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J Biol Chem, Vol. 274, Issue 26, 18428-18437, June 25, 1999
From the Department of Cancer Biology, M.D. Anderson Cancer Center,
Houston, Texas 77030
Since c-src overexpression increases
colonic cell invasiveness and because both Src activity and urokinase
receptor protein are elevated in invasive colon cancers, the present
study was undertaken: 1) to determine if a constitutively active Src
regulates urokinase receptor expression and 2) to identify required
cis-elements and trans-acting factors. SW480
colon cancer cells transfected with an expression plasmid (c-srcY527F)
encoding a constitutively active Src protein manifested increased
urokinase receptor gene expression and Src activity. Treatment of the
src transfectants with a Src-inhibitor (PD173955) reduced
urokinase receptor protein levels and laminin degradation. Inasmuch as
we recently implicated an upstream region of the urokinase receptor
promoter ( The urokinase receptor
(u-PAR),1 a heavily
glycosylated, 45-60-kDa cell surface protein, binds the serine
protease urokinase specifically and with high affinity
(KD ~0.5 nM) (1, 2). The u-PAR (1)
is comprised of three similar repeats approximately 90 residues each
(3, 4) with the amino-terminal domain binding the plasminogen activator
and the carboxyl-terminal domain tethering the binding protein to the
cell surface via a glycosyl phosphatidylinositol anchor (4).
The u-PAR plays an important role in many physiological and
pathological functions including wound healing, tissue remodeling, and
tumor cell invasion and metastasis (5-8). The ability of the u-PAR to
promote these biological effects is a consequence of its diverse
function. First, urokinase bound to the u-PAR activates plasminogen
much more efficiently than the fluid-phase plasminogen activator (9),
thereby facilitating basement membrane degradation. Second, u-PAR
clears urokinase-inhibitor complexes from the extracellular space via
an The spread of a cancer to distant sites is characterized by extensive
tissue remodeling in which surrounding normal tissue and extracellular
matrix are proteolytically degraded. Several lines of evidence have
implicated the u-PAR in this process. Thus, the overexpression of a
human u-PAR cDNA increases the invasion of human osteosarcoma cells
through an extracellular matrix-coated porous filter (16), whereas
down-regulating u-PAR levels using either antisense expression
constructs, oligonucleotides, or synthetic compounds reduces the
ability of divergent invasive cancers to invade in vitro and
in vivo (17-19). Finally, clinical studies on colon and
gastric cancers have revealed a correlation between high u-PAR
expression and short survival times (20, 21).
The level of surface-u-PAR is controlled mainly via the regulation of
transcription of the seven-exon u-PAR gene located on chromosome 19q13
(22, 23), although altered mRNA stability and receptor recycling
may represent other means of control (24-26). Regarding
transcriptional regulation, Soravia et al. (27) reported that the basal expression of the gene was regulated via Sp1-binding motifs located proximal ( Since the u-PAR is a key protein in promoting tissue remodeling, it is
of essential importance to identify molecules regulating its
expression. One possible candidate is the c-src gene, which encodes the pp60c-src protein-tyrosine kinase,
since (a) its specific activity is higher in distant
metastases as compared with the primary colonic tumors (30) and
(b) this protein-tyrosine kinase stimulates in
vitro invasion of rat colonic cells (31). Therefore, we conducted the present study with the following objectives: 1) to determine whether a constitutively active Src regulates u-PAR gene expression, and 2) to elucidate the molecular mechanisms by which this occurs. Our
data show, for the first time, that the urokinase-receptor gene is
transcriptionally regulated by this protein-tyrosine kinase and that
this induction requires an upstream sequence ( Vectors, Reagents, and Antibodies--
The u-PAR CAT reporter
consists of 449 base pairs of sequence stretching from Cell Lines and Cell Culture--
SW480 colon adenocarcinoma
cells were grown in McCoy's 5A medium supplemented with 10% fetal
bovine serum. Stable constitutively active Src-expressing SW480 clones
were generated by transfecting SW480 cells with c-srcY527F using
LipofectAMINE (34). G418-resistant clones were generated and propagated
in the presence of 400 µg/ml G418.
Preparation of Nuclear Extracts and EMSA--
Nuclear extracts
and EMSA were carried out as described elsewhere (28). EMSA was carried
out as described (28, 29) using 10 µg of nuclear extract, 0.6 µg of
poly(dI-dC), and 2 × 104 cpm of a
[ Nuclear Run-on Experiments--
These were as described by us
previously (35). Nuclei from approximately 6 × 107
cells (SW480, 1D8, and 2C8) were isolated and incubated in the presence
of [ CAT Assays--
Cells were transfected at 60% confluence using
poly-L-ornithine as described previously (36). CAT assays
were performed as described by us previously (28). Where indicated,
transient transfections were performed in the presence of an RSV-driven luciferase expression vector (4 µg) for the determination of
transfection efficiencies. The amount of acetylated
[14C]chloramphenicol was determined using a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.
Luciferase Assays--
Luciferase assays for determining the
effect of c-srcY527F on the Determination of u-PAR Protein Amounts--
Western blotting was
performed as described previously (29, 37). Briefly, cells were
extracted into a Triton X-100-containing buffer supplemented with
protease inhibitors. Insoluble material was removed by centrifugation
and the cell extract immunoprecipitated with a polyclonal anti-u-PAR
antibody. The immunoprecipitated material was subjected to standard
Western blotting and the blot probed with 5 µg/ml of an anti-u-PAR
monoclonal antibody (3931; American Diagnostica, Greenwich, CT) and an
horseradish peroxidase-conjugated goat anti-mouse IgG. Bands were
visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).
For the determination of u-PAR by ELISA, resected tissue was prepared
and assayed as described by the manufacturer (American Diagnostica).
Laminin Degradation Assays--
These were carried out as
described previously (38). Cells were harvested with 3 mM
EDTA/phosphate-buffered saline (PBS), 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-PAR were
saturated by incubating the cells at 37 °C for 30 min with 5 nM urokinase and unbound plasminogen activator 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 (38).
Generation of Å5, a Urokinase Receptor Binding
Antagonist--
A urokinase receptor binding antagonist (Å5) was
developed based on the sequence of amino acids 20-30 of its ligand
(urokinase). This peptide was synthesized and cyclized using a covalent
linker, as described previously (39). Å5 was tested for its ability to
inhibit the binding of 125I-DFP-urokinase to RKO cells.
Cells (2 × 104/well) were seeded in a 48-well plate
and allowed to attach overnight at 37 °C. The cells were chilled to
4 °C, washed three times with cold PBS, and Å5 (diluted in
PBS/0.1% bovine serum albumin) added to the wells at various
concentrations (0.1-1000 nM).
125I-DFP-urokinase (0.5 nM final concentration)
was then added to each well. Unlabeled DFP-urokinase was used as a
positive control to calibrate the assay. The plate was incubated for
2 h at 4 °C, and the cells washed extensively, after which they
were lysed using 1 M NaOH and radioactivity in each lysate
counted using a Immune Complex Kinase Assays for Determination of Src
Activity--
These assays were performed as described elsewhere (34).
Briefly, cells were rinsed twice with 4 °C phosphate-buffered saline (PBS) and lysed in 250-500 µl of standard radioimmune precipitation buffer, and tumor and mucosal tissues were homogenized in radioimmune precipitation buffer using a Polytron homogenizer (Brinkmann
Instruments, Westbury, NY). Lysates were additionally homogenized using
an 18-gauge needle and clarified by centrifugation at 10,000 × g. 250 µg of protein was reacted with monoclonal antibody
327 (Oncogene Science Inc., Cambridge, MA) for 1 h for
immunoprecipitation of Src. Immune complexes were formed using 6 µg
of rabbit-anti-mouse IgG (Oreganon Teknika, Durham, NC) for 1 h,
followed by 50 µl of formalin-fixed Pansorbin
(Staphylococcus aureus, Cowan strain; Calbiochem,
La Jolla, CA) for 30 min. Pellets were washed three times in 0.1%
Triton X-100, 150 mM NaCl, and 10 mM sodium
phosphate. The kinase reaction was initiated by adding 10 µCi of
[
The amount of Src protein was determined by immunoblotting. Aliquots
(50 µg of protein) of cell lysate were resolved in an 8%
SDS-polyacrylamide gels and transferred to a nitrocellulose filter.
After blocking, the filter was incubated for 18 h at 4 °C with
1 µg/lane of mAb 327 (Oncogene Science Inc., Cambridge, MA) and
washed subsequently. Then, the filter was incubated with rabbit
anti-mouse IgG and reactive bands visualized with chemiluminescence (see "Determination of u-PAR Protein Amounts").
Statistical Analysis--
Statistical comparison of ELISA and
CAT results was done using a two-sided Student's t test
(SPSS for Windows statistical software, release 6.1.3, SPSS Inc.,
Chicago, IL). The correlation between u-PAR amounts and specific or
relative Src activity in tumor and colonic mucosa tissues was
determined by linear regression analysis and Pearson's correlation
coefficient. Statistical significance was defined at p < 0.05.
U-PAR Promoter Activity Is Increased by Transient Transfection of
SW480 Cells with an Expression Construct Encoding a Constitutively
Active Src Protein--
Since both Src activity (30) and u-PAR
mRNA/protein (20, 35) amounts are elevated in invasive colon
cancers, we hypothesized that this protein-tyrosine kinase could
regulate the expression of this receptor. To test this hypothesis, we
first transiently co-transfected SW480 cells with a CAT reporter driven
by 398 base pairs of 5'-flanking sequence of the u-PAR promoter and
increasing amounts of an expression construct encoding a constitutively
active Src (c-srcY527F) (Fig.
1A). A significant induction
of promoter activity was observed with 0.1-10 µg of c-srcY527F,
whereas the empty expression construct (pcDNA3) had minimal effect
on reporter activity. To exclude the possibility that the effect of the
constitutively active Src on u-PAR promoter activity was due to a
general induction of transcriptional activity, we performed a control
experiment comparing the effect of c-srcY527F transfection on a
luciferase reporter regulated by either the u-PAR or
To rule out the possibility that the induction of u-PAR expression by
the constitutively active Src was due to the introduction of a
transforming gene, we performed an experiment in which the u-PAR
promoter CAT reporter was co-transfected into SW480 cells with a
v-mos expression construct (33). A CAT reporter driven by
the urokinase promoter, which previously has been shown to be activated
by this construct, served as a positive control (Fig. 2). In contrast to the urokinase
reporter, which was activated up to 4-fold, no effect of this
non-membrane protein kinase on u-PAR promoter activity was observed.
Thus, we consider it unlikely that the induction of u-PAR promoter
activity in SW480 cells is a mere effect of expressing a transforming
gene.
u-PAR Gene Transcription and u-PAR-mediated Extracellular Matrix
Degradation Are Up-regulated in SW480 Clones Stably Transfected with a
Plasmid Encoding a Constitutively Active Src--
We then asked
whether the stable expression of the constitutively active Src induced
endogenous u-PAR gene expression. Toward this end, we compared the
amount of u-PAR protein in SW480 cells, characterized by its very low
Src-activity, with two derived clones (1D8, 2C8) stably expressing an
exogenous constitutively active Src (Fig.
3A). The growth rates of the
untransfected and Src-transfected cells were indistinguishable.
Analysis of cell extracts by ELISA indicated that the 2C8 clone
expressing the highest level of Src activity (Fig. 3A) had
8-fold more u-PAR protein (32 ± 5 ng of u-PAR protein/mg of
cellular protein) compared with the parental SW480 cells (4 ± 1 ng of u-PAR protein/mg of cellular protein) (Fig. 3B). The
1D8 clone, characterized by its intermediate Src activity, had an
intermediate amount of u-PAR protein (10 ± 2 ng of u-PAR
protein/mg of cellular protein). The ELISA data measuring u-PAR protein
were corroborated by Western blotting (Fig. 3C) in which the
highest and lowest amount of u-PAR protein was evident in the 2C8 clone
and the non-transfected SW480 cells, respectively. The diffuse nature
of the immunoreactive bands is probably due to the heavily glycosylated
state of the u-PAR protein (40).
To determine if the increased u-PAR protein was due to a higher
transcription rate, we performed nuclear run-on experiments comparing
parental SW480 cells and SW480 cells stably expressing either neo
(SW480 neo) or the constitutively active Src (1D8 and 2C8) (Fig.
3D). Hybridization of radioactive mRNA from isolated nuclei to a nylon filter-immobilized u-PAR cDNA yielded a signal with 2C8 cells, which was dramatically higher than that achieved with
SW480 cells transfected with nothing (SW480) or the neo gene (SW480
neo). Thus, u-PAR gene transcription is induced by an activated Src.
One of the functions of u-PAR is to accelerate
plasminogen-dependent proteolysis via receptor-bound
urokinase. Accordingly, we used laminin degradation as a biological end
point to measure the effect of the src transfection on the
display of the urokinase binding site. Toward this end, we performed
laminin degradation assays comparing the activity of SW480, 1D8, and
2C8 cells. SW480 and 2C8 cells in serum-free medium (
These data suggested that the stable transfection of a low
u-PAR-expressing colon cancer cell line with a plasmid encoding a
constitutively active Src results in transcriptional activation of the
u-PAR gene, the latter in proportion to the amount of Src activity
detected in the individual clones.
A Specific Src Inhibitor Reduces u-PAR Expression and
u-PAR-mediated Extracellular Matrix Degradation--
The results
presented so far suggested that the expression of the u-PAR gene is
transcriptionally induced by expressing a constitutively active Src
cDNA in SW480 colon cancer cells. As a corollary to these
experiments, we asked whether the inhibition of Src activity in the
SW480 cells stably expressing the constitutively active Src would
result in a decrease in u-PAR protein amounts, promoter activity, and
laminin degradation. 2C8 cells (the SW480 transfected clone showing the
highest Src activity) were treated with varying amounts of PD173955
(41), which specifically targets Src family members, and assayed for
u-PAR protein by ELISA. Increasing amounts of the PD173955 led to a
dose-dependent decrease in the amount of the urokinase
receptor (Fig. 5A). Similarly,
an identical concentration range of the Src inhibitor repressed u-PAR
promoter activity (Fig. 5B). We did not observe changes in
growth rate or morphological signs of cell toxicity using this
concentration range (data not shown). These results were paralleled by
a dose-dependent attenuation in endogenous Src activity
(Fig. 5C) but not in Src protein levels (Fig. 5D)
as demonstrated by immunocomplex kinase assay. We also used laminin
solubilization as a biological end point for measuring u-PAR display.
Treatment of 2C8 cells with concentrations of PD 173955 that inhibited
u-PAR protein amounts blocked laminin solubilization. Similarly, a
specific u-PAR inhibitor (Å5) effectively countered (Fig.
6) laminin degradation brought about by
2C8 cells incubated with plasminogen, indicating that the laminin
degradation achieved was indeed due to cell surface u-PAR display. The
effect of the PD173955 on laminin degradation was greater than would be
expected based on the reduction in u-PAR protein (Fig. 5A).
We can only speculate that this is due to the repression of endogenous
urokinase, which is also regulated by v-src (42). Thus, the
reduced synthesis of the protease would be expected to result in fewer
u-PAR molecules bound with the endogenous enzyme despite the
preincubation of cells with exogenous urokinase (since the half-life of
receptor-bound urokinase is shorter than the duration of the laminin
degradation assay (140 (Ref. 43) and 240 min, respectively).
Nevertheless, these data provide further support for the contention
that the u-PAR gene expression is regulated by a constitutively active
Src.
The Induction of u-PAR Promoter Activity by the Constitutively
Active Src Requires an Upstream Region (
SW480 cells were transiently transfected with a CAT reporter driven by
either the u-PAR promoter (u-PAR CAT) or a thymidine kinase (tk)
minimal promoter flanked by the
Since the promoter region
Supershifting experiments were performed to confirm the identity of the
slowest migrating band (Fig. 8B). In the absence of antibody, the intensity of the slowest migrating band (marked with a
bracket) was again substantially higher in nuclear extracts from the src transfectants (1D8 and 2C8) compared with the
untransfected SW480 cells (compare lanes 1,
8, and 15). Addition of an anti-Sp1 antibody
(lanes 4, 11, and 18)
supershifted this complex (indicated with a line) and at the
same time abolished the shifted band (indicated with
bracket) (Fig. 8B), indicating that the DNA-bound
complex contains largely Sp1. On the other hand, antibodies to Sp2,
Sp3, and Sp4 had no effect in the EMSA.
We next determined the requirement of the Sp1-like binding motif in
this region of the u-PAR promoter for the induction by src.
Toward this end, the effect of the constitutively active src
expression construct on the activity of a CAT reporter driven by either
the wild type u-PAR promoter or the promoter in which nucleotide
substitutions (29) abolishing the binding of the Sp1-transcription
factor was determined. We found in duplicate experiments (Fig.
9) that the promoter, that was unable to
bind Sp1 in the region spanning The Amount of u-PAR Protein Correlates with Src Activity in
Resected Colon Cancer--
Since our data were generated using a
cultured colon cancer cell line, it was possible that the increased
u-PAR expression brought about by src was unique to a tissue
culture environment. To address this possible criticism, we analyzed
homogenates of human colon carcinomas, each of them paired with its
corresponding adjacent non-malignant tissue, for endogenous Src
specific activity (ratio of Src autophosphorylation/Src protein level)
and the amounts of endogenous u-PAR protein. A representative
autoradiograph of Src kinase activity and corresponding u-PAR protein
amounts is shown in Fig.
10A. For patients 1, 5, 6, and 10 (Fig. 10A), both Src activity and u-PAR protein were
higher in the resected tumors compared with the normal adjacent tissue
(Fig. 10A). In contrast, in patients 7 and 8, Src activity
and u-PAR protein amounts were low in both the tumor and the normal
tissue. There was a significant correlation between the amounts of
endogenous u-PAR protein and Src specific activity when analyzing both
tumor and normal tissue (correlation coefficient 0.8258, p < 0.001) by linear regression, Fig. 10B).
These data indicate that src-dependent induction
of u-PAR gene expression in cultured colon cancer cell lines is a reproducible observation in resected colon cancer.
The u-PAR plays a critical role in cellular invasion in normal
tissues undergoing remodeling and in malignancy for infiltrative cancer. In the present study, we have made a novel finding that a
constitutively active Src transcriptionally induces the expression of
the u-PAR. Further, we show for the first time that the inductive effect of the constitutively active Src on the expression of a gene is
mediated, at least in part, via increased Sp1 binding to the promoter.
Interestingly, in addition to modulating u-PAR expression, this
protein-tyrosine kinase up-regulates the production of several proteases. Thus, Sato et al. (44) demonstrated that
v-src elevated the expression of the 92-kDa type IV
collagenase and in a separate report, Ishidoh et al. (45)
found that cathepsin L, was produced in larger amounts in rat
fibroblasts transformed with this oncogene. Equally important,
v-src also increases the expression of urokinase, which is
the ligand for the u-PAR, and this induction requires the catalytic
activity of the protein-tyrosine kinase and its plasma membrane
localization (42, 46). The observation that src can
up-regulate the u-PAR as well as several proteases is of particular
interest, since it suggests a coordinate means of regulating a set of
genes required for the invasive phenotype.
Considering the evidence presented herein implicating the
constitutively active Src in regulating u-PAR expression, how then is
this achieved? It was apparent from the current study that Sp1 bound to
the u-PAR promoter sequence Considering the pivotal role of this transcription factor in mediating
the inductive effect of Src on u-PAR gene expression, how is the Sp1
altered to effect this stimulation? Our data showed that nuclear
extracts from the src-transfected SW480 cells demonstrated increased binding of the Sp1 to region The finding that region We recently demonstrated the requirement of an AP-2 Interestingly, while we provide strong evidence for a role of a
constitutively active src in the regulation of u-PAR
expression, there is ample evidence for a reciprocal interaction at the
protein level. Thus, in the fibrosarcoma cell line HT 1080, Src family members co-immunoprecipitate with u-PAR, and one of these,
hck, is activated by receptor-bound urokinase (54).
Association of Src family members and tyrosine phosphorylation after
urokinase stimulation was also reported for monocytes (55). In bovine endothelial cells, it has been demonstrated that focal adhesion kinase
is tyrosine-phosphorylated upon urokinase stimulation of u-PAR
localized at focal cell contacts, the former of which is an effector of
Src kinases. All of these findings suggest that Src family members play
an important role in u-PAR signaling. Our findings now indicate, that
in addition to Src being a downstream target of u-PAR, there is a
reciprocal relationship in which the latter is regulated by the former.
In conclusion, we have shown that a constitutively active Src induces
the expression of the u-PAR gene. Equally important, we have also
demonstrated the involvement of the Sp1 transcription factor in the
regulation of src-dependent gene expression.
Since other invasion-promoting genes, including cathepsin L, type IV collagenase, and urokinase, are also regulated by src, our
results raise the exciting possibility that antagonizing this
non-receptor protein-tyrosine kinase could coordinately repress their
expression resulting in a suppression of the metastatic phenotype.
The urokinase CAT reporter was kindly
provided by Dr. Francesco Blasi, University of Milan, Italy. H. A. thanks Professor Dr. Dr. h. c. F. W. Schildberg and Dr. M. M. Heiss (Dept. of Surgery, Klinikum Grosshadern, Ludwig-Maximilians
University of Munich, Munich, Germany), as well as Dr. Ernst R. Lengyel
(Department of Gynecology and Obstetrics, Technical University of
Munich, Munich, Germany), for their continuous invaluable support and input. We thank Hector D. Avila, Parham Khanbolooki, and Barbara Young
for excellent technical assistance. We thank Dr. Doris Siwak, Kayo
Nakano, and Nila Parikh for invaluable discussions and technical help.
*
This work was supported by National Institutes of Health
Grants R01 CA58311, R01 DE10845, and P01 DE11906 and a Physician's Referral Service grant (all to to D. B.); National Institutes of
Health Grants CA65527 and 63617 (to G. G.); and a fellowship from
the Dr. Mildred Scheel Cancer Foundation (Deutsche Krebshilfe, Bonn,
Germany) (to H. A.).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.
§
Present address: Parke-Davis Pharmaceutical Research (Div. of
Warner-Lambert Co.), Ann Arbor, MI 48105.
¶
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.
The abbreviations used are:
u-PAR, urokinase-type plasminogen activator receptor;
AP-1, activator
protein-1;
AP-2, activator protein-2;
CAT, chloramphenicol
acetyltransferase;
DFP, diisopropyl fluorophosphate;
EMSA, electrophoretic mobility shift assay;
ELISA, enzyme-linked
immunosorbent assay;
PBS, phosphate-buffered saline;
RSV, Rous sarcoma
virus;
tk, thymidine kinase.
Transcriptional Induction of the Urokinase Receptor Gene by a
Constitutively Active Src
REQUIREMENT OF AN UPSTREAM MOTIF (
152/135) BOUND WITH
Sp1*
,,
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152/135) in constitutive urokinase receptor expression,
we determined its role for the induction by src. Whereas
the activity of a CAT reporter driven by this region was stimulated by
c-srcY527F, the u-PAR promoter mutated at the Sp1-binding motif in the
152/135 region was not. Nuclear extracts from the src
transfectants demonstrated increased Sp1 binding to region 152/135
compared with those from SW480 cells. Finally, endogenous urokinase
receptor protein amounts in 10 colon cancers and corresponding normal
colon correlated with Src specific activity. These data suggest that
urokinase receptor gene expression is regulated by Src partly via
increased Sp1 binding.
INTRODUCTION
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2-macroglobulin receptor-dependent
endocytotic mechanism (10, 11). Third, the u-PAR interacts with the
extracellular domain of integrins allowing association with the
cytoskeleton, thereby mediating cell adhesion and migration (12-14).
Fourth, the u-PAR is chemotactic for human monocytes and mast cells,
and this may contribute to inflammatory and tissue remodeling
processes, which are also often observed in tumor infiltrated areas (6, 15).
110/24) to the transcriptional start site.
Our laboratory showed that both the constitutive and phorbol 12-myristate 13-acetate-inducible expression of the gene required a
footprinted region (190/171) of the promoter containing an AP-1
motif (28). A second region of the promoter (152/135) bound with an
AP-2-related protein was also demonstrated to be required for the
constitutive u-PAR gene expression and moreover for u-PAR mediated
extracellular matrix degradation (29).
152/135) bound with Sp1.
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398 to +51
(relative to the transcription start site) cloned into the
XbaI site of the pCAT-Basic vector (Promega, Madison, WI).
The u-PAR-firefly luciferase reporter was generated by cloning the
aforementioned sequence into the SmaI site of pGL3 (Promega). The R2 CAT reporter construct, consisting of an
oligonucleotide spanning nucleotides 154/128 of the u-PAR promoter,
was as described previously (29). The urokinase CAT reporter included
2345 base pairs of 5'-flanking region of the urokinase promoter fused
directly to the reporter. The c-srcY527F construct contained the
c-src coding sequence (32) harboring a tyrosine to
phenylalanine substitution at codon 527 and cloned into the
HindIII/BamHI cloning site of pcDNA3. The
-actin-renilla-luciferase reporter plasmid was kindly provided by
Dr. Menashe Bar-Eli (Department of Cancer Biology, M. D. Anderson Cancer Center, Houston, TX). The v-mos expression construct was as described elsewhere (33). Antibodies to Sp1, Sp2, Sp3,
and Sp4 were purchased from Santa Cruz Biotechnology. Oligonucleotides
were supplied by Genosys Biotechnologies, The Woodlands, TX.
-32P]ATP T4 polynucleotide kinase-labeled oligonucleotide.
-32P]UTP in transcription buffer (150 mM KCl, 5 mM MgCl2, 1 mM MnCl2, 20 mM HEPES, pH 7.9, 10%
glycerol, 5 mM dithiothreitol). Nuclei were then treated
with DNase I and proteinase K, and the RNA extracted with
phenol/chloroform and precipitated. Radioactive RNA (6.6 × 107 cpm) was hybridized to nylon-immobilized u-PAR
cDNA, glyceraldehyde-3-phosphate dehydrogenase cDNA, and the
linearized empty vector for the u-PAR cDNA (pBC12B1) as a control.
-actin promoter were performed using the
Dual Luciferase Reporter Assay System by Promega according to the
manufacturer's protocol.
counter. These experiments indicated the
IC50 of Å5 to be approximately 11 nM.
-32P]ATP, 10 mM Mg2+, 10 µg
of rabbit muscle enolase (Sigma) as an exogenous substrate, and 100 µM sodium orthovanadate in 20 mM HEPES. After
10 min, reactions were terminated by adding SDS sample buffer. Products were separated in an 8% polyacrylamide gel and exposed to an x-ray film overnight.
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-actin promoter
(Fig. 1B). -Actin is a principal component of
microfilaments and abundantly expressed in non-muscle cells. In
contrast to a strong dose-dependent induction of u-PAR
promoter activity, the activity of the -actin promoter was unchanged
by the expression of the c-srcY527F construct. Thus, it is unlikely
that the increased u-PAR promoter activity brought about by the
src expression construct is due to a general effect on
transcription. The dose dependence of the src expression construct on u-PAR promoter activity was more evident using the luciferase reporter compared with the CAT reporter. This is probably a
consequence of substrate depletion of the radioactive chloramphenicol in the latter assays.
View larger version (34K):
[in a new window]
Fig. 1.
u-PAR promoter activity is induced by
transient transfection of SW480 cells with a plasmid encoding a
constitutively active Src. Panel A, SW480
cells were transiently transfected with a CAT reporter regulated by 398 base pairs of the urokinase receptor promoter (u-PAR
CAT) and equal amounts of either the empty vector
(pcDNA3) or the c-srcY527F plasmid (Src
Y527). Parallel dishes of SW480 cells were transfected with
pRSV CAT or pSV0CAT as positive and negative transfection controls.
After 48 h, cells were harvested and assayed for CAT activity.
Chloramphenicol conversions are given as mean ± S.D. Differences
in chloramphenicol-conversion were significant (p < 0.05) for the paired experiments indicated (*). Panel
B, SW480 cells were transiently co-transfected with a
luciferase-reporter driven by either the -actin promoter or the
u-PAR promoter and the indicated amounts of empty vector (
) or
c-srcY527F (
). Control transfections were performed in parallel
dishes with the promoterless luciferase reporters for the -actin
reporter (pRLnull) and the u-PAR reporter (pGL3). The experiment was
done at least twice.
View larger version (42K):
[in a new window]
Fig. 2.
The u-PAR promoter induction brought about by
c-srcY527F is not a general effect of expressing a transforming
gene. SW480 cells were transiently co-transfected with either a
CAT reporter driven by the urokinase (urokinase
CAT) or the u-PAR (u-PAR CAT) promoters and
either the empty vector (pkSV10) or v-mos
expression plasmid (wt mos pkSV10) in the amounts indicated.
Positive (RSV CAT) and negative
(pSV0CAT) transfection controls were performed with parallel
dishes. The experiment was carried out twice. The range of
chloramphenicol conversions is indicated.
View larger version (26K):
[in a new window]
Fig. 3.
Endogenous u-PAR gene expression is elevated
in SW480 clones stably expressing a constitutively active Src.
Panel A, cellular extracts (equal protein) were
assayed for Src activity by an immunocomplex kinase assay.
Panel B, cells, at 90% confluence, were
harvested and lysates assayed for endogenous u-PAR protein by ELISA.
The data represent three different experiments. Differences in amounts
between SW480 and 1D8 as well as between SW480 and 2C8 are significant
at p < 0.05 (Student's t test).
Panel C, cells were immunoprecipitated with an
anti-u-PAR antibody and assayed for u-PAR protein by Western blotting.
+ve Control represents extract of the RKO cell line known to
express high amounts of u-PAR (28). The immunoprecipitating antibody is
indicated. The experiment was done twice. Panel
D, nuclei were isolated and incubated with radioactive UTP.
Radioactive RNA (107 cpm/reaction) was incubated with
nylon-immobilized cDNAs corresponding to u-PAR,
glyceraldehyde-3-phosphate dehydrogenase, and empty vector. The
experiment was performed twice.
, 
)
demonstrated minimal solubilization of laminin in the absence of the
zymogen, plasminogen (Fig. 4). After
addition of plasminogen, SW480 cells showed a moderate degradation of
laminin (50,000 cpm/106 cells) in the culture supernatant
after 240-min incubation (×). However, addition of plasminogen to the
src transfectants resulted in a much stronger increase in
laminin solubilization indicating plasmin-dependent
proteolysis. In this respect, the 2C8 clone, characterized by the
highest Src activity, showed the highest amount of solubilized laminin
in the supernatant (> 200,000 cpm/106 cells after 240 min;
) with the 1D8 clone being intermediate (130,000 cpm/106
cells; ).
View larger version (18K):
[in a new window]
Fig. 4.
Increased laminin degradation by SW480
clones expressing a constitutively active Src. SW480, 1D8, and 2C8
cells plated in serum-free medium on radioactive laminin-coated dishes
were subjected to a laminin degradation assay. At the indicated times,
aliquots of the culture supernatant was harvested and counted for
radioactivity. After 240 min, cells were enumerated. The data are
typical of duplicate experiments. 
, serum-free medium, plasminogen
treatment; , SW480 control; , 2C8 control; ×, SW480, plasminogen
treatment; , ID8, plasminogen treatment; , 2C8, plasminogen
treatment.
View larger version (36K):
[in a new window]
Fig. 5.
A specific Src inhibitor counters u-PAR gene
expression in 2C8 cells. Panel A, u-PAR
protein amounts (ng/mg protein) as measured by ELISA in 2C8 cells
treated with increasing amounts of PD173955 for 48 h.
Panel B, 2C8 cells were transiently transfected
with the u-PAR promoter CAT reporter and subsequently treated with
either the carrier (DMSO) or the Src inhibitor PD173955 in
the amounts indicated for 48 h. Cells were harvested and analyzed
for CAT activity. The range of chloramphenicol conversions as measured
by a phosphorimager are indicated. Panels C and
D, 2C8 cells were treated with PD173955 as described in
panel A and assayed for Src activity
(panel C) and protein (panel
D) by immunocomplex kinase assay and Western blotting,
respectively. The experiments were done twice.
View larger version (18K):
[in a new window]
Fig. 6.
PD173955 inhibits u-PAR mediated laminin
degradation. 2C8 cells treated for 48 h with
Me2SO ( ); 75 (), 150 (), or 750 (
)
nM amounts of PD173955; or specific u-PAR inhibitor Å5 at
50 (×) and 100 () nM were subjected to a laminin
degradation assay in the presence of plasminogen and as described for
Fig. 4. The data are representative of two different experiments.
152/135) Bound with
Sp1--
We next sought to determine which cis-elements of
the u-PAR promoter and transcription factors were involved in the
transcriptional induction of u-PAR gene expression by c-srcY527F. In a
recent report (29), we demonstrated that constitutive u-PAR promoter activity in another cell line (RKO) is, at least in part, due to a
DNase I-footprinted region of the promoter spanning 152/135 (relative to the transcription start site). This region of the promoter
contained overlapping non-canonical Sp1 and AP-2 motifs (29). To
determine the potential role of this region in mediating the induction
of u-PAR expression in response to the constitutively active Src, the
following experiments were carried out.
152/135 u-PAR promoter region
(R2-CAT) with, or without, the constitutively active src expression plasmid (Fig. 7). In the absence of the expression construct, the activity of the R2-CAT was lower than that achieved with
the u-PAR CAT. This is to be expected, since we have found that
transactivation of the u-PAR gene requires multiple cis
elements (28). More importantly, the c-srcY527F caused an increased
activity of the CAT reporter driven by either the 152/135 region
(R2-CAT) or the wild type u-PAR promoter (u-PAR CAT) (Fig.
7). This increase in activity of the
R2-CAT reporter was not due to an activation of the minimal tk-promoter
since the expression construct encoding the constitutively active Src
did not increase CAT activity in cells co-transfected with the tk-CAT
reporter.
View larger version (37K):
[in a new window]
Fig. 7.
Stimulation of a CAT reporter regulated by
region 152/135 of the u-PAR promoter. SW480 cells were
transiently co-transfected with a CAT reporter regulated by either 398 base pairs of 5'-flanking (upstream of the main transcriptional start
site) sequence (u-PAR CAT) or by a sequence of
the u-PAR promoter spanning nucleotides 154/128 (R2
CAT) and the indicated amount of empty vector
(pcDNA3) or an expression vector bearing the
constitutively active Src (SrcY527). After 48 h, cells
were harvested and assayed for CAT activity. As a positive control,
SW480 cells were transfected with pRSVCAT. The data are typical of
triplicate experiments and are given as mean ± S.D. The
inductions achieved with the src expression construct were
significant (p < 0.05) for the paired experiments
indicated (*).
152/135 contained putative Sp1 and AP-2
binding motifs, we considered the possibility that the corresponding
transcription factors were bound. To explore this possibility, we
performed EMSA experiments comparing equal protein amounts of nuclear
extracts from SW480, SW480 neo, 1D8, and 2C8 cells for transcription
factor binding to an oligonucleotide spanning this region of the u-PAR
promoter. At least three bands of slower mobility, which could be
competed by a 100-fold excess of unlabeled oligonucleotide, were
observed with extracts of parental SW480 cells and
src-transfected clones (Fig.
8A). However, 2C8 cells (which
contain the highest Src activity) demonstrated an increased intensity
(lane 5) of the slowest migrating complex (marked
with a bracket) when compared with that achieved with SW480
cells (lane 7) or SW480 neo cells
(lane 1). 1D8 cells, characterized by their intermediate Src activity, showed a band (bracket) intensity
(lane 3) between the 2C8 and the SW480/SW480 neo
cells (Fig. 8A). On the other hand, the intensity of the
fastest-migrating band (marked with an *), presumed to be the
AP-2-related factor (29), was similar (compare lanes
1, 3, and 5) between the SW480 neo
cells and the src-transfected derivatives (1D8 and 2C8). As
a control, nuclear extract from 2C8 cells was incubated with the
radioactive 154/128 probe and a 100-fold excess of an
oligonucleotide (394/378) corresponding to a sequence of the u-PAR
promoter further upstream. This unlabeled competitor had no discernible
effect (compare lanes 9 and 11) on the
shifted bands (Fig. 8A).
View larger version (47K):
[in a new window]
Fig. 8.
Nuclear extracts from
src-expressing SW480 cells demonstrate increased
binding of Sp1 to an oligonucleotide spanning region 152/135.
Panel A, equal protein amounts of nuclear extract
were incubated with an end-labeled oligonucleotide (154/128 u-PAR)
in the presence, or absence, of a 100-fold excess of the unlabeled
154/128 or 394/378 competitor sequences. The experiment was
carried out three times. Panel B, nuclear
extracts were treated as above with the exception that the indicated
antibodies or oligonucleotide competitors were added and complexes
subsequently analyzed by gel electrophoresis. Data are representative
of three different experiments.
152/135 (u-PARSp1 mt CAT), had
almost lost the ability to be induced by the src expression
construct. In contrast, the wild type-u-PAR promoter was stimulated up
to 6-fold. Similarly, we were unable to detect stimulation of the u-PAR
promoter harboring nucleotide substitutions (u-PARAP-2/Sp1 mt CAT),
which simultaneously prevented the binding of the AP-2-related factor and Sp1. We have been unable at the present time to generate a
mutation that destroys the binding of the AP-2-related factor but
preserves Sp1 binding, this presumably reflecting the fact that these
motifs are overlapping (29). Nevertheless, taken together, these
experiments suggest that a region of the u-PAR promoter spanning
152/135 and bound with Sp1 is required and sufficient for the
induction of the gene by the constitutively active src.
View larger version (44K):
[in a new window]
Fig. 9.
The abolition of Sp1 binding to region
152/135 counters u-PAR promoter induction by the constitutively
active Src. SW480 cells were transiently transfected with the wild
type (u-PAR CAT) or Sp1-mutated (u-PARSp1 mt CAT)
or AP-2/Sp1 mutated (u-PARAP-2/Sp1 mt CAT) u-PAR promoter
with, or without, the empty expression vector (pcDNA3)
or c-srcY527F (Src Y527). After 48 h, the
cells were harvested, lysed, and assayed for CAT activity. Parallel
dishes were transfected with RSV CAT and pSV0CAT as positive and
negative controls, respectively. Chloramphenicol conversions are shown
as range of duplicate experiments.
View larger version (61K):
[in a new window]
Fig. 10.
Correlation of u-PAR protein amounts
with Src activity in resected colon cancer. Panel
A, resected colonic tumors (T) or adjacent
non-malignant mucosa (N) were assayed for Src activity and
u-PAR protein by immunocomplex kinase assay and ELISA, respectively.
Autoradiographs of kinase assays show autophosphorylation of the 60-kDa
Src and phosphorylation of enolase its 44-kDa substrate.
Panel B, linear regression analysis correlating
u-PAR protein amounts with Src specific activity (ratio of Src
autophosphorylation to Src protein. Both parameters were quantified by
densitometry). The data include, but are not limited to, patient data
from panel A. The positive correlation between
u-PAR amounts and Src activity is significant with p < 0.001 and Pearson's correlation coefficient = 0.8258.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
152/135 plays a critical role in the
src-dependent induction of u-PAR expression.
This finding was initially surprising, since this transcription factor is known more for its role in regulating the constitutive expression of
housekeeping genes. As mentioned above, v-src up-regulates several genes, including urokinase, the 92-kDa type IV collagenase, cathepsin L, and prostaglandin synthase 2 (42, 44, 45, 47). However, in
those studies, there was little evidence for a role of Sp1 in the
regulation of gene expression. Thus, Sato and co-workers (44) reported
that the mutation of an Sp1 binding site in the 92-kDa type IV
collagenase had little effect on the induction of this
metalloproteinase by v-src. Further, while
v-src-expressing fibroblasts contained Sp1-like factors
bound to the cathepsin L promoter, the amounts complexed were similar
using nuclear extracts from the non-transfected counterparts (45) and
mutational analysis was not carried out to determine the role of these
factors in mediating the inductive effect of the protein-tyrosine
kinase. Nevertheless, there are an increasing number of reports in
which Sp1 is required for inducible gene expression brought about by other stimuli. For example, platelet thromboxane receptor expression is
elevated in response to the phorbol ester phorbol 12-myristate 13-acetate, and this induction has been ascribed to a 14-nucleotide response element bound with Sp1 and located over 1900 base pairs upstream from the transcriptional start site (48). Likewise, Sp1
cooperates with AP-1 to up-regulate the expression of CD11c expression
in myeloid cells (49) and the disruption of two Sp1 binding sites in
the promoter of the 12-lipoxygenase gene reduces the epidermal growth
factor-induced expression of this gene (50). Thus, taken together, our
data indicate a novel role for Sp1 as a regulator of inducible gene
expression in response to src.
152/135 of the u-PAR promoter when compared with nuclear extracts from the parental cells.
This augmented binding could be a result of increased synthesis of the
Sp1 or could reflect a higher DNA binding affinity. While we are unable
to distinguish between these two possibilities at the present time, it
is worth noting that TGF- stimulates 2 (I) collagen expression by
increasing the affinity of an Sp1-containing protein complex for its
cognate DNA-binding site (51). On the other hand, to the knowledge of
the authors, there are no studies to date showing increased production
of the transcription factor brought about by Src.
152/135 of the u-PAR promoter was
stimulated by the constitutively active Src in reporter assays is
consistent with the notion that this region is sufficient to mediate
the inductive effect of the src on u-PAR expression. Indeed, we observed up to an 8-fold induction in activity of a CAT reporter construct regulated by this region compared with an ~10-fold
elevation in u-PAR transcription as judged by nuclear run-on
experiments. Nevertheless, it is still possible that additional
cis-elements also contribute to elevated u-PAR expression
brought about by src. For example, since it is well
established that Src up-regulates the activity and/or binding of AP-1
transcription factors (46, 52, 53) and that the expression of the u-PAR
gene is modulated by an AP-1 motif located at 184 (28), it is very
plausible that the inductive effect of Src on u-PAR expression also
requires AP-1-binding transactivators. Indeed, we did see an inhibition of src-mediated u-PAR promoter induction when the promoter
was mutated at the AP-1 consensus motif described previously (data not
shown). Thus, it is more likely that elevated u-PAR expression brought
about by the constitutively active Src is the result of the concerted
action of multiple transcription factors including Sp1 binding to a
region of the promoter spanning 152/135.
-related factor
bound to the 152/135 region of the u-PAR promoter for the
constitutive expression of the binding site in the RKO colon cancer
cell line (29). Although it was apparent from EMSA that this
transcription factor was bound to the u-PAR promoter, it does not
appear to have a major role in the induction of u-PAR expression by
src. Thus, the amount bound in mobility shift assays did not
differ between the src transfectants and the untransfected SW480 cells. Further, site-directed mutagenesis of the u-PAR promoter, which maintained the binding of this AP-2-related factor but which
destroyed Sp1 binding, practically abolished promoter stimulation by
src. We can only speculate that the utilization of different transcription factors reflects the different molecular mechanisms by
which u-PAR expression is regulated in the constitutive setting and in
response to src expression. Alternatively, it may be that this difference is a consequence of the separate cell lines used in the
two studies.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Ångstrom Pharmaceuticals, San Diego, CA 92121.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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