From Cancer Research Laboratories, Botterell Hall, Queen's University, Kingston, Ontario, K7L 3N6 Canada
Received for publication, November 28, 2000, and in revised form, January 2, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously shown coexpression of
hepatocyte growth factor (HGF) and its receptor Met in the
invasive tumor front of human breast carcinomas. We have also
demonstrated secretion of HGF, constitutive activation of Met, and
increased invasion in a murine breast carcinoma cell line, SP1. These
observations suggest the presence of an HGF autocrine loop in some
breast carcinoma cells, which confers increased survival, growth, and
invasiveness during tumor progression and metastasis. c-Src tyrosine
kinase, which is critical in regulating the expression of many genes, is activated in SP1 carcinoma cells, as well as in most human breast
cancers. We therefore examined the role of c-Src kinase in HGF
expression in breast carcinoma cells. Expression of activated c-Src in
SP1 cells increased transcription from the HGF promoter and
expression of HGF mRNA and protein, while dominant negative c-Src
had the opposite effect. Using deletion analysis, we showed that the
region between Scatter factor, also known as hepatocyte growth factor
(HGF),1 is a multifunctional
cytokine. Through binding to its receptor (Met), HGF can induce cell
survival (1), growth (2), differentiation (3), and motility (4). It has
been shown that both HGF and Met are essential for embryo development.
Disruption of HGF expression in mice results in lethality in early
development (5), while deletion of Met causes underdevelopment of limb
buds (6). During development of the mammary gland, HGF is expressed by
stromal cells, whereas epithelial cells express Met, but not HGF (7). Paracrine stimulation of normal breast epithelium with HGF, in cooperation with other growth factors (e.g. neuregulin),
promotes branching morphogenesis (8). The tissue-specific suppression of HGF expression in normal epithelial cells provides a tightly controlled regulation of mammary ductal morphogenesis (9).
In contrast to normal breast epithelium, HGF and Met are frequently
overexpressed in breast carcinomas (10-12) as well as many other
cancer types (10, 11, 13, 14). This high level of HGF and Met
expression has been identified as a possible independent predictor of
poor survival in breast cancer patients (11). Our laboratory has
previously shown that invasive human carcinoma cells coexpress HGF and
Met, particularly at the migrating tumor front (12). We have also found
that breast carcinoma cell lines frequently express HGF and Met,
whereas most nonmalignant epithelial cell lines express Met, but not
HGF.2 Furthermore,
overexpression of HGF or a constitutively active mutant form of Met
(Tpr-Met) in transgenic mice (15, 16) or in transformed cell lines (17,
18) promotes tumorigenesis and metastasis. Together, these results
suggest that establishment of an autocrine HGF loop and sustained
activation of the Met signal transduction pathway in carcinoma cells
may promote tumor progression. However, the mechanisms leading to
aberrant expression of HGF in carcinoma cells are not known.
A number of signaling molecules, such as c-Src (19), Grb2/Ras (17), and
phosphatidylinositol 3-kinase (1), have been shown to be part of
the HGF/Met signaling pathway. Activation of Met through binding of HGF
causes autophosphorylation of two specific tyrosine residues in the
cytoplasmic tail of the receptor tyrosine kinase (20). These
phosphorylated tyrosine residues act as multifunctional docking sites
that bind the SH2 domain of specific cytoplasmic signaling molecules
and causes their activation. The c-Src nonreceptor tyrosine kinase is
expressed in many cell types, and its activity is increased in response
to HGF and binding to Met (19). Increased activation of the tyrosine
kinase c-Src occurs in many human cancer cells, and c-Src plays a
critical role in breast cancer. Overexpression of an activated form of c-Src in transgenic mice induces mammary hyperplasia (21). Furthermore, c-Src kinase is required in polyoma middle T-induced mammary
tumorigenesis in transgenic mice (22). We have shown previously that
c-Src kinase is constitutively activated in a mouse breast carcinoma cell line, SP1, which expresses both HGF and tyrosine-phosphorylated Met and which exhibits spontaneous invasion through matrigel (19, 23,
24). Furthermore, c-Src kinase activity is required for HGF-dependent cell motility and anchorage-independent
growth of SP1 cells (19). Collectively, these findings indicate that
c-Src kinase is an important requirement, but is not sufficient, for mammary tumorigenesis.
Activation of c-Src kinase can lead to increased expression of many
genes, including growth factors such as vascular endothelial growth
factor (25, 26) and parathyroid hormone-related peptide (27). We
therefore hypothesized that elevated c-Src activity can promote
increased HGF expression and the establishment of an HGF autocrine loop
in SP1 cells. We observed that the c-Src tyrosine kinase inhibitor PP2
causes a 2-fold reduction in HGF transcription in SP1 cells.
In addition, expression of a dominant negative mutant of c-Src (SRC-RF)
in SP1 cells leads to similar levels of reduction in HGF mRNA and
functional protein. Using deletion mutants of the HGF
promoter, we have located a region (between Antibodies and Reagents--
Rabbit anti-c-Src IgG was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody
EC10 against chicken c-Src was a gift from Dr. S. Parsons. Rabbit
anti-sheep IgG conjugated with horseradish peroxidase was from Jackson
ImmunoResearch Laboratories (West Grove, PA). Sheep anti-HGF IgG was a
gift from Genentech (San Francisco, CA). Rabbit anti-HGF antibody was
generated against recombinant glutathione
S-transferase-HGF-(1-120) protein in our laboratory
at Queen's University, this antibody recognizes only the N-terminal
portion of HGF (data not shown). Anti-Stat1, -Stat3, -Stat5A, and
-Stat5B and anti-phospho-Stat3 (Y705) antibodies were obtained from
Upstate Biotechnology (Lake Placid, NY). c-Src family kinase inhibitor
PP2 was obtained from Calbiochem (La Jolla, CA).
Plasmid Construction--
c-Src expression plasmids were
constructed by subcloning activated (Y527F) and dominant negative
(K295R,Y527F) chicken c-src cDNAs (gift from Drs. J. Brugge and D. Shalloway) into the EcoRI site of DNA
polymerase I (Klenow fragment)-treated pBabePuro plasmid to generate
pBabe Y527F and pBabe Src-RF. A reporter construct containing the
full-length HGF promoter region fused to luciferase (2.7 HGF-luc) was constructed by ligating the
HindIII/XbaI fragment (treated with DNA
polymerase I (Klenow fragment)) of 2.8 HGF-CAT (gift from Dr. R. Zarnegar) into the HindIII site of pGL2-Basic (Promega),
also treated with DNA polymerase I (Klenow fragment). Further deletions
were constructed by cutting 2.7 HGF-luc with SmaI,
SacI, and BglII, followed by re-ligation to
generate 0.5 HGF-luc, 0.3 HGF-luc, and 0.1 HGF-luc, respectively. The
1.2 HGF-luc was constructed by ligating the 1.4-kb SalI
fragment from 2.7 HGF-luc into the XhoI site of pGL2-Basic.
An internal deletion mutant 0.5 Tissue Culture and Cell Lines--
The SP1 tumor cell line is
derived from a spontaneous poorly metastatic murine mammary intraductal
adenocarcinoma, and expresses both HGF and Met. The characterization of
the SP1 cell line has been described previously (19, 23, 24).
Maintenance medium for SP1 cells was RPMI 1640 supplemented with 7%
fetal bovine serum. HC11 is a mammary epithelial cell line (32) and was
maintained in RPMI 1640 medium supplemented with 10% fetal bovine
serum, insulin (5 µg/ml), and epidermal growth factor (10 ng/ml).
Cell Transfection--
All transfections were carried out with
LipofectAMINE Plus reagent (Canadian Life Technology, Burlington, ON,
Canada) according to manufacturer's instructions. Cells (15,000) were
seeded in a 24-well plate and transfected with 0.4 µg of reporter
plasmid, 0.1 µg of pSG5
To obtain stably transfected cells, SP1 cells were plated at 70%
confluence in 60-mm plates and transfected with 2 µg of plasmids expressing various mutants of c-Src. Puromycin (2 µg/ml, Sigma, Oakville, ON) was added to cells 24 h following transfection, and
was maintained until all cells in the mock transfection were killed.
Puromycin-resistant cells were then collected and used as pooled cell
lines. Expression and activity of c-Src mutants in transfected cells
were checked using Western blotting analysis and a c-Src kinase assay.
Total c-Src protein was immunoprecipitated with an excess amount of
anti-c-Src (pan) antibody to maximize the amount of antibody-protein
complex formed. We have previously found that these c-Src mutants are
quite effective, and that relatively small levels of expression can
result in significant phenotypes (19).
RNA Isolation and RT-PCR--
Cells grown to 80% confluence on
a 100-mm dish were washed and lysed with TriZol reagent (Canadian Life
Technology). Phase separation was achieved by addition of chloroform
and centrifugation at top speed in a microcentrifuge for 10 min.
Aqueous phase containing total RNA was removed to a new tube and
precipitated with an equal volume of isopropyl alcohol for 10 min at
room temperature. The RNA pellet was recovered by centrifugation and
washed with 70% ethanol. After brief drying, the RNA pellet was
resuspended in diethyl pyrocarbonate-treated water. RNA concentration
was determined by spectrophotometry. An aliquot (1 µg) of total RNA
was used for reverse transcription with avian myeloblastosis reverse
transcriptase at 42 °C for 15 min. One-tenth of the reaction was
used in PCR analysis with end-labeled oligonucleotides specific for
HGF (5'-TGTCGCCATCCCCTATGCAG-3' and
5'-GGAGTCACAAGTCTTCAACT-3') and Copper Affinity Column Chromatography--
Conditioned media
were collected and HGF was partially purified using copper (II)
affinity column chromatography, as described previously (34). Cells
were grown to 80% confluence. The cell monolayer was washed with fresh
Dulbecco's modified Eagle's medium and incubated in serum-free
Dulbecco's modified Eagle's medium for 24 h. Conditioned media
were collected, and cell debris was removed by centrifugation.
Conditioned medium (10 ml) from each cell line was then loaded onto a
copper (II) affinity column. The copper (II) affinity column was
prepared by chelating Cu2+ ions on a 1-ml HiTrap Chelating
column (Amersham Pharmacia Biotech, Baie d'Urfe, PQ), and equilibrated
with equilibration buffer (20 mM sodium phosphate, pH 7.2, 1 M NaCl, 1 mM imidazole). The conditioned medium was recycled through the column 5 times to ensure binding of all
HGF proteins, and the column was washed thoroughly with 15 volumes of
equilibration buffer. HGF protein was eluted from the column with
equilibration buffer containing 80 mM imidazole at a flow
rate of 1 ml/min. Fractions of 1 ml each were collected; previous
experiments have determined that essentially all HGF was eluted in
fraction 2 (Ref. 34 and data not shown). The fraction containing HGF
was concentrated by centrifugation with Microcon centrifugal filter
devices (Millipore Corp., Bedford, MA) with a 10-kDa molecular
mass cut off. The samples were analyzed on a denaturing 10%
SDS-PAGE gel, followed by Western blotting with anti-HGF antibody.
Immunoprecipitation, Western Blotting Analysis, and c-Src Kinase
Assay--
Cells were grown to confluence and treated as indicated.
After three washes with cold phosphate-buffered saline, cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM
Na3VO4, 50 mM NaF, 2 mM
EGTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride. Cell debris was removed by
centrifugation and protein concentrations were determined by a
bicinchoninic acid protein assay (Pierce, Rockford, IL). For
immunoprecipitation, equal amounts of lysate were incubated with the
indicated antibodies at 4 °C for 2 h or overnight.
Immunoprecipitates were collected on protein A-Sepharose (Amersham
Pharmacia Biotech), washed three times with lysis buffer, separated by
SDS-PAGE gel, and transferred to a nitrocellulose membrane. Western
blotting analysis was performed as described previously (19).
In vitro c-Src kinase assays were performed as described
previously (19). Briefly, each lysate was immunoprecipitated with anti-c-Src IgG (Santa Cruz Biotechnology) as described above. One-half
of each immunoprecipitate was subject to SDS-PAGE under nondenaturing
conditions and Western blot analysis to confirm the amount of c-Src
protein present. The other half of each immunoprecipitate was assayed
for c-Src kinase activity by incubating with 10 µl of reaction buffer
(20 mM PIPES, pH 7.0, 10 mM MnCl2,
10 µM Na3VO4), 1.4 µg of
freshly prepared acid-denatured enolase (Sigma), and 10 µCi of
[ Oligonucleotides and Probe Labeling--
Oligonucleotides used
for electrophoretic mobility shift assay binding were Stat3-110F
(5'-GGGCTGTTGTTAAACAGT-3'), Stat3-110R (5'-AGAACTGTTTAACAACAG-3'),
Stat3-149F (5'-GGGGTTGAGGAAAGGAAG-3'), and Stat3-149R
(5'-CCCCTTCCTTTCCTCAAC-3'). Complementary oligonucleotides were
annealed by boiling equal molar amounts of each oligonucleotide for 10 min and then cooling slowly to room temperature. The annealed oligonucleotides (20 pmol) were labeled by a filling-in reaction with
Klenow enzyme and [ Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays--
Nuclear extracts were prepared as described
previously (35). Briefly, 107 cells were washed once with
phosphate-buffered saline before resuspension in cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM sodium
orthovanadate). Cells were allowed to swell on ice for 10 min before
lysis by brief vortexing. Nuclei were pelleted and resuspended in
buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25%
glycerol, 0.5 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM sodium
orthovanadate). High salt extraction was performed by incubation on ice
for 30 min in buffer C and centrifugation at 4 °C. The protein
content of the supernatant (nuclear extract) was determined using a
Bradford protein assay (Bio-Rad, Mississauga, ON).
Electrophoretic mobility shift assays were performed as described by
Mohan et al. (36). Briefly the binding reaction was performed by incubating 5 µg of nuclear extracts with 0.1 pmol of
32P-labeled oligonucleotide probe in the presence of
binding buffer (10 mM HEPES, pH 7.9, 60 mM KCl,
0.1 mM EDTA, 1 mM dithiothreitol), 9%
glycerol, and 4 µg of poly(dI-dC) (Amersham Pharmacia Biotech). Binding was allowed to proceed at room temperature for 10 min before
analysis on 5% nondenaturing PAGE gel in Tris glycine buffer (40 mM Tris-HCl, pH 8.4, 266 mM glycine). When
unlabeled oligonucleotides were added, 10-fold molar excess was
included in the binding reaction. For supershifting experiments,
nuclear extracts were incubated with 2 µg of the indicated antibody
at room temperature for 20 min prior to the binding reaction. After
electrophoresis, the gel was fixed in 7% acetic acid, 40% methanol
for 30 min, and dried under vacuum. The gel was then exposed to a
PhosphorImager screen, and analyzed using a Storm PhosphorImager.
Inhibition of Activity of c-Src Family Kinases Impairs HGF mRNA
Expression--
To study the regulation of HGF expression in breast
carcinoma cells, we used the mouse mammary carcinoma cell line SP1,
which coexpresses HGF and tyrosine-phosphorylated Met (23).
Semi-quantitative RT-PCR was performed to determine the levels of HGF
mRNA in SP1 cells. We first examined the dose-dependent
effect of an inhibitor of c-Src family kinases, PP2 (37). Total RNA was
isolated from SP1 cells treated with different concentrations of PP2
and used for cDNA synthesis by reverse transcription. Relative HGF
mRNA levels were determined by RT-PCR with HGF-specific primers,
and each sample was normalized to the expression of a housekeeping gene
c-Src Kinase Activity Regulates HGF Expression at Both mRNA and
Protein Levels--
We further investigated the role of c-Src tyrosine
kinase in HGF expression by transfecting chicken c-Src mutants
(SRC-Y527F and SRC-RF) with altered kinase activity into SP1 cells. The
SRC-Y527F mutant contains a phenylalanine substitution at tyrosine 527 which results in constitutive kinase activity (39, 40). The SRC-RF mutant contains a double substitution at tyrosine 527 to phenylalanine and at lysine 295 to arginine, which produces a dominant negative phenotype (26). We have previously shown that expression of a similar
dominant negative form of murine c-Src in SP1 cells reduces endogenous
c-Src kinase activity and also impairs anchorage-independent growth in
soft agar (19). As predicted, expression of the dominant negative form
of chicken c-Src (SRC-RF) also decreased total c-Src kinase activity in
SP1 cells, when compared with untransfected cells (Fig.
2, top panel). In addition,
expression of the activated form of c-Src (SRC-Y527F) dramatically
increased total c-Src kinase activity in SP1 cells. Expression of the
chicken c-Src mutants was detected by an antibody (EC10) specific for
avian c-Src (Fig. 2, bottom panel).
To assess the effect of c-Src kinase activity on HGF mRNA
expression, RT-PCR analysis was carried out on RNA extracted from SP1
cells expressing the different c-Src mutants, or treated with the PP2
inhibitor (Fig. 3A).
Expression of the dominant negative SRC-RF mutant or treatment with PP2
reduced the HGF mRNA level in SP1 cells by ~60%. Conversely,
expression of the constitutively active SRC-Y527F mutant increased HGF
mRNA expression by about 2-fold. In a parallel approach, the level
of secreted HGF protein was compared in conditioned media collected
from the same cells and under the same conditions described in Fig.
3A. Our laboratory has previously shown that HGF is a
Cu(II)-binding protein, which can be purified with high recovery from
conditioned media with copper (II) affinity chromatography (34) and
analyzed on a denaturing SDS-PAGE gel (Fig. 3B). Using this
method, we showed that expression of the dominant negative SRC-RF
mutant or treatment with PP2 significantly decreased the amount of HGF
protein secreted by SP1 cells. In contrast, expression of activated
c-Src (SRC-Y527F) increased the amount of secreted HGF protein.
Together these data suggest that HGF expression (both at the mRNA
and protein levels) is regulated by c-Src kinase activity.
c-Src Kinase Activity Induces HGF Expression through a Specific
cis-Acting Region on the HGF Promoter--
To determine the effect of
c-Src kinase mutants on HGF promoter activity, we
constructed a series of reporter plasmids with the luciferase gene
linked to different fragments of the 2.7-kb region 5' of the
HGF transcriptional start site (Fig.
4B). These reporter constructs
were co-transfected into SP1 cells with a control vector, or vectors
expressing the SRC-Y527F or SRC-RF mutants of chicken c-Src kinase, and
luciferase activity of the transfected cells was compared (Fig.
4A). The results show that expression of activated c-Src
increased up to 2-fold the activity of the 2.7-kb HGF
promoter, whereas dominant negative c-Src had the opposite effect.
Deletions of up to
To confirm the importance of the regions of the promoter responsive to
activated c-Src, several internal deletion mutants were constructed.
Full-length reporter constructs missing Stat3 Activates HGF Transcription in Cooperation with Activated
c-Src--
Examination of the c-Src responsive region of the
HGF promoter revealed several Stat3-binding sites. This
consensus sequence is highly conserved among mouse, rat, and human
(100% identity), while this conservation is lost in regions upstream
of
The nonmalignant mammary epithelial cell line, HC11, shows at least a
15-fold lower level of HGF transcription and no detectable HGF protein, compared with SP1 carcinoma cells (data not shown). We
therefore determined whether coexpression of c-Src and Stat3 can
activate HGF transcription in HC11 cells. Expression of
activated c-Src induced expression by about 4-fold (Fig.
5B). In contrast to SP1 cells, expression of Stat3 alone in
HC11 cells did not significantly induce HGF transcription.
However, when activated c-Src and Stat3 were coexpressed,
HGF transcription was synergistically induced up to 17-fold.
Similarly, two clones of HC11 cells, stably transfected with Stat3
followed by transient expression of activated c-Src, showed up to a
20-fold increase in HGF promoter activity (data not shown).
Together, these results suggest that increased c-Src kinase activity
and Stat3 expression can override the repression of HGF
transcription in nonmalignant mammary epithelial cells.
To determine whether the c-Src responsive region of the HGF
promoter is involved in the observed cooperative effect between c-Src
and Stat3, the transcriptional activity of a mutant HGF reporter lacking the c-Src responsive region ( c-Src Kinase Regulates Tyrosine 705 Phosphorylation and DNA Binding
Activity of Stat3--
Previous reports have found that c-Src
activates Stat3 by inducing tyrosine phosphorylation of Stat3 and
increasing its DNA binding affinity (42, 44). We therefore examined the
effect of c-Src kinase activity on Stat3 tyrosine 705 phosphorylation in SP1 cells. We found that expression of activated c-Src induced Stat3-specific tyrosine 705 phosphorylation, while expression of
dominant negative c-Src had the opposite effect (Fig.
7). c-Src kinase activity similarly
affected the nuclear protein binding affinity of the Stat3 consensus
sites on the HGF promoter (Fig. 8). We used electrophoretic mobility
shift assays to examine the Stat3 consensus DNA binding affinity of
nuclear protein extracts from cells expressing different mutants of
c-Src. Radiolabeled oligonucleotide probes with DNA sequences
corresponding to the two Stat3 consensus binding sites in the region
between
Although there is a strong indication of Stat3 being the transcription
factor binding to the c-Src responsive region of the HGF
promoter, other Stat proteins (such as Stat1, Stat5A, and Stat5B) can
also bind to a Stat3 consensus site, albeit at lower levels (28, 45,
46). Therefore, antibodies against specific Stat proteins were used in
supershift experiments to determine the composition of the DNA binding
complex (Fig. 9). Nuclear extracts from
SP1 cells were preincubated with antibodies against Stat1, Stat3,
Stat5A, or Stat5B prior to the addition of the radiolabeled probe. Both
During normal breast development, HGF is expressed primarily by
mesenchymal cells, while its receptor Met is expressed by epithelial
cells (7). However, HGF is expressed in regions of human invasive
breast carcinoma, and in various breast carcinoma cell lines (11-13).
During tumorigenesis HGF stimulates angiogenesis, invasion, and
metastasis (47, 48). Our laboratory (1) and others (49) have shown that
HGF can stimulate survival of carcinoma cells. Therefore, acquired HGF
expression leading to an HGF autocrine loop in breast carcinoma cells
may be an important step during mammary tumorigenesis. However, the
regulation of HGF expression in breast carcinoma cells is not very well
understood, although some studies have been done in fibroblasts
(50-54). In the present study, we examined the role of c-Src kinase,
which shows increased activity in human breast cancer (55), in
controlling HGF expression in breast carcinoma cells.
We previously described a mammary breast carcinoma cell line, SP1,
which expresses both HGF and activated Met (23). In SP1 cells, several
downstream signaling molecules, such as phosphatidylinositol 3-kinase,
phospholipase C In this report, we showed that inhibition of c-Src kinase activity in
SP1 cells, through either the presence of c-Src kinase inhibitors or
the expression of a dominant negative mutant of c-Src, caused a
decrease in HGF mRNA and protein levels. Expression of an activated
c-Src kinase had the reverse effect. This finding suggests that c-Src
is important in regulating the basal level of HGF
transcription in epithelial and carcinoma cells, and can induce
elevated expression of HGF. However, since inhibition of c-Src kinase activity cannot completely eliminate HGF basal expression, other transcription factors may play roles in maintaining HGF basal
expression. Indeed, in our system, the Sp1 transcription factor is
essential in maintaining HGF basal level transcription, but has no
effect on c-Src-induced HGF expression (data not shown). Furthermore,
aggregates of SP1 cells expressing the activated form of c-Src, in
which HGF protein level was high, showed spontaneous scattering when
plated on plastic, compared with the parent cell line which required
addition of exogenous HGF.3
The higher level of endogenous HGF expression in SP1 cells expressing the activated form of c-Src may be sufficient to induce spontaneous scattering of these cells. Together, these findings suggest that c-Src
kinase activity is important in regulating HGF expression.
By using deletion mutants of the HGF promoter, we
mapped the c-Src responsive element to In the c-Src responsive region of the HGF promoter
there are two consensus binding sites for Stat3 (at Stat2, -4, and -6 are not normally expressed in mammary tissues
(60-63), and are therefore unlikely to be involved in the formation of
DNA-protein complexes in SP1 cells. Both Stat1 and Stat3 have been
shown to be activated by c-Src in fibroblast cells when they are
stimulated with various growth factors (30, 45, 64), while Stat5 is
expressed and activated during mammary development (60). Moreover, both
Stat3 and Stat5 have been found to be constitutively active in cells
transformed by v-Src, v-Abl, and other oncoproteins (42-44, 59,
65-67). Therefore, other Stat proteins cannot be ignored as part of
the complex. Supershift studies with antibodies against specific Stat
proteins allowed us to identify Stat3, and exclude Stat1, -5A, or -5B,
as a component of the DNA-protein complex. Furthermore, since there is
only one DNA complex formed with each probe and each probe can
effectively abolish DNA-protein complex formation with the other (data
not shown), the same DNA-binding protein(s) must be involved in binding
to each of these regions. Since Stat3 protein binds as dimers to its
binding sites, it is reasonable to assume that Stat3 dimers are binding
to both sites in the c-Src responsive region. Together, these
observations suggest that c-Src kinase may regulate
Stat3-dependent transcriptional activation through direct
or indirect tyrosine phosphorylation of Stat3, resulting in increased
DNA binding ability.
In contrast to SP1 carcinoma cells, the nonmalignant mammary epithelial
cell line, HC11, showed a very low level of HGF
transcription with no detectable HGF protein. Furthermore, expression
of activated c-Src (Y527F) had very little effect on HGF
transcription in HC11 cells, possibly due to the presence of the
epithelial cell type-specific repressor (9). However, coexpression of
Stat3 and activated c-Src caused a strong synergistic induction of
HGF transcription in HC11 cells, implying that the lack of
c-Src kinase activity and the low level of activated Stat3 may be
limiting for HGF transcription in HC11 cells. Increased
activities of these proteins can possibly override the repression by
the cell type-specific repressor and allow expression of HGF in
epithelial cells. Interestingly, we found that fibroblast cells, which
normally express HGF, also require c-Src kinase activity to regulate
HGF expression, and that this regulation of HGF is dependent on the
same region of the HGF promoter as our breast carcinoma cell
model (data not shown). These results suggest a similar regulation
pattern between fibroblast cells that express HGF endogenously, and
carcinoma cells, which acquire the ability to express HGF. It is
possible that during epithelial-mesenchymal transition, epithelial
cells acquire different genetic mutations leading to the activation of
c-Src kinase. For example, increased expression and activity of
HER2/Neu, an epidermal growth factor-like receptor tyrosine kinase, in
breast carcinoma cells has been shown to activate c-Src kinase (64,
68). Activation of c-Src, in turn, may lead to a de-repression of HGF
expression, giving these cells a growth advantage compared with
nontransformed epithelial cells. This step may be an important initial
step in tumorigenesis.
Here, we have reported that c-Src kinase and Stat3 act
cooperatively in stimulating HGF gene expression in breast
carcinoma cells, most likely via regulation of
Stat3-dependent transcriptional activation of the
HGF promoter. Although many reports have indicated that
increased Src kinase activity (particularly through the expression of
v-Src) can activate gene expression via Stat3, in this study we
identify a target region (254 and
70 base pairs was required for c-Src
responsiveness of the HGF promoter. This region contains two putative consensus sequences (at
110 and
149 base pairs) for
the Stat3 transcription factor, which bind protein complexes containing
Stat3 (but not Stat1, -5A, or -5B). Coexpression of activated c-Src and
Stat3 synergistically induced strong HGF promoter activity
in SP1 cells, as well as in a nonmalignant epithelial cell line, HC11
(HGF negative). c-Src kinase activity correspondingly increased the
tyrosine 705 phosphorylation and DNA binding affinity of Stat3 (but not
Stat1, -5A, or -5B). Collectively, our data indicate a cooperative
effect of c-Src kinase and Stat3 in the activation of HGF
transcription and protein expression in breast carcinoma cells. This
process may be important in overriding the strong repression of
HGF expression in nonmalignant epithelium, and thereby
promote tumorigenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
254 and
70) of the
HGF promoter responsive to increased c-Src kinase activity
in SP1 cells. This region contains two putative consensus binding sites
for Stat3. Stat3 is a transcription factor originally described as the
target of interferon receptors (28), but recent reports have indicated
that Stat3 can be activated by c-Src kinase via platelet-derived growth
factor (29) and HGF receptors (30), and is important in mammary
differentiation (30). We therefore examined the role of Stat3 in
c-Src-dependent regulation of HGF transcription.
The results indicate that while expression of Stat3 alone increased
HGF promoter activity, simultaneous expression of Stat3 and
activated c-Src led to strong cooperative activation of HGF
transcription in both nonmalignant epithelial and carcinoma cells.
Expression of mutant c-Src kinases in breast carcinoma cells altered
both the tyrosine phosphorylation status and DNA binding activity of
Stat3. While activated c-Src induced Stat3 tyrosine phosphorylation and
DNA binding activity, a dominant negative mutant of c-Src reduced
tyrosine phosphorylation and DNA binding. Together these data suggest
that c-Src kinase and Stat3 act cooperatively in the activation of HGF
expression in breast carcinoma cells, and may be important in
overriding the strong repression of HGF expression in nonmalignant
epithelial cells.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
HGF-luc was constructed by digestion
of 0.5 HGF-luc with PvuII/BglII and treatment
with DNA polymerase I (Klenow fragment) before re-ligation. The
1
HGF-luc was constructed by ligating the SmaI fragment of 2.7 HGF-luc into the same site of 0.5
HGF-luc. The
2 HGF-luc was
constructed by ligating the SmaI fragment of 2.7 HGF-luc
into 0.8 HGF-luc. The
HGF-luc was made by ligating the
SmaI fragment of
2 HGF-luc into the same site of 0.5
HGF-luc. For normalization of transfection efficiency of each sample,
pSG5
gal (a gift from Dr. M. Petkovich) or pCHC
gal (a gift from
Dr. F. Kern) (31), which expresses
-galactosidase under the control of SV40 and cytomegalovirus promoters, respectively, was used.
gal, and up to 0.4 µg of expression
plasmids (such as c-Src) as indicated. After 48 h, transfected
cells were harvested and lysed. One-fifth of the cell lysate was used
to assay for
-galactosidase activity, an equal amount of lysate was
used for a luciferase assay using PharMingen Luciferase Substrates (BD PharMingen, Mississauga, ON). Luciferase activity was measured using a
luminometer with wavelength at 562 nm. Luciferase activity of each
sample was normalized to the corresponding
-galactosidase activity.
For immunoprecipitation and in vitro c-Src kinase assays, 2.5 × 105 cells were seeded in a 100-mm tissue
culture plate and transfected with 4 µg of reporter plasmid, 1 µg
of pSG5-
-galactosidase, and up to 4 µg of expression plasmids as
indicated. One-tenth of the cells was used for a luciferase assay, and
the remaining cells were lysed and used for immunoprecipitation.
-glucuronidase
(GUSB) sequences, as previously described (33). The
PCR reaction conditions were 2 min at 95 °C, followed by 25 cycles
of 1 min at 95 °C, 1 min at 55 °C, 1 min at 72 °C, and a final
cycle of 10 min at 72 °C. The reaction was then analyzed on a 2%
agarose gel by electrophoresis. The bands corresponding to the
HGF and GUSB products were excised and the
amount of radioactivity was determined by scintillation counting.
-32P]ATP. After a 10-min incubation at 30 °C,
reactions were terminated by the addition of 2 × SDS sample
buffer, and samples were subjected to 8% SDS-PAGE. Serine and
threonine phosphorylations were hydrolyzed by incubating the acrylamide
gel in 1 M KOH at 45 °C for 30 min, followed by fixing
in 45% MeOH and 10% acetic acid for 30 min at room temperature. The
gel was dried under vacuum. Autoradiograms were produced and analyzed
with a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
-32P]dCTP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-glucuronidase (GUSB) (33). The results
showed that the PP2 inhibitor reduced HGF mRNA expression in a
dose-dependent manner up to 40% of untreated cells (Fig.
1A). In addition, we examined
the level of transcription of the HGF gene using a reporter plasmid. A plasmid containing a 2.7-kb fragment 5' of the
HGF transcriptional start site ligated to the firefly
luciferase gene was transiently transfected into SP1 cells. Bell
et al. (38) have previously shown that this 2.7-kb fragment
of the HGF promoter contains all the necessary sequence to
direct HGF expression and mimics the expression pattern of the
endogenous HGF gene in transgenic mice. Following
transfection, these cells were treated with different concentrations of
the PP2 inhibitor under conditions used in Fig. 1A. After a
24-h incubation, the cells were lysed and luciferase activity in each
sample was determined and compared with control cells. The results show
a similar dose-dependent reduction of HGF
transcription following PP2 treatment (Fig. 1B). These
findings suggest that the activity of c-Src kinase family members is
important in the regulation of HGF transcription and
mRNA expression.
View larger version (26K):
[in a new window]
Fig. 1.
Treatment with the c-Src family kinase
inhibitor PP2 decreases HGF mRNA level and transcription.
Panel A, prestarved SP1 cells were incubated with the Src
family kinase inhibitor PP2 at the concentrations indicated. After
24 h, cells were lysed and total RNA was extracted. The amount of
HGF mRNA in each sample was quantitated using RT-PCR with
HGF-specific primers and primers for GUSB (see "Materials and
Methods"). The amount of HGF mRNA was normalized to GUSB
mRNA, and the level of HGF mRNA expression in each group was
expressed as a percentage of that in untreated (control) cells. Values
represent the mean of two experiments ± range. Panel
B, SP1 cells were transfected with a reporter plasmid containing
the 2.7-kb fragment of the HGF promoter driving expression
of the luciferase gene (2.7 HGF-luc). A -galactosidase expression
plasmid was co-transfected in each group for normalization to account
for differences in transfection efficiency. After 24 h of
incubation, PP2 was added at the concentrations indicated, and the
cells were incubated for an additional 24 h, lysed, and assayed
for luciferase activity. Luciferase activity of each sample was
expressed as a percentage of control (untreated) cells. Values
represent the mean ± S.D. of triplicate samples. The experiment
was done twice with similar results.
View larger version (29K):
[in a new window]
Fig. 2.
Ectopic expression of c-Src kinase mutants in
SP1 cells. SP1 cells were transfected with expression vectors
containing activated c-Src (SRC-Y527F) or dominant negative c-Src
(SRC-RF) or an empty expression vector (SP1). After 48 h, cells
were lysed. Equal amounts of the cell lysates were immunoprecipitated
with anti-c-Src (pan) antibody at excess antibody concentration. Half
of the immunoprecipitates was used to detect c-Src kinase activity
using enolase as a substrate (top panel). The other half was
subjected to Western blotting with anti-Src (pan) antibody to confirm
equal amounts of total c-Src protein in the immunoprecipitates
(middle panel), and then reprobed with monoclonal
anti-chicken c-Src (EC10) antibody to detect the relative level of
ectopic expression of each c-Src mutant (bottom panel). The
amount of chicken c-Src compared with total c-Src may be relatively
low, and cannot be directly inferred from these results, since
different antibodies and exposure times were used for each Western
blot.
View larger version (16K):
[in a new window]
Fig. 3.
c-Src kinase activity modulates HGF mRNA
and protein levels in SP1 cells. Panel A, SP1 cells
transfected with dominant negative Src (SRC-RF) or activated Src
(SRC-Y527F) or empty vector (control) were prestarved overnight. PP2
(40 µM) was added to one plate of SP1 cells and incubated
for an additional 24 h. A nonmalignant breast epithelial cell line
HC11 was used as a negative control. Total RNA was isolated, and the
amount of HGF mRNA in each sample was quantitated using RT-PCR and
normalized to GUSB mRNA as described in the legend to Fig. 1. The
level of HGF mRNA expression in each group was expressed as a
percentage of that in untreated (control) cells. Values represent the
mean of two experiments ± range. Panel B, serum-free
conditioned media were collected for 24 h from HC11 cells,
PP2-treated SP1 cells, and SP1 cells transfected as in Panel
A. HGF protein from the conditioned media was purified using
copper (II) affinity chromatography (34). The fraction containing HGF
protein was concentrated in Microcon concentrators and subjected to
denaturing SDS-PAGE. Recombinant HGF (100 ng) was included in one lane
as a control. After electrophoresis, the proteins were transferred onto
nitrocellulose and the blot was probed with anti-HGF antibody.
Immunoreactive bands were revealed using Enhanced Chemiluminescence
kit.
538 bp (0.5
HGF) had no significant effect on
the c-Src dependent activity of the HGF promoter, although
some fluctuations in basal activity of the promoter were apparent. A
further deletion of
273 bp (0.3 HGF-luc) significantly reduced the
basal HGF promoter activity, while some c-Src dependent
activity remained. The remaining c-Src kinase responsiveness was
eliminated when all but 72 bp (0.1 HGF-luc) of the HGF
promoter was removed. This suggests that a cis-acting element responsive to c-Src kinase activity is located within
273 and
70 bp of the HGF promoter. An internal deletion construct lacking the
70 to
254-bp region (named 0.5
HGF-luc) was used to
confirm the c-Src responsiveness of this region. As predicted, the
0.5
HGF-luc reporter did not respond to expression of SRC-Y527F, although basal activity remained. A similar pattern of repression of
the luciferase activity among all the HGF promoter deletion mutants used was seen when dominant negative c-Src (SRC-RF) was coexpressed with the HGF-luc constructs.
View larger version (32K):
[in a new window]
Fig. 4.
c-Src kinase responsiveness of HGF
transcription requires the 254 to
70 bp region of the
HGF promoter. Panel A, the 2.7-kb
HGF-luciferase reporter (2.7 HGF-luc), or reporter constructs
containing various deletions of the HGF promoter (see Panel
B), were co-transfected into SP1 cells with activated c-Src
(SRC-Y527F), dominant negative c-Src (SRC-RF), or an empty expression
vector (control). Luciferase activity of each sample was determined,
and normalized to the empty vector control value within each group as
described in the legend to Fig. 1B. Values represent
mean ± S.D. of triplicate samples. The experiments were done
three times using two different preparations of plasmid DNA with
similar results. Panel B, schematic representation of the
wild-type HGF reporter construct and the corresponding
internal deletion mutants used in Panel A is shown. The name
of each construct refers to the full-length (2.7 kb) or truncated
promoter sequences (1.2, 0.8, 0.5, 0.3, and 0.1 kb) upstream of the
transcriptional start site (indicated by arrow). In
addition, constructs containing the 0.5-kb sequence with an internal
deletion of the region between
254 and
70 (0.5
), or the
full-length sequence containing internal deletion of regions between
254 and
70 (
1),
1231 and
755 (
2), or both (
) were
used.
273 to
70 bp (
1),
1231
to
755 bp (
2), or both regions (
) of the HGF
promoter were transfected into SP1 cells in the presence or absence of
the SRC-Y527F and SRC-RF mutants (Fig. 4A). As predicted,
1 and
deletion mutants exhibited neither induction nor
repression of HGF promoter activity when activated c-Src or
dominant negative c-Src was expressed, respectively. In contrast, the
2 mutant showed strong induction of HGF promoter activity
corresponding to expression of the activated SRC-Y527F mutant, and
strong repression of HGF promoter activity when the SRC-RF
mutant was expressed. This finding shows that only the region between
254 and
70 bp of the HGF promoter is
important for c-Src responsiveness of HGF expression in SP1
cells. We will refer to this region as the c-Src responsive region.
500 bp of the HGF promoter (41). Since Stat3 activation
by Src induces specific gene expression and is required for cell
transformation (42, 43), we examined whether expression of Stat3 in the
presence or absence of the activated c-Src mutant (SRC-Y527F) has any
effect on HGF promoter activity. A reporter plasmid
containing the
2.7-kb full-length HGF promoter was
co-transfected with a constant amount of the SRC-Y527F, and varying
amounts of Stat3, expression plasmids. Expression of activated c-Src
(SRC-Y527F) alone increased HGF transcription by about
2-fold (Fig. 5A). Likewise,
expression of Stat3 alone increased HGF transcription by
about 2-fold, and maintained a plateau value with even 0.05 µg of
plasmid DNA. However, in cells coexpressing both the activated c-Src
mutant and increasing amounts of Stat3, HGF transcription
increased up to 5-fold. This result indicates that there is a
cooperative effect between c-Src kinase activity and Stat3 protein in
the regulation of HGF transcription.
View larger version (37K):
[in a new window]
Fig. 5.
Stat3 induces HGF
transcription in cooperation with activated c-Src. SP1
carcinoma cells (Panel A) and HC11 mammary epithelial cells
(Panel B) were co-transfected with the 2.7 HGF-luc reporter
and activated c-Src (SRC-Y527F) or an empty vector
(control), in combination with varying amounts of Stat3.
Luciferase activity was determined and expressed as a percentage of
that in control cells as described in the legend to Fig. 1B.
Values represent the mean ± S.D. of triplicate samples. The
experiment was done twice with similar results.
1 HGF-luc) was compared with that of the full-length (2.7 HGF-luc) HGF
reporter. Each reporter construct was transfected into SP1 cells alone, or in combination with Stat3, and the activated c-Src (SRC-Y527F) mutant, expression plasmids. Expression of the activated c-Src mutant
induced activation of the full-length HGF promoter, but not
of the deletion mutant (
1 HGF-luc) (Fig.
6). Similarly, Stat3 expression increased
the activity of the full-length HGF promoter, and only
marginally affected that of the deletion mutant (
1 HGF-luc), this
result suggests that Stat3 activates the HGF promoter. The level of induction due to Stat3 expression is even higher than that due
to activated c-Src alone. This effect is probably due to a limiting
amount of endogenous Stat3 in SP1 cells. When both Stat3 and activated
c-Src were coexpressed, HGF promoter activity in the
full-length construct was strongly induced, this effect was not seen in
the deletion mutant (
1 HGF-luc). These results show a cooperative
effect between Stat3 and activated c-Src in the induction of
HGF transcription, and imply the presence of specific
Stat3-binding sites on the HGF promoter.
View larger version (21K):
[in a new window]
Fig. 6.
The cooperative effect of Stat3 and activated
c-Src on induction of HGF transcription requires the
c-Src responsive region of the HGF promoter. SP1 cells were
co-transfected with the 2.7 HGF-luciferase reporter, an internal
deletion mutant ( 1 HGF-luc), and a combination of activated Src
(SRC-Y527F) and Stat3 as indicated. Transfections and luciferase assays
were performed as described in the legend to Fig. 1B. Values
represent the mean ± S.D. of triplicate samples. The experiment
was done four times with similar results.
254 to
70 of the HGF promoter were used to
detect putative Stat3 binding (Fig. 8). Binding of probes corresponding
to each Stat3 consensus site (
110 or
149) was detected in nuclear
protein extracts of SP1 cells (lane 1 in Fig. 8,
A and B, respectively). These DNA binding
activities were specific since the presence of the corresponding unlabeled probes abolished the binding (second lane), while
a probe with an unrelated DNA sequence had no effect (third
lane). In addition, when comparing first, fourth, and
seventh lanes (Fig. 8), it is apparent that there
was less specific DNA binding in nuclear extracts from SP1 cells
expressing dominant negative c-Src than in control cells expressing no
exogenous c-Src. Moreover, nuclear extracts from SP1 cells expressing
activated c-Src had higher binding activity than that from
untransfected cells. This finding indicates that the expression of
dominant negative c-Src reduces nuclear protein binding to the Stat3
consensus sites, whereas activated c-Src has the opposite effect. Thus
specific binding of nuclear protein to the Stat3 consensus sites
correlates with phosphorylation at tyrosine 705 of Stat3 in these
cells.
View larger version (19K):
[in a new window]
Fig. 7.
c-Src kinase activity regulates
phosphorylation of tyrosine residue 705 of Stat3. SP1 cells
transfected with SRC-RF or SRC Y527F, or untransfected SP1 cells, were
lysed. Equal amounts of proteins from each cell lysate were subjected
to denaturing SDS-PAGE. The proteins were then transferred onto
nitrocellulose and the blot was probed with antibody specific for
phosphotyrosine 705 of Stat3 (Panel A). The blot was
subsequently reprobed with anti-Stat3 (pan) antibody (Panel
B).
View larger version (31K):
[in a new window]
Fig. 8.
c-Src kinase activity regulates nuclear
protein binding to the Stat3 consensus sites (at positions 110 and
149) of the HGF promoter. Nuclear extracts were
prepared from SP1 cells transfected with SRC-RF, SRC-Y527F, or
untransfected cells. Equal amounts of each nuclear extract were used in
binding studies with radiolabeled probes containing either the
110
(Panel A) or the
149 region (Panel B) of the
HGF promoter. 10-fold molar excess of an unlabeled probe
containing the
110,
149 or a nonspecific sequence (NS),
respectively, was included in the binding reaction where indicated. The
gel was fixed, dried, and analyzed using a Storm PhosphorImager as
described under "Experimental Procedures." The arrow
indicates the position of the protein-DNA complex.
110 (Fig. 9A) and
149 (Fig. 9B) probes formed
DNA-protein complexes when nuclear extracts were added. However, only
anti-Stat3 antibody could efficiently bind to these complexes to form a
supershift band. Antibodies to Stat1 (data not shown), Stat5A, or
Stat5B did not retard the DNA-protein complex further, despite the fact that these transcription factors were present in SP1 cells (data not
shown). This observation suggests that Stat3 is preferentially involved
in the DNA-protein complexes which bind to the c-Src responsive
elements in the HGF promoter.
View larger version (26K):
[in a new window]
Fig. 9.
Stat3 forms part of the DNA-protein complex
at both the 110 and
149 consensus sites. Nuclear extracts were
prepared from SP1 cells as described under "Experimental
Procedures." For supershift assays, nuclear extracts were incubated
with anti-Stat3, Stat5A, or Stat5B antibody on ice for 30 min prior to
electrophoretic mobility shift assay analysis. After incubation with
labeled
110 (Panel A) or
149 (Panel B)
probes, the reaction was subjected to nondenaturing PAGE. The
asterisk indicates the position of supershift band.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and focal adhesion kinase, are constitutively phosphorylated on tyrosine residues in SP1 cells, consistent with the
presence of an autocrine loop (1, 23). We have also found that c-Src
tyrosine kinase in SP1 cells is constitutively active and is required
for several HGF-dependent processes, such as cell motility
and anchorage-independent growth (19).
254 to
70 bp. Since there is
significant homology among the mouse, rat, and human HGF
promoter sequences between
500 and +1 (41), the regulation of
HGF expression by c-Src kinase through this element is
probably conserved among these species. Previous studies in fibroblast
cells have demonstrated several transcription factors which regulate
HGF expression: C/EBP (
4 bp) (50), an epithelial
cell-specific repressor (
16 bp) (9), Sp1/Sp3 (
318 bp) (52),
estrogen receptor (
872 bp) (51), and chicken ovalbumin upstream
promoter-transcription factor (
860 bp) (51). Transgenic mouse studies
showed that 0.7 kb of the HGF promoter exhibited the same
expression pattern as the full-length (2.7 kb) promoter (38). Although
in our system we observed that Sp1/Sp3 maintain the basal level
expression of HGF in breast carcinoma cells, these sites are not
responsible for c-Src induced expression of HGF (data not shown). The
C/EBP site appeared to have no transcriptional activity in
vivo (38). Binding sites for estrogen receptor and chicken
ovalbumin upstream promoter-transcription factor are likely to be
involved in estrogen-induced expression of HGF since the upstream
sequence between
2.7 and
0.7 kb has been shown to be necessary for
maximal inducibility of the HGF promoter (such as after
partial hepatectomy) (38). However, the c-Src responsive region (
254
to
70 bp) described here has not been previously reported.
110 and
149),
both of which are completely conserved among human, mouse, and rat. Our
results showed that Stat3, in cooperation with c-Src kinase, can
activate HGF promoter, this activation is completely
dependent on the presence of these Stat3-binding sites and implies a
role of Stat3 as a downstream effector of c-Src kinase. We therefore examined the mechanism by which c-Src regulates Stat3 activity in SP1
carcinoma cells. Stat3 has been shown to be regulated by both tyrosine
and serine phosphorylations (56-58). Although there is no direct
evidence that Stat3 is phosphorylated directly by c-Src, some reports
suggest that c-Src and Stat3 interact physically (30, 59). Therefore,
it is possible that c-Src regulates Stat3 through tyrosine
phosphorylation. Our results showed that expression of a dominant
negative form of c-Src reduced tyrosine phosphorylation of Stat3 and
the expression of constitutively active c-Src mutant had the opposite
effect. In addition, we found that the formation of a DNA-protein
complex with the two Stat3-binding sites in the c-Src responsive
elements was dependent on the level of c-Src kinase activity in the
cells. An apparently greater effect of activated c-Src on the binding
activity of the
149 Stat3 site compared with the
110 Stat3 site was
observed. This difference could potentially represent different binding
affinities, or interaction with other transcription factors.
254 to
70 bp) of the HGF
promoter responsive to elevated activity of c-Src kinase in breast
carcinoma cells. There is recent evidence suggesting that an HGF
autocrine loop can provide selective survival and growth advantage to
carcinoma cells and that overexpression of HGF can be a reliable
indicator of poor survival of breast cancer patients (11). Our findings therefore provide an important link between breast cancer progression and HGF expression, and suggest that the c-Src/Stat3 pathway regulating HGF expression can be a potential target for therapy in breast cancer treatment.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. J. Brugge, P. Greer, R. Jove, C. Mueller, S. Parsons, M. Petkovich, D. Shalloway, and R. Zarnegar for generous gifts of plasmids and reagents. E. Tremblay provided excellent technical assistance, and Drs. P. Greer and C. Mueller provided valuable discussion and comments on the preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by U.S. Army Medical Research Materiel Command Grant DAMD17-96-I-6251 (to B. E.) and the Medical Research Council of Canada (to B. E.).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.
Recipient of Postdoctoral Fellowship Award DAMD17-98-I-8330 from
the U.S. Army Medical Research Materiel Command. Current address: Div.
of Cancer Biology, Sunnybrook & Women's College Health Sciences
Centre, Rm. S207, 2075 Bayview Ave., Toronto, Ontario M4N 3M5, Canada.
§ To whom correspondence should be addressed: Cancer Research Laboratories, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2825; Fax: 613-533-6830; E-mail: Elliottb@post.queensu.ca.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M010715200
2 W. Hung, J. Gin, and B. Elliott, unpublished results.
3 B. Elliott, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HGF, hepatocyte growth factor;
kb, kilobase pair(s);
RT-PCR, reverse
transcriptase-polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis;
bp, base pair(s);
GUSB, -galactosidase;
PIPES, 1,4-piperazinediethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Qiao, H.,
Saulnier, R.,
Patrzykat, A.,
Rahimi, N.,
Raptis, L.,
Rossiter, J. P.,
Tremblay, E.,
and Elliott, B. E.
(2000)
Cell Growth Differ.
11,
123-133 |
2. | Rubin, J. S., Chan, A. M., Bottaro, D. P., Burgess, W. H., Taylor, W. G., Cech, A. C., Hirschfield, D. W., Wong, J., Miki, T., Finch, P. W., and Aaronson, S. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 415-419[Abstract] |
3. | Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991) Cell 67, 901-908[Medline] [Order article via Infotrieve] |
4. | Gherardi, E., Gray, J., Stoker, M., Perryman, M., and Furlong, R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5844-5848[Abstract] |
5. | Schmidt, C., Bladt, F., Goedecke, S., Brinkmann, V., Zschiesche, W., Sharpe, M., Gherardi, E., and Birchmeier, C. (1995) Nature 373, 699-702[CrossRef][Medline] [Order article via Infotrieve] |
6. | Bladt, F., Riethmacher, D., Isenmann, S., Aguzzi, A., and Birchmeier, C. (1995) Nature 376, 768-771[CrossRef][Medline] [Order article via Infotrieve] |
7. | Andermarcher, E., Surani, M. A., and Gherardi, E. (1996) Dev. Genet. 18, 254-266[CrossRef][Medline] [Order article via Infotrieve] |
8. | Yang, Y. M., Spitzer, E., Meyer, D., Sachs, M., Niemann, C., Hartmann, G., Weidner, K. M., Birchmeier, C., and Birchmeier, W. (1995) J. Cell Biol. 131, 215-226[Abstract] |
9. | Liu, Y., Beedle, A. B., Lin, L., Bell, A. W., and Zarnegar, R. (1994) Mol. Cell. Biol. 14, 7046-7058[Abstract] |
10. | Ghoussoub, R. A. D., Dillon, D. A., D'Aquila, T., Rimm, B. E., Fearon, E. R., and Rimm, D. L. (1998) Cancer 82, 1513-1520[CrossRef][Medline] [Order article via Infotrieve] |
11. | Yamashita, J., Ogawa, M., Yamashita, S., Nomura, K., Kuramoto, M., Saishoji, T., and Shin, S. (1994) Cancer Res. 54, 1630-1633[Abstract] |
12. | Tuck, A. B., Park, M., Sterns, E. E., Boag, A., and Elliott, B. E. (1996) Am. J. Pathol. 148, 225-232[Abstract] |
13. | Toi, M., Taniguchi, T., Ueno, T., Asano, M., Funata, N., Sekiguchi, K., Iwanari, H., and Tominaga, T. (1998) Clin. Cancer Res. 4, 659-664[Abstract] |
14. | Di Renzo, M. F., Poulsom, R., Olivero, M., Comoglio, M., and Lemoine, N. R. (1995) Cancer Res. 55, 1129-1138[Abstract] |
15. |
Liang, T. J.,
Reid, A. E.,
Xavier, R.,
Cardiff, R. D.,
and Wang, T. C.
(1996)
J. Clin. Invest.
97,
2872-2877 |
16. |
Takayama, H.,
LaRochelle, W. J.,
Sharp, R.,
Otsuka, T.,
Kriebel, P.,
Anver, M.,
Aaronson, S. A.,
and Merlino, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
701-706 |
17. |
Fixman, E. D.,
Holgado-Madruga, M.,
Nguyen, L.,
Kamikura, D. M.,
Fournier, T. M.,
Wong, A. J.,
and Park, M.
(1997)
J. Biol. Chem.
272,
20167-20172 |
18. | Jeffers, M., Rao, M. S., Rulong, S., Reddy, J. K., Subbarao, V., Hudson, E., Vande Woude, G. F., and Resau, J. H. (1996) Cell Growth Differ. 7, 1805-1813[Abstract] |
19. |
Rahimi, N.,
Hung, W.,
Saulnier, R.,
Tremblay, E.,
and Elliott, B.
(1998)
J. Biol. Chem.
273,
33714-33721 |
20. |
Fixman, E.,
Fournier, T.,
Kamikura, D.,
Naujokas, M.,
and Park, M.
(1996)
J. Biol. Chem.
271,
13116-13122 |
21. | Webster, M. A., Cardiff, R. D., and Muller, W. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7849-7853[Abstract] |
22. | Guy, C. T., Muthuswamy, S. K., Cardiff, R. D., Soriano, P., and Muller, W. J. (1994) Genes Dev. 8, 23-32[Abstract] |
23. | Rahimi, N., Tremblay, E., McAdam, L., Park, M., Schwall, R., and Elliott, B. E. (1996) Cell Growth Differ. 7, 263-270[Abstract] |
24. |
Rahimi, N.,
Tremblay, E.,
and Elliott, B. E.
(1996)
J. Biol. Chem.
271,
24850-24855 |
25. | Mukhopadhyay, D., Tsiokas, L., and Sukhatme, V. P. (1995) Cancer Res. 55, 6161-6165[Abstract] |
26. | Mukhopadhyay, D., Tsiokas, L., Zhou, X. M., Foster, D., Brugge, J. S., and Sukhatme, V. P. (1995) Nature 375, 577-581[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Karaplis, A. C.,
Lim, S. K.,
Baba, H.,
Arnold, A.,
and Kronenberg, H. M.
(1995)
J. Biol. Chem.
270,
1629-1635 |
28. |
Leaman, D. W.,
Leung, S.,
Li, X.,
and Stark, G. R.
(1996)
FASEB J.
10,
1578-1588 |
29. | Wang, Y., Wharton, W., Garcia, R., Kraker, A. J., Jove, R., and Pledger, W. (2000) Oncogene 19, 2075-2085[CrossRef][Medline] [Order article via Infotrieve] |
30. | Boccaccio, C., Ando, M., Tamagnome, L., Bardelli, A., Michieli, P., Battistini, C., and Comoglio, P. (1998) Nature. 391, 285-288[CrossRef][Medline] [Order article via Infotrieve] |
31. | McLeskey, S. W., Kurebayashi, J., Honig, S. F., Zwiebel, J., Lippman, M. E., Dickson, R. B., and Kern, F. G. (1993) Cancer Res. 53, 2168-2177[Abstract] |
32. | Doppler, W., Groner, B., and Ball, R. K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 104-108[Abstract] |
33. | Ivanchuk, S. M., Myers, S. M., and Mulligan, L. M. (1998) Oncogene. 16, 991-996[CrossRef][Medline] [Order article via Infotrieve] |
34. | Rahimi, N., Etchells, S., and Elliott, B. (1996) Protein Expression Purif. 7, 329-333[CrossRef][Medline] [Order article via Infotrieve] |
35. | Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499[Medline] [Order article via Infotrieve] |
36. |
Mohan, W. S.,
Chen, Z. Q.,
Zhang, X.,
Khalili, K.,
Honjo, T.,
Deeley, R. G.,
and Tam, S. P.
(1998)
J. Lipid Res.
39,
255-267 |
37. |
Hanke, J. H.,
Gardner, J. P.,
Dow, R. L.,
Changelian, P. S.,
Brissette, W. H.,
Weringer, E. J.,
Pollok, B. A.,
and Connelly, P. A.
(1996)
J. Biol. Chem.
271,
695-701 |
38. |
Bell, A. W.,
Jiang, J. G.,
Chen, Q.,
Liu, Y.,
and Zarnegar, R.
(1998)
J. Biol. Chem.
273,
6900-6908 |
39. | Cobb, B. S., and Parsons, J. T. (1993) Oncogene 8, 2897-2903[Medline] [Order article via Infotrieve] |
40. | Superti-Furga, G. (1995) FEBS Lett. 369, 62-66[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Liu, Y.,
Michalopoulos, G. K.,
and Zarnegar, R.
(1994)
J. Biol. Chem.
269,
4152-4160 |
42. |
Turkson, J.,
Bowman, T.,
Garcia, R.,
Caldenhoven, E.,
De Groot, R. P.,
and Jove, R.
(1998)
Mol. Cell. Biol.
18,
2545-2552 |
43. |
Bromberg, J. F.,
Horwath, C. M.,
Besser, D.,
Lathem, W. W.,
and Darnell, J. E., Jr.
(1998)
Mol. Cell. Biol.
18,
2553-2558 |
44. | Yu, C. L., Meyer, D. J., Campbell, G. S., Larner, A. C., Carter-Su, C., Schwartz, J., and Jove, R. (1995) Science 269, 81-83[Medline] [Order article via Infotrieve] |
45. | Cirri, P., Chiarugi, P., Marra, F., Raugei, G., Camici, G., Manao, G., and Ramponi, G. (1997) Biochem. Biophys. Res. Commun. 239, 493-497[CrossRef][Medline] [Order article via Infotrieve] |
46. | Lu, L., Zhu, J., Zheng, Z., Yan, M., Xu, W., Sun, L., Theze, J., and Liu, X. (1998) Eur. J. Immunol. 28, 805-810[CrossRef][Medline] [Order article via Infotrieve] |
47. | Beviglia, L., Matsumoto, K., Lin, C. S., Ziober, B. L., and Kramer, R. H. (1997) Int. J. Cancer 74, 301-309[CrossRef][Medline] [Order article via Infotrieve] |
48. | Meiners, S., Brinkmann, V., Naundorf, H., and Birchmeier, W. (1998) Oncogene 16, 9-20[CrossRef][Medline] [Order article via Infotrieve] |
49. | Fan, S., Wang, J. A., Yuan, R. Q., Rockwell, S., Andres, J., Zlatapolskiy, A., Goldberg, I. D., and Rosen, E. M. (1998) Oncogene 17, 131-141[CrossRef][Medline] [Order article via Infotrieve] |
50. | Jiang, J.-G., and Zarnegar, R. (1997) Mol. Cell. Biol. 17, 5758-5770[Abstract] |
51. |
Jiang, J. G.,
Bell, A.,
Liu, Y.,
and Zarnegar, R.
(1997)
J. Biol. Chem.
272,
3928-3934 |
52. | Jiang, J. G., Chen, Q., Bell, A., and Zarnegar, R. (1997) Oncogene 14, 3039-3049[CrossRef][Medline] [Order article via Infotrieve] |
53. | Liu, Y., Bell, A. W., Michalopoulos, G. K., and Zarnegar, R. (1994) Gene (Amst.) 144, 179-187[Medline] [Order article via Infotrieve] |
54. | Liu, Y., Michalopoulos, G. K., and Zarnegar, R. (1993) Biochim. Biophys. Acta 1216, 299-303[Medline] [Order article via Infotrieve] |
55. | Ottenhoff-Kalff, A. E., Rijksen, G. A. U., Hennipman, A., Michels, A. A., and Staal, G. E. (1992) Cancer Res. 52, 4773-4778[Abstract] |
56. | Kuroki, M., and O'Flaherty, J. T. (1999) Biochem. J. 341, 691-696[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Jenab, S.,
and Morris, P. L.
(1997)
Endocrinology
138,
2740-2746 |
58. |
Wen, Z.,
and Darnell, J. E. J.
(1997)
Nucleic Acids Res.
25,
2062-2067 |
59. | Cao, X., Tay, A., Guy, G. R., and Tan, Y. H. (1996) Mol. Cell. Biol. 16, 1595-1603[Abstract] |
60. |
Karpf, A. R.,
Peterson, P. W.,
Rawlins, J. T.,
Dalley, B. K.,
Yang, Q.,
Albertsen, H.,
and Jones, D. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14007-14012 |
61. | Kazansky, A. V., Raught, B., Lindsey, S. M., Wang, Y. F., and Rosen, J. M. (1995) Mol. Endocrinol. 9, 1598-1609[Abstract] |
62. | Smith, P. D., and Crompton, M. R. (1998) Biochem. J. 331, 381-385[Medline] [Order article via Infotrieve] |
63. | Akira, S. (2000) Oncogene 19, 2607-2611[CrossRef][Medline] [Order article via Infotrieve] |
64. |
Olayioye, M. A.,
Beuvink, I.,
Horsch, K.,
Daly, J. M.,
and Hynes, N. E.
(1999)
J. Biol. Chem.
274,
17209-17218 |
65. | Danial, N. N., Pernis, A., and Rothman, P. (1995) Science 269, 1875-1877[Medline] [Order article via Infotrieve] |
66. | Migone, T. S., Lin, J. X., Cereseto, A., O'Shea, J. J., Franchini, G., and Leonard, W. E. (1995) Science 269, 79-81[Medline] [Order article via Infotrieve] |
67. | Garcia, R., Yu, C. L., Hudnall, A., Catlett, R., Nelson, K. L., Smithgall, T., Fujit, E. J., Ethier, S. P., and Jove, R. (1997) Cell Growth Differ. 8, 1267-1276[Abstract] |
68. | Muthuswamy, S. K., Siegel, P. M., Dankork, D. L., Webster, M. A., and Muller, W. J. (1994) Mol. Cell. Biol. 14, 735-743[Abstract] |