4 molecule. The cell suspension was then run through VS+
separation columns using the VarioMACS magnet according to the
manufacturer's protocol.
Western Blotting and Enzyme-linked Immunosorbent Assay for u-PAR
Protein--
RKO cells were extracted into a buffer (10 mM
Tris, pH 7.4, 0.15 M NaCl, 1% Triton X-100, 0.5% Nonidet
P-40, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride, 1 mM EGTA, 1 mM EDTA) for 10 min at
4 °C. Insoluble material was removed by centrifugation, and 750 µg
protein of cell extract was immunoprecipitated at 4 °C for 16 h
with 0.25 µg of a polyclonal anti-u-PAR antibody and protein
A-agarose beads. The polyclonal antibody (kindly provided by Dr. Andrew
Mazar, Angstrom Pharmaceuticals, San Diego, CA) was raised in rabbits
against amino acids 1-281 of the human u-PAR and purified on a
Sepharose-immobilized u-PAR column. The immunoprecipitated material was
subjected to standard Western blotting (42), and the blot was probed
with 5 µg/ml of an anti-u-PAR monoclonal antibody (catalog no. 3931, American Diagnostica, Greenwich, CT) and a horseradish
peroxidase-conjugated goat anti-mouse IgG. Bands were visualized by
enhanced chemiluminescence (Amersham Pharmacia Biotech).
For the determination of u-PAR by enzyme-linked immunosorbent assay,
resected tissue was prepared as described by the manufacturer (American Diagnostica).
Laminin Degradation Assays--
These were carried out as
described previously (43). RKO cells were harvested with 3 mM EDTA/phosphate-buffered saline, washed twice, and seeded
(500,000 cells) on radioactive laminin-coated (2 µg/dish) dishes. The
cells were allowed to attach overnight. Subsequently, cell surface
u-PARs were saturated by incubating the cells at 37 °C for 30 min
with 5 nM urokinase, and unbound plasminogen activator was
removed by washing. The cells were then replenished with serum-free
medium with or without 10 µg/ml plasminogen (final concentration).
After varying times at 37 °C, aliquots of the culture medium were
withdrawn and counted for radioactivity. Solubilized laminin represents
the degraded glycoprotein (43).
 |
RESULTS |
Region II of the u-PAR Promoter Footprinted by Nuclear
Extract from a High u-PAR-expressing Cell Line Is Bound with Sp1, Sp3, and an AP-2
-related Factor
We previously reported (22) that nuclear extract from a high (3 × 105 binding
sites/cell) u-PAR-expressing colon cancer cell line (RKO) footprinted a
region (referred to as region II) of the u-PAR promoter (nucleotides
148/
124). As a first step to identifying transcription factor(s)
bound to this region, EMSA was carried out using an oligonucleotide
spanning nucleotides
154/
128 (Fig.
1A). The oligonucleotide was
not extended to the 3'-end (
124) of the footprinted region II, since
preliminary EMSA utilizing a probe that included sequences 3' of
128
had failed to reveal any specific binding complexes. Employing the
154/
128 oligonucleotide, three slower migrating bands (indicated by
a brace, arrow, and asterisk) were apparent (Fig. 1B, lanes 2 and
8) with a 100-fold excess of the nonradioactive
oligonucleotide eliminating (lane 3) all of these bands. Computer analysis of this region of the u-PAR promoter revealed
the presence of putative AP-2- (
142/
134) and Sp1/Sp3- (
147/
138)
binding motifs all bearing one mismatch with the corresponding canonical (AP-2, GCCNNNGGC; Sp1, RYYCCGCCCM) sequences. The addition of
a 100-fold excess of a consensus AP-2-containing oligonucleotide (Fig.
1B, lane 7 and 11) from the
human metallothionein IIa promoter sequence (44) eliminated one of the
shifted bands (*). In contrast, substitution of this oligonucleotide at
the AP-2 motif (CC to TT) prevented it from competing for the shifted
bands (Fig. 1B, lane 10). Increasing
amounts of a nonradioactive oligonucleotide bearing a consensus Sp1
motif caused a dose-dependent decrease in the intensity of
two of the shifted bands (indicated with a brace and
arrow) (Fig. 1C) while having little effect on
the band (*) competed with the AP-2 motif-containing
oligonucleotide.

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Fig. 1.
Binding of Sp1, Sp3, and an AP-2-related
factor to footprinted region II of the u-PAR promoter.
A, schematic representation of the footprinted region II of
the u-PAR promoter (22) and the oligonucleotide used in EMSA in this
figure. B, RKO nuclear extract (8 µg) was
incubated at 21 °C for 20 min with an end-labeled oligonucleotide
( 154/ 128 u-PAR) in the presence or absence of a 100-fold excess of
competitor sequences. After this time, 2 µg of the indicated
antibodies were added, and complexes were subsequently analyzed by gel
electrophoresis. C, EMSA was carried out as described for
B with the exception that the amount of the Sp1 consensus
sequence was varied. D and E, RKO nuclear extract
was incubated where indicated with an anti-AP-2 A, an anti-AP-2 ,
or an anti-AP-2 antibody (2 µg) or an equivalent amount of an
unspecific IgG for 50 min at 4 °C followed by protein A-agarose
beads. Beads were subsequently removed by centrifugation, and treated
and untreated nuclear extract was analyzed by EMSA as described for
B. The data are typical of duplicate experiments.
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The ability of the consensus Sp1 motif to compete for the binding of
nuclear-extracted proteins to the u-PAR promoter footprinted region II
oligonucleotide suggested that transcription factors recognized by
these motifs were bound to the u-PAR promoter. To examine this
possibility, "supershifting" experiments were carried out. The
addition of an anti-Sp1-specific antibody to the RKO nuclear extract
resulted (Fig. 1B, lane 4) in a slower
migrating band (indicated with a line) with a concomitant
decreased intensity of the complex (indicated by a brace)
competed with the Sp1 consensus sequence. On the other hand, the
addition of an antibody against Sp2 had no effect (lane
5) on the migration pattern, while an antibody directed at
the DNA-binding domain of Sp3 completely abolished (Fig. 1B,
lane 6) the shifted band with the intermediate mobility (arrow). These data suggested that the region of
the u-PAR promoter footprinted with nuclear extract from a high
u-PAR-expressing cell line (22) is bound with Sp1 and Sp3.
Since the fastest migrating complex evident in the EMSA (indicated by
an asterisk) was competed with an AP-2 consensus motif (Fig.
1B), we hypothesized that the bound protein was an AP-2 isoform. To test this hypothesis, two sets of experiments were carried
out. First, RKO nuclear extract was mixed with 2 µg of an
anti-AP-2
antibody or an equal amount of IgG and subsequently with
protein A-agarose beads. The supernatant (depleted of
AP-2
-immunoreactive proteins) was then used in band shifting
experiments. The fastest migrating band (*), which was competed with an
excess of the AP-2 consensus, was practically abolished (Fig.
1D) by treatment of the nuclear extract with the
anti-AP-2
antibody, whereas the bands recognized by the Sp1
(brace) and Sp3 (arrow) antibodies were
unaffected. Further, the IgG control had no effect on the intensity or
mobility of any of the shifted bands and antibodies specific for
AP-2
, or AP-2
failed to deplete the RKO nuclear extract of the
binding factor (Fig. 1E). Second, we determined if authentic
AP-2
could bind to the u-PAR promoter region II. In EMSA using an
oligonucleotide spanning
154/
128, authentic AP-2
(Promega) gave
rise to a shifted band (line) (Fig.
2), which could be competed with an
excess of either the u-PAR region II oligonucleotide (
154/
128) or a
consensus AP-2 motif. The authentic AP-2
bound to the
154/
128
probe had a different mobility from the retarded band (which was
removed by immunoprecipitation with the anti-AP-2
antibody; Fig.
1D) observed using RKO nuclear extract (*).

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Fig. 2.
Footprinted region II of the u-PAR promoter
binds authentic AP-2 . RKO nuclear extract (8 µg) or purified AP-2 (10 ng) was incubated at 21 °C for 20 min
with radioactive oligonucleotides corresponding to nucleotides
154/ 128 of the u-PAR promoter in the presence or absence of a
100-fold excess of the indicated unlabeled competitor sequences. After
this time, 2 µg of an anti-AP-2 antibody or an equivalent amount
of an unspecific IgG was added to the reaction mixture where indicated.
Binding complexes were subsequently analyzed by EMSA. The data are
typical of duplicate experiments.
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|
To delineate the minimal sequence of footprinted region II required for
the binding of Sp1/Sp3 and the factor recognized by the anti-AP-2
antibody, band-shifting experiments were carried out with the u-PAR
promoter oligonucleotide truncated from either the 5'- or 3'-end (Fig.
3A). The removal of two base
pairs at the 5' terminus (generating oligonucleotide
152/
128) had
little effect on the intensities of the Sp1- (brace) and
Sp3- (arrow) bound complexes when compared with probe
154/
128. However, a severe attenuation in the binding of these
factors was apparent with further truncation from the 5'-end as evident
with probe
150/
128. These data suggested that nucleotides 5' of the
nonconsensus Sp1 motif (
147/
138) are required for the optimal
binding of Sp1 and Sp3 to region II of the u-PAR promoter. On the other
hand, the fastest migrating band (*), which is recognized by the
anti-AP-2
antibody, was unaffected by the removal of 5' nucleotides
with binding maintained with probe
143/
128. These data rule out the possibility that an AP-2-like motif located further upstream
(
151/
143) is mediating the binding of this factor to region II of
the u-PAR promoter. While the binding of Sp1/Sp3 and the AP-2
antibody-reactive factor demonstrated different 5' requirements, the
binding of these transcription factors showed identical requirements
for 3' sequences. Thus, the removal of up to 5 base pairs from the 3'
terminus of oligonucleotide
154/
130 (generating oligonucleotide
154/
135) had little effect on transcription factor binding. However, the deletion of an additional 3 base pairs from the 3'-end (oligonucleotide
154/
138) completely abolished the binding of these
three transcription factors. Thus, sequences in the u-PAR promoter
extending 3' to
135 are required for the optimal binding of Sp1, Sp3,
and the factor recognized by the anti-AP-2
antibody.

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Fig. 3.
Identification of minimal sequences of
footprinted region II required for binding of
nucleus-derived transcription factors from RKO cells.
A, RKO nuclear extract (8 µg) was incubated at 21 °C
for 20 min with end-labeled oligonucleotides with (+), or without ( )
a 100-fold excess of the indicated competitor or an oligonucleotide
containing a consensus AP-2 motif (AP-2 consensus). Binding complexes
were resolved by EMSA as described in the legend to Fig. 1. The
experiments were carried out three times. B, the nucleotide
sequence of the u-PAR promoter, including the footprinted region II is
shown. Underlined and overlined nucleotides
indicate putative motifs identified by the Genetics Computer Group
program (Madison, WI). C, RKO nuclear extract (8 µg) or
AP-2 (10-20 ng) was incubated at 21 °C for 20 min with a
radioactive oligonucleotide corresponding to nucleotides 147/ 128 of
the u-PAR promoter in the presence or absence of a 100-fold excess of
the unlabeled competitor sequences. After this time, 2 µg of the
indicated antibody was added to the reaction mixture where indicated.
Binding complexes were subsequently analyzed by EMSA. The data are
typical of duplicate experiments.
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While the anti-AP-2
antibody "supershifted" (arrow)
authentic AP-2
bound to the u-PAR promoter oligonucleotide
154/
128 (Fig. 2), in contrast, we were unable to detect a
"supershift" of the fastest migrating band (using RKO nuclear
extract) with this antibody (data not shown). However, this could be
due to the presence of multiple complexes evident in the EMSA masking a
"supershifted" band. Thus, we repeated the EMSA using an
oligonucleotide spanning
147/
128 (Fig. 3C). This probe
is unable to bind Sp1/Sp3 as demonstrated from Fig. 3A. The
addition of RKO nuclear extract to this probe yielded a slower
migrating band (indicated with an asterisk), which was
abolished with an excess of the unlabeled AP-2 consensus
oligonucleotide. However, the addition of the anti-AP-2
antibody,
effective in supershifting authentic AP-2
bound to the
147/
128
probe (arrow), failed to produce a "supershifted" complex. Likewise, antibodies to other AP-2
isoforms did not yield a
"supershift" of the RKO-derived nuclear factor. Another possible
explanation for the lack of a "supershift" using RKO nuclear
extract is that there may be an inhibitor present in the extract.
However, this is unlikely, since the addition of nuclear extract from
this cell line to authentic AP-2
did not prevent a supershift of the
latter in EMSA employing the
154/
128 probe (data not shown). These
data suggest that a DNA-binding factor related to AP-2
(hereafter
referred to as AP-2
-related factor), derived from RKO nuclear
extract, is bound to region II of the u-PAR promoter sequence.
Binding of Authentic Sp1 and AP-2
to the u-PAR Promoter Region
II Is Mutually Exclusive--
There is substantial overlap between the
Sp1/Sp3 (
147/
138) and AP-2 motifs (
142/
134) in the footprinted
region II of the u-PAR promoter. Thus, we considered it unlikely that
Sp1 and the AP-2
-related factor were simultaneously binding to the
u-PAR oligonucleotide probe. Nevertheless, to examine this possibility, the u-PAR oligonucleotide spanning
154/
128 was incubated with authentic Sp1 and authentic AP-2
proteins alone or in combination. Binding of the oligonucleotide with the individual purified proteins resulted in slower migrating complexes (Fig.
4). However, the simultaneous addition of
both purified proteins to oligonucleotide
154/
128 did not yield a
ternary complex and in fact resulted in a decrease in binding of either
protein. The topmost band represents material remaining in the well and
failed to resolve as a third band even with continuous electrophoresis.
These data would suggest that the binding of Sp1 and AP-2
to the
u-PAR promoter sequence spanning
154/
128 is mutually exclusive.
However, it should be emphasized that this conclusion is based on the
use of authentic AP-2
rather than the AP-2
-related factor present
in RKO nuclear extract.

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Fig. 4.
The binding of authentic
AP-2 and Sp1 to region II of the u-PAR
promoter is mutually exclusive. Authentic AP-2 A (10 ng) and Sp1
(10 ng) were incubated alone or in combination at 21 °C for 20 min
with an end-labeled oligonucleotide ( 154/ 128) with or without a
100-fold excess of the unlabeled oligonucleotide. After this time,
binding complexes were resolved by EMSA as described in the legend to
Fig. 1. The data are representative of two separate experiments.
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Effect of Inhibiting Sp1/Sp3 Binding to Region II on u-PAR Promoter
Activation--
To determine whether u-PAR promoter activation was
dependent on the binding of Sp1/Sp3 to region II, G nucleotides at
positions
148,
147,
144, and
142 (within the Sp1-like motif)
were substituted for T (Fig.
5A). EMSA using this
substituted oligonucleotide indicated that the slower migrating bands
identified as Sp1 (brace) and Sp3 (arrow) by
their reactivity with specific antibodies (see Fig. 1B) were
markedly diminished in their intensity when compared with an
oligonucleotide corresponding to the wild type u-PAR promoter sequence
(Fig. 5B). In contrast, the intensity of the complex (*)
recognized with the anti-AP-2
antibody (see Fig. 1D) was not decreased by this change. RKO cells were then transfected with a
CAT reporter driven by either the wild type u-PAR promoter (u-PAR CAT)
or the promoter harboring the mutations that reduced Sp1 and Sp3
binding to region II. In duplicate experiments, the activity of the
mutated u-PAR promoter was similar to that achieved with the wild type
u-PAR promoter (Fig. 5C).

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Fig. 5.
Effect of interfering with the binding of
Sp1/Sp3 to footprinted region II on the constitutive activity of the
u-PAR promoter. A, substituted nucleotides in the u-PAR
promoter region II are indicated with asterisks.
Underlined and overlined nucleotides indicate
putative motifs identified by the Genetics Computer Group program.
B, RKO nuclear extract (8 µg) was incubated with an
oligonucleotide spanning nucleotides 154/ 128 of the u-PAR promoter
( 154/ 128) or the corresponding sequence that had been
substituted (G to T) at the indicated nucleotides. EMSA was performed
as described in the legend to Fig. 1. C, RKO cells were
transiently transfected with the wild type (u-PAR CAT) or mutated
(Sp1/Sp3mt u-PAR CAT) u-PAR promoter (see A for nucleotide
substitutions). After 48 h, the cells were harvested, lysed, and
assayed for CAT activity. Parallel dishes were transfected with pRSV
CAT and pSV0CAT as positive and negative controls, respectively. The
data are representative of duplicate experiments.
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Elimination of Sp1/Sp3 and AP-2
-related Factor Binding to u-PAR
Region II Abolishes Constitutive Promoter Activation--
We were
unable to interfere with the binding of the AP-2
-related factor to
region II of the u-PAR promoter without having a deleterious effect on
Sp1/Sp3 binding, and this was consistent with the large overlap of
these motifs and band shifting experiments (see Fig. 3A),
which revealed identical 3' nucleotide requirements. We therefore
determined the effect of eliminating the binding of these three
transcription factors on constitutive u-PAR promoter activity.
Nucleotide substitutions (Fig.
6A) of the Sp1-like and AP-2-like motifs, which prevented (Fig. 6B) these
transcription factors binding to oligonucleotide
154/
128, were
engineered into the u-PAR promoter CAT reporter construct. The reporter
construct was then compared with the wild type promoter for activation
in the u-PAR-overexpressing RKO cells. We found in duplicate
experiments that the activity of the mutated u-PAR promoter was reduced
by over 90% in comparison with the wild type promoter construct (Fig. 6C). These data, combined with the observation that the
mutation of region II, which prevented the binding of Sp1/Sp3 (but not the AP-2
-related factor), had minimal effect on the activity of the
u-PAR promoter (see Fig. 5C), implies that binding of the AP-2
-related factor to this region is critical for the constitutive activity of this promoter in RKO cells.

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Fig. 6.
Interfering with the binding of the
AP-2-related factor and Sp1/Sp3 to region II of the u-PAR promoter
abolishes its constitutive activity in RKO cells. A,
substituted nucleotides in region II of the u-PAR promoter sequence are
indicated with asterisks. Underlined and
overlined nucleotides indicate putative motifs identified by
the Genetics Computer Group program. B, EMSA was carried out
using oligonucleotides corresponding to the wild type u-PAR sequence
(nucleotides 154/ 128) or the mutated sequence (AP-2/Sp1/Sp3 mutant)
and RKO nuclear extract. C, RKO cells were, at 60%
confluence, transfected with a RSV-luciferase expression construct and
a CAT reporter regulated by the wild type or mutated u-PAR (as
indicated in A) promoter sequence. Both promoter sequences
were in the context of 398 base pairs of 5'-flanking sequence. After
48 h, the cell were harvested, lysed, and assayed for CAT activity
after normalization for transfection efficiency. The amount of
acetylated chloramphenicol was determined using a Storm 840 PhosphorImager. The data are typical of duplicate experiments.
|
|
Down-regulation of u-PAR Promoter Activity by the Co-expression of
either a Dominant Negative or an Antisense AP-2--
As indicated
above, any conclusions drawn as to the role of the AP-2
-related
factor in the regulation of u-PAR promoter activity were confounded by
the fact that nucleotide substitutions of region II affecting the
binding of this factor also disrupted the binding of Sp1 and Sp3. To
circumvent this problem, we determined the effect of interfering with
AP-2
on u-PAR promoter activity. RKO cells were transiently
transfected with an AP-2
B expression construct. AP-2
B is an
alternatively spliced product of AP-2
and acts as a negative
regulator of AP-2 transcriptional activity (39). In three separate
experiments, increasing amounts of the AP-2
B expression construct
caused a dose-dependent repression of u-PAR promoter
activity (Fig. 7). An input of 0.5 µg
of the dominant negative expression construct brought about an 81 ± 4% inhibition of u-PAR promoter activity compared with the empty
expression vector (pSG5). Increasing the amount of AP-2
B DNA to 5 µg was marginally more effective, resulting in a 93 ± 5%
inhibition of u-PAR promoter activity.

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Fig. 7.
The expression of a negative regulator of
AP-2 down-regulates u-PAR promoter activity in
RKO cells. RKO cells were transiently transfected with a CAT
reporter regulated by 398 base pairs of upstream sequence of the
urokinase receptor promoter (u-PAR CAT) and the indicated amounts of an
expression vector (pSG5) bearing AP-2 B (AP-2 B pSG5). Parallel
dishes of RKO cells were transfected with pRSV CAT or pSV0CAT as
positive and negative transfection controls. CAT assays were carried
out as described in the legend to Fig. 5. Representative data of
triplicate experiments are shown. Significant differences are indicated
as follows: *, p < 0.05; **, p < 0.01 for comparison of CAT activities achieved with the AP-2 B when
compared with the equivalent amount of the empty expression
construct.
|
|
As an alternative to using the dominant negative expression construct,
parallel experiments were also carried out using an antisense AP-2
expression vector. Similar to the results achieved with the AP-2
B,
the antisense expression construct caused an inhibition of u-PAR
promoter activity (data not shown), with 10 µg of DNA yielding a
90 ± 14% repression of u-PAR promoter activity when compared
with the vector backbone (pSG5). To rule out the possibility that
interfering with AP-2
activity was having a general suppressive
effect on transcription, RKO cells were transiently co-transfected with
the AP-2
B expression construct and a CAT reporter driven by the
urokinase promoter. The AP-2
B expression construct failed to repress
the urokinase reporter construct using 1 µg of the effector an amount
that inhibited u-PAR promoter activity by over 90 ± 7% (data not
shown). Thus, it is unlikely that the effect of AP-2
B on u-PAR
promoter activity is due to a general shut down of transcription.
To confirm that the repressive effect of AP-2
B on u-PAR promoter
activity was mediated via the footprinted region II, two experiments
were carried out. First, RKO cells were transiently co-transfected with
an AP-2
B expression construct and a CAT reporter regulated by either
148 or 105 base pairs of 5'-flanking sequence of the u-PAR gene. Based
on band shifting experiments (see Fig. 3A) the
148 u-PAR
promoter fragment can, via region II, bind the AP-2
-related factor,
whereas the u-PAR promoter regulated by only 105 base pairs of upstream
sequence cannot. It should be noted that Sp1/Sp3 binding is preserved
in the
148 u-PAR construct due to the contribution of plasmid
sequences. The AP-2
B expression construct caused a marked reduction
(80%) in promoter activity using the reporter construct flanked by 148 base pairs of upstream sequence while bringing about only a modest
repression (20%) of the reporter construct regulated by 105 base pairs
of the u-PAR regulatory sequence (data not shown). Second, RKO cells
were co-transfected with the AP-2
B expression construct and a CAT
reporter regulated by either an oligonucleotide (
154/
128) spanning
region II (R2 CAT) of the u-PAR promoter or 398 base pairs of
5'-flanking sequence (u-PAR CAT). The CAT reporter regulated by region
II (R2 CAT) was activated (Fig. 8) in RKO
cells, albeit to a lesser extent than that achieved with 398 base pairs
of flanking sequence (u-PAR CAT). In two separate experiments, the
activity of the R2 CAT reporter construct was inhibited by over 75% by
the co-expression of the AP-2
B when compared with an equivalent
amount of the empty expression construct (pSG5). This inhibition was
quantitatively similar to that achieved using the u-PAR CAT reporter.
Taken together, these data suggest that the u-PAR promoter activity is
indeed regulated by an AP-2
-related factor and possibly Sp1/Sp3 in
RKO cells and that this occurs via region II of the promoter.

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Fig. 8.
Repression of u-PAR promoter activity by
AP-2 B is mediated through footprinted region
II. RKO cells were transiently co-transfected with a CAT reporter
regulated by either 398 base pairs of flanking sequence (u-PAR CAT) or
by a sequence of the u-PAR promoter spanning nucleotides 154/ 128
(R2 CAT) and the indicated amount of an expression vector (pSG5)
bearing AP-2 B (AP-2 B pSG5). CAT assays were performed as stated
in the legend to Fig. 5. RKO cells were transfected with pRSV CAT and
pSV0CAT as positive and negative controls, respectively. The CAT assay
of a representative experiment is shown with the range of
chloramphenicol conversions of duplicate experiments indicated at the
top.
|
|
Nuclear Extract from u-PAR-rich Cells Demonstrate Increased Binding
of the AP-2
-related Factor to the u-PAR Promoter Region II Compared
with Nuclear Extract from Low u-PAR-expressing Cells--
Considering
the evidence implicating the AP-2
-related factor in the regulation
of u-PAR expression in RKO cells, we speculated that nuclear extract
derived from cells rich in u-PAR protein would contain more of this
transcription factor bound to region II of the u-PAR promoter compared
with nuclear extract derived from cells characterized by their low
u-PAR protein. Toward this end, we made use of another colon cancer
cell line (GEO), which displays 10-fold fewer u-PARs compared with RKO
as a consequence of reduced transcription of the gene (22). Nuclear
extracts were generated from each cell line, and equal protein amounts were incubated with the radioactive oligonucleotide
154/
128 in the
presence or absence of excess oligonucleotide competitors (Fig.
9). Binding complexes were then analyzed
by electrophoresis. Nuclear extract from GEO cells gave rise to a
retarded band with a mobility similar to that identified as the
AP-2
-related factor (*) using RKO nuclear extract. An excess of the
AP-2 consensus sequence reduced the intensity of this band, consistent
with the notion that it represents an AP-2
-related factor-bound
complex. Interestingly, the intensity of the complex (*) was reduced in nuclear extract from the low u-PAR-expressing GEO cells when compared with RKO cells, the latter of which display over 10-fold more u-PAR.
Equally important, treatment of GEO cells with PMA, which increases
u-PAR gene transcription in this cell line (22, 45), brought about a
dramatic increase in the amount of this complex (*). In contrast, the
intensity of the retarded band, which was indistinguishable from Sp3
(arrow), was decreased by PMA treatment of GEO cells.

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Fig. 9.
Nuclear extract from cells expressing high
u-PAR levels intrinsically (RKO) or as a consequence of PMA treatment
(GEO) contains more AP-2 -related factor bound
to the u-PAR footprinted region II compared with nuclear extract from
the untreated GEO cells, which have few u-PAR. Nuclear extract (8 µg) from 90% confluent cells treated with, or without, PMA (100 nM) for 60 min was incubated with a radioactive
oligonucleotide spanning nucleotides 154 to 128 of the u-PAR
promoter. Where indicated, a 100-fold excess of nonradioactive
oligonucleotide competitors was included. Binding complexes were then
analyzed by EMSA. The data are representative of duplicate
experiments.
|
|
To determine if the altered amounts of the region II-bound factors were
required for the stimulation of u-PAR promoter activity by PMA, GEO
cells were co-transfected with a CAT reporter flanked by the wild type
or mutated u-PAR promoter and a vector bearing AP-2
B and
subsequently treated with the phorbol ester. Treatment of the cells
with PMA caused a strong (10-20-fold) induction of the wild type
(u-PAR CAT) u-PAR promoter, which was prevented in cells made to
co-express AP-2
B (Fig. 10). Mutation
of the u-PAR promoter to prevent Sp1/Sp3 binding (Sp1/Sp3mt u-PAR CAT)
reduced the stimulation by PMA. This stimulation was further reduced
when nucleotide substitutions of the u-PAR promoter were undertaken to
abolish the binding of the AP-2
-related factor as well as Sp1/Sp3
(AP-2/Sp1/Sp3mt u-PAR CAT). Interestingly, the ability of the AP-2
B
expression construct to repress u-PAR promoter activity was diminished
by mutations that prevented the binding of Sp1/Sp3 (Sp1/Sp3mt u-PAR
CAT). Taken together, these data would suggest that PMA stimulation of
u-PAR gene expression in GEO cells requires, at least in part, the
binding of the AP-2
-related factor as well as Sp1/Sp3 to region II
of the promoter.

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Fig. 10.
The binding of the
AP-2 -related factor and Sp1/Sp3 to region II
are required for the stimulation of u-PAR promoter activity by PMA in
GEO cells. GEO cells were co-transfected with a CAT reporter
driven by the wild type u-PAR promoter (u-PAR CAT) or the promoter that
had nucleotide substitutions in region II to prevent the binding of
Sp1/Sp3 (Sp1/Sp3mt u-PAR CAT) or AP-2/Sp1/Sp3 (AP-2/Sp1/Sp3mt u-PAR
CAT) and an expression vector (pSG5) encoding AP-2 B (AP-2 B pSG5).
Following transfection, cells were replenished with fresh medium
supplemented with or without PMA and cultured for an additional 2 days.
Cells were then harvested, lysed, and assayed for CAT activity as
described in the legend to Fig. 5. The data are typical of two separate
experiments.
|
|
Stimulation of u-PAR Promoter Activity in the Low u-PAR-expressing
GEO Cell Line by the Expression of Exogenous AP-2
A--
While the
above experiments suggested a requirement for the AP-2
-related
factor in the stimulation of u-PAR gene expression by phorbol ester, it
was not clear as to whether this transcription factor alone was
sufficient to augment u-PAR promoter activity. To address this issue,
GEO cells, which have low u-PAR protein, were co-transfected on three
separate occasions with a u-PAR promoter-regulated CAT reporter and an
expression vector bearing the full-length form of AP-2
(AP-2
A).
The activity of the promoter alone was below the detection limit of the
assay (Fig. 11), consistent with the
low expression of the u-PAR gene in this cell line. However, the
co-transfection of AP-2
A into these cells caused a
dose-dependent increase in u-PAR promoter activity with up
to 0.05 µg of the effector plasmid. This amount of the expression
construct brought about over a 13-fold stimulation of promoter
activity. Higher amounts of the AP-2
A diminished u-PAR promoter
stimulation, presumably as a consequence of squelching (46). The
AP-2
A-dependent stimulation of the u-PAR promoter was
greater than that achieved with 2 µg of a JunD expression vector,
which we had shown previously (22) to stimulate u-PAR expression via a
separate footprinted region (
190/
171) of the promoter containing a
classical AP-1 motif. The transfection of GEO cells with JunD and
AP-2
A together stimulated u-PAR promoter activity to an extent
greater than the sum of the individual expression constructs,
indicative of synergism. Thus, it is likely that that these
transcription factors cooperate with each other to regulate u-PAR
expression. Notwithstanding these observations, our data indicate that
the expression of AP-2
A is sufficient to up-regulate u-PAR gene
expression in GEO cells.

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Fig. 11.
Stimulation of u-PAR promoter activity in
GEO cells made to co-express AP-2 A. GEO
cells were transiently co-transfected with a u-PAR promoter-regulated
CAT and an expression vector (pSG5) bearing either JunD or AP-2 A.
Parallel cultures were transfected with pSV0CAT or pRSV CAT as negative
and positive controls, respectively. After 48 h, the cells were
lysed and assayed for CAT activity as described in the legend to Fig.
5. The data are representative of triplicate experiments.
Chloramphenicol conversions are shown as average values ± S.D.
for the three experiments. An asterisk indicates that
differences in chloramphenicol conversions achieved by the expression
of the JunD and/or the AP-2 A when compared with the empty expression
construct (pSG5) were statistically significant (p < 0.05).
|
|
Expression of AP-2
B Decreases Endogenous u-PAR Protein Amount in
RKO Cells--
We then determined if the expression of AP-2
B, which
acts as a negative regulator of AP-2 function, reduces the expression of the endogenous u-PAR gene in RKO cells. Cells were co-transfected with an expression vector encoding a mutated CD4 and varying amounts of
an expression construct bearing AP-2
B. Cells were harvested 48 h later, and transfected cells were enriched with magnetic beads coated
with an anti-mutated CD4 antibody (which is non-cross-reactive with
wild type CD4) and assayed for u-PAR protein by Western blotting (Fig.
12A). A band whose molecular
mass was indistinguishable from that of u-PAR (55 kDa) (47) was
detected in the immunoblot. Increasing amounts of the AP-2
B
expression vector caused a dose-dependent decrease in the
amount of this protein. The reduced amount of u-PAR protein was
associated with the attenuated activity of a CAT reporter regulated by
three tandem AP-2 motifs upstream of a thymidine kinase minimal
promoter (Fig. 12B). Thus, the expression of a negative
regulator of AP-2 reduces the expression of the endogenous u-PAR gene
in RKO cells.

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Fig. 12.
Reduced amount of u-PAR protein and laminin
degradation by RKO cells made to express
AP-2 B. A, RKO cells were
co-transfected with the indicated amount of the expression vector
encoding AP-2 B or the vector backbone (pSG5) alone along with an
expression construct bearing a mutated CD4. After 48 h, the cells
were harvested and assayed for u-PAR protein by Western blotting.
ve Control, no cell extract. B, RKO cells were
co-transfected with an expression vector (pSG5) bearing AP-2 B
(AP-2 B) along with a CAT reporter regulated either by three AP-2
tandem repeats upstream of a thymidine kinase minimal promoter (AP-2
pBLCAT2) or the minimal promoter alone (pBLCAT2). After 48 h, the
cells were lysed and assayed for CAT activity. C, RKO cells
transfected and harvested as described for A were plated in
serum-free medium on radioactive laminin-coated dishes. After cell
attachment, surface u-PAR were saturated with exogenous urokinase, and
the cultures were washed extensively and then replenished with
serum-free medium supplemented with or without plasminogen. Aliquots of
the culture supernatant were withdrawn at the indicated times
thereafter and counted for radioactivity. After 2 h, cells were
enumerated. The data are typical of duplicate experiments. ,
serum-free medium only; -, serum-free medium plus
plasminogen; , RKO-serum-free medium; ,
RKO-pSG5-plasminogen-serum-free medium; , RKO-AP-2B-serum-free
medium; , RKO-AP-2B-plasminogen-serum-free medium.
|
|
Inhibition of u-PAR-directed Laminin Degradation in RKO Cells Made
to Express AP-2
B--
One of the functions of the u-PAR is to
accelerate plasminogen-dependent proteolysis (8), and this
is a requirement for the invasive potential of a divergent set of
cancers (31, 32, 48). We were therefore interested in determining
whether interfering with the transcriptional activation of the gene
leading to reduced u-PAR synthesis would diminish extracellular matrix
degradation. RKO cells in serum-free medium (RKO-SF)
demonstrated minimal solubilization of laminin (Fig. 12C).
However, the addition of plasminogen to the RKO cells transfected with
the vector backbone (RKO-pSG5-Pl-SF) resulted in a strong
time-dependent increase in laminin degradation, indicating
plasmin-dependent proteolysis. After a 2-h incubation in
the presence of zymogen, nearly 500,000 cpm of solubilized laminin was
evident in the culture supernatant. In contrast, RKO cells transfected
with the AP-2
B expression construct (RKO-AP-2B-Pl-SF) showed markedly reduced plasminogen-dependent degradation
(80% reduction) of this glycoprotein after a 2-h incubation. These data suggest that reduced u-PAR synthesis brought about by interfering with the AP-2
-dependent transcriptional activation of
the u-PAR gene in RKO cells attenuates
plasminogen-dependent proteolysis.
 |
DISCUSSION |
The u-PAR plays a critical role in extracellular matrix
degradation and tumor invasion (34, 49). We have identified an important regulatory region in the u-PAR promoter (
152/
135) that is
trans-activated by an AP-2
-related factor. Further, our finding that the expression of AP-2
B, which interferes with the transcriptional activation of the u-PAR gene by the AP-2
-related factor and Sp1/Sp3, diminishes u-PAR protein amounts and laminin degradation serves to illustrate that transcriptional studies of a
target gene can be utilized to bring about a more indolent phenotype of
cancer at least in an experimental setting.
Interestingly, in a previous report, Soravia et al. (26),
using HeLa nuclear extracts, failed to detect, by EMSA, the binding of
AP-2 to the urokinase receptor promoter. The reason for this difference
is unclear at the present time. One possibility is that urokinase
receptor expression is regulated differently in the separate cell lines
used in the two studies.
The identity of the AP-2
-related factor bound to the u-PAR promoter
region II is unknown at the present time. Certainly, it is strongly
related to AP-2
, since depletion of nuclear extract with an
anti-AP-2
antibody and protein A-agarose beads severely diminished
the intensity of the retarded band in EMSA and since the u-PAR promoter
region II can clearly bind authentic AP-2
. On the other hand, in
EMSA it had a distinct mobility from authentic AP-2
, and the
anti-AP-2
antibody was unable to supershift the RKO nuclear factor
bound to a region II oligonucleotide while effecting a supershift with
authentic AP-2
bound to this oligonucleotide. We were also unable to
supershift or immunodeplete the AP-2
-related factor from RKO nuclear
extract using recently available antibodies (Santa Cruz Biotechnology)
to AP-2
or AP-2
, making it unlikely that the RKO-derived nuclear
factor was one of these isoforms. Lee et al. (50) also noted
an AP-2
-related factor in THP-1 monocytes that regulated the
expression of the B2 subunit of the V-ATPase. Similar to our study, the
THP-1-derived AP-2
-related factor was not supershifted in EMSA and
had a different size from authentic AP-2
based on its mobility in
gel retardation studies. It remains to be determined whether the
AP-2
-related factor found in the current study and in the study by
Lee et al. (50) are the same.
In addition to the AP-2
-related factor recognized by footprinted
region II of the u-PAR promoter, both Sp1 and Sp3 were also bound, as
evident in EMSA using nuclear extract from the high u-PAR-expressing
cell line RKO. However, we found that mutations of the u-PAR promoter
that prevented the binding of either of these transcription factors had
a negligible effect on promoter activation, arguing against a role for
these factors in regulating at least the constitutive u-PAR expression
in RKO cells.
The requirement of the u-PAR promoter region II for the
PMA-dependent elevation of u-PAR gene expression (53)
merits discussion. We had shown previously (22) that the induction by
this phorbol ester required an intact AP-1 motif (at
184) in a
separately footprinted region (
190/
171) of the u-PAR promoter.
However, several observations in the current study would indicate that the induction of the u-PAR promoter by PMA requires, in addition, other
transcription factor binding sites including the motif bound with the
AP-2
-related factor. Thus, nucleotide substitutions of the u-PAR
promoter that prevented the binding of the AP-2
-related factor
substantially reduced the stimulation of the promoter by the phorbol
ester. Further, the co-expression of AP-2
B, which is a negative
regulator of AP-2 function, completely ablated the stimulation of the
u-PAR promoter by PMA. Finally, an increased amount of the
AP-2
-related factor bound to the u-PAR promoter region II was
evident using nuclear extract from PMA-treated GEO cells (compared with
nuclear extract from untreated GEO cells). Our results are reminiscent
of other studies in which the stimulation of the PAC-1
(phosphatase of activated cells)
phosphatase and neuropeptide tyrosine genes by PMA was shown to be
mediated partly through an AP-2-related site and coincided with induced
DNA binding of AP-2 (54, 55). It was also clear that interfering with the binding of Sp1/Sp3 (but not affecting the binding of the
AP-2
-related factor) to region II of the u-PAR promoter had a
deleterious effect on the activation of the u-PAR promoter by PMA. A
similar requirement for Sp1 has been reported for the stimulation of
thromboxane receptor gene expression by PMA (56), although, in contrast
to that study, we did not detect increased binding of this
transcription factor. Taken together, it is likely that the stimulation
of the u-PAR gene expression by phorbol ester is complex, requiring the
interactions of multiple transcription factors with their cognate
binding sites in the upstream sequence of this gene.
The mechanism by which the AP-2
B alters u-PAR promoter activity
deserves comment. Initially, we assumed that this was through a direct
antagonism of the AP-2
-like factor bound to the u-PAR promoter
region II, presumably as a consequence of heterodimerization of the two
proteins (39). However, one experiment suggested that this proposal
might not be entirely accurate. Thus, the ability of the AP-2
B to
counter the stimulation of u-PAR promoter activity by PMA was
diminished by mutations in the u-PAR promoter that prevented the
binding of Sp1/Sp3. These data suggested that the expressed AP-2
B
was mediating its effect, at least in part, through the Sp1/Sp3-binding motif.
Our data based on mobility shift assays using purified proteins
strongly suggested that the binding of at least AP-2
A and Sp1 to the
u-PAR promoter region II is mutually exclusive, and this is consistent
with the considerable (about 50%) overlap of the Sp1/Sp3 and AP-2
motifs. Thus, it is unlikely that the AP-2
-related factor and Sp1
are physically interacting on the u-PAR promoter region II sequence.
The biological significance of having two overlapping transcription
factor binding motifs in the u-PAR promoter region II, which can be
bound with only one transcription factor at any one time, can only be
speculated on. Certainly, for the regulation of keratin 3 gene
expression in corneal epithelial cells, overlapping AP-2 and Sp1 motifs
act as a switch to up-regulate expression of this gene in response to
the latter transcription factor (57). For the u-PAR promoter, since
this motif can be bound with Sp3, which for some genes is a
trans-repressor (58), it may be that this represents a
mechanism for either up- or down-regulating u-PAR expression.
In conclusion, we have shown that the constitutive and PMA-inducible
expression of the u-PAR gene requires, at least in part, the
trans-activation of a region of the promoter spanning
152/
135 by an AP-2
-related factor. A high u-PAR protein level in
colon cancer, which promotes the invasive phenotype (31, 32) and portends a poor patient outcome (30), is probably partly a consequence of trans-activation of this gene by a greater amount and/or
binding activity of the AP-2
-related factor. Further, interfering
with the trans-activation of the promoter by the
AP-2
-related factor reduced endogenous u-PAR expression and
diminished laminin degradation. These findings raise the exciting
possibility that interfering with the AP-2
-related
factor-dependent trans-activation of the u-PAR
gene may represent a novel means of diminishing extracellular matrix
degradation and consequently reducing colon cancer invasion and metastasis.
 |
ACKNOWLEDGEMENTS |
Expression constructs encoding AP-2
A,
AP-2
B, the antisense AP-2, and the AP-2 CAT reporter were kindly
provided by Dr. Michael Tainsky (Department of Tumor Biology, M.D.
Anderson Cancer Center, Houston, TX). The urokinase CAT reporter was
kindly provided by Dr. Francesco Blasi (University of Milan, Italy). We
thank Drs. Michael Tainsky, Ernest Lengyel (Department of Gynecology
and Obstetrics, Technical University of Munich, Germany), Christian Simon (Department of Ears, Nose, and Throat, University of Tubingen, Germany), and Jose Juarez (Department of Urology, University of California, Davis School of Medicine) for excellent intellectual and
technical input. We thank Prof. F. W. Schildberg (Department of
Surgery, Klinikum Grosshadern, Ludwig-Maximilians University of Munich,
Germany) for input. Finally, we are grateful to Drs. Tim Schaeffer and
Francois Claret for critical appraisal of the manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants R01 CA58311, R01 DE10845, and P01 DE11906; a Physician's
Referral Service grant (to D. B.); and fellowships from the Dr.
Mildred Scheel Cancer Foundation (Deutsche Krebshilfe, Bonn, Germany) and the Deutsche Forschungsgemeinschaft (Bonn, Germany) (to H. A. and
R. B., respectively).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
This paper is in tribute to Barbara Young for her dedicated work.
To whom all correspondence should be addressed: Dept. of
Cancer Biology, Box 108, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-8953; Fax: 713-794-0209; E-mail: dboyd{at}notes.mdacc.tmc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
u-PAR, urokinase-type plasminogen activator receptor;
AP-1 and -2, activator
protein-1 and -2, respectively;
CAT, chloramphenicol acetyltransferase;
EMSA, electrophoretic mobility shift assay;
PMA, phorbol 12-myristate
13-acetate;
RSV, Rous sarcoma virus.
 |
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