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INTRODUCTION |
egr-1 was originally identified as an immediate early
growth response gene that exhibits a rapid and transient burst of
transcription without de novo protein synthesis, following
stimulation by a variety of mitogens (1). EGR-1 is a nuclear
phosphoprotein that contains three zinc finger motifs in the C-terminal
portion of the molecule, which confer specific DNA-binding properties (2-4). EGR-1 preferentially but not exclusively binds GC-rich regulatory elements (GCE)1 with a
consensus sequence of 5'-GCG(T/G)GGGCG-3' (5). For some GCEs in certain
genes, other transcription factors also bind and influence
transcription. Examples include Sp-1 binding to the GC-rich
region of murine adenosine deaminase gene promoter (6) or the Wilm's
tumor-1 (WT-1) suppression factor binding to a GCE of the
TGF-
1 gene which therefore competes with EGR-1 (7).
Several groups have reported that EGR-1 has functional effects in
regulating cell growth, differentiation, and development (reviewed in
Ref. 8). Furthermore, the expression of EGR-1 in a variety of human
tumor cell lines has revealed growth-inhibiting and -suppressing roles.
Conversely, loss or deletion of chromosome region 5q31 containing the
EGR-1 locus is seen frequently in patients with
myelodysplastic syndrome and acute myelogenous leukemia (9-10) and
small cell lung carcinoma (11). Decreased or absent EGR-1 expression
occurs in non-small cell lung carcinoma (12) as well as in human breast
carcinoma (13).
The importance of EGR-1 in the regulation of cell proliferation and
tumor formation was demonstrated in experiments that showed that stable
overexpression of EGR-1 inhibited transformation in model cells and
human tumor lines (13-15). Stable expression of EGR-1 inhibited
growth, focus formation, and soft-agar growth in platelet-derived
growth factor v-sis-transformed NIH-3T3 cells (15).
Moreover, expression of antisense egr-1 RNA in the parental NIH-3T3 cells completely eliminated expression of the endogenous EGR-1
(15) and tended to have the opposite morphological effects (14-15),
indicating that endogenous EGR-1 levels may mediate a growth
regulatory role. Kieser et al. (16) reported that protein kinase C-
reverted v-raf-transformed NIH-3T3 cells by
induction of egr-1 and Jun-B. Constitutive
expression of either egr-1 alone or Jun-B alone
suppressed anchorage-independent growth, but maximum suppression
required both egr-1 and Jun-B.
Similarly, overexpression of protein kinase C-
inhibited invasion
and metastasis of Dunning R-3327 MAT-Lylu rat prostate cancer cells in
syngeneic rats (17). Stable overexpression of EGR-1 in human tumor
lines such as fibrosarcoma HT1080, osteosarcoma Saos2, glioblastomas
U251 and U373, and breast carcinoma ZR75-1 led to decreased DNA
synthesis and growth albeit to variable extents (14). The most striking
effects were observed in human fibrosarcoma HT1080 cells where
proliferation was reduced by 50%, and tumorigenicity was reduced by
40.3% (14). The inhibition of proliferation was highly correlated with
the level of EGR-1 expression (14).
An approach to understanding the underlying molecular basis for these
EGR-1 functions has been to study signal transduction events associated
with the expression of EGR-1. Previous studies of the monkey kidney
epithelial cell line, CV-1, showed that WT-1 bound to two GCEs of the
human TGF-
1 promoter, leading to strong suppression of
transcription and that expression of EGR-1 reversed this effect (7). In
HT1080 cells, transient expression of egr-1 strongly
activated a TGF-
1 minimal promoter reporter containing the GCE consensus sequences, whereas coexpression of WT-1 inhibited this effect (18). Similarly, TGF-
1 reporter construct is
strongly activated in HT1080 cells that stably expressed EGR-1 (18). Addition of a specific TGF-
1 antibody completely reversed the transformation suppressive effect associated with stable expression of
EGR-1, whereas addition of rhTGF-
1 to parental cells suppressed growth. These results indicated that EGR-1 binds to two potential GCE
sites of the human TGF-
1 promoter, enhances the
expression and secretion of functional TGF-
1 by EGR-1-expressing
cells, inhibits cell proliferation, and restores
anchorage-dependent growth (18-19).
TGF-
1 belongs to the TGF superfamily of cytokines that have been
implicated in the regulation of growth, differentiation, development,
and apoptosis (20-21). TGF-
1 is a potent growth-inhibitory protein
in many cell types, including epithelial cells, endothelial cells,
lymphocytes, and hematopoietic progenitor cells. Signal transduction by
TGF-
1 has been studied intensively. TGF-
1 stimulates the
synthesis and accumulation of several extracellular matrix (ECM)
proteins, such as FN and several types of collagen and their receptors,
respectively (22-24). In addition, TGF-
1 influences the function of
pericellular proteases, such as by induction of plasminogen activator
inhibitor-1 (PAI-1), which acts on urokinase and inhibits the
fibrinolytic pathway thereby stabilizing the ECM (25-26). PAI-1 also
promotes cell adhesion and spreading and acts as a molecular bridge
between the cell surface and the ECM (27). The function of FN has been
studied broadly. FN plays an important role in anchoring cells to the
extracellular matrix. Inhibition of FN expression leads to a loss of FN
from the cell surface and causes oncogenic transformation in
vitro (28-30) and tumorigenic and metastatic phenotypes in
vivo (31-32). Conversely, the addition of plasma FN to cultures
of transformed fibroblasts restores a normal phenotype to cells (31).
Indeed, overexpression of an intact form of recombinant FN in HT1080
human fibrosarcoma cells suppresses the transformed phenotype, reduces
cell migration on the substratum, and suppresses tumor growth in
vivo (33).
These observations suggest that TGF-
1 may suppress transformation of
HT1080 or other tumor cells bearing functional TGF-
1 receptors by
induction of one or more of the ECM proteins thereby enhancing the
regulatory role of the ECM. We have investigated the production of ECM,
specifically FN and PAI-1. Our results show that EGR-1 not only
stimulates the expression of TGF-
1, but also greatly stimulates the
accumulation of the ECM proteins including FN and PAI-1. We provide
evidence that EGR-1 directly transactivates the TGF-
1
gene and binds and regulates the FN gene. In contrast,
induction of the PAI-1 gene is a secondary effect of EGR-1
and is regulated by the TGF-
1 (40) transduction pathway. Addition of
recombinant FN or PAI-1 proteins to parental HT1080 cells or addition
of specific inhibitors of FN or PAI-1 to EGR-1-expressing HT1080 cells
shows that all proteins are functional in mediating growth control with
enhancing cell adhesion. These results indicate that EGR-1 function to
initiate a coordinated program of gene expression leading to increased
extracellular matrix formation with enhanced cellular regulation.
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MATERIALS AND METHODS |
Cells and Cell Culture--
Fibrosarcoma HT1080 subclone H4
cells, EGR-1-expressing transfectants (H4E2, H4E3, H4E9),
neomycin-resistant control cells (H4N), and EGR-1-null cells (H4E4,
H4E6) were prepared by transfection of H4 cells with expression vectors
for mouse wild-type egr-1 (pCMV-egr-1) as
described (14) and were maintained in DMEM supplemented with 5% fetal
bovine serum and grown in the presence of penicillin and streptomycin,
and 200 µg/ml G-418 for all transfectants. Cell numbers were
determined by direct cell counting (Coulter Electronics Inc., Hialeah,
FL) similar to previous studies (14).
Protein Preparation and Western Blot--
Cells were plated at
the density of 4 × 104 cells/cm2,
incubated overnight, washed twice with ice-cold phosphate-buffered
saline (PBS), and lysed by scraping from the plates with RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mM aprotinin, 1 mM sodium orthovanadate). The lysates were passed through a
21-gauge needle to shear the DNA, incubated for 60 min on ice, and
centrifuged at 12,000 × g for 20 min. The protein
concentrations were determined using Bio-Rad protein assay reagent
(Bio-Rad). 100 µg of protein were resolved by 7% SDS-polyacrylamide
gel electrophoresis (SDS-PAGE), electrophoretically transferred onto
polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA), and
incubated with rabbit polyclonal anti-Egr-1 (Santa Cruz Biotechnology,
Santa Cruz, CA). Immunoreactive bands were visualized by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech). The intensity of
EGR-1-containing bands was determined by image analysis using a Kodak
Digital ScienceTM 1D image analysis system (Eastman Kodak
Co.).
Cell Labeling, Extracellular Matrix Preparation, and
Immunoprecipitation--
For the plasminogen activator inhibitor
(PAI-1) assay, 2 × 105 cells were plated in 6-well
tissue culture plates in DMEM supplemented with 5% fetal bovine serum
and incubated overnight. Then cells were subjected to
cysteine/methionine-free DMEM in the presence or absence of various
doses of recombinant human TGF-
1 (rhTGF-
1, R&D Systems Inc.,
Minneapolis, MN) in the range 0.001 to 100 ng/ml or 30 µg/ml
monoclonal mouse anti-TGF-
1,2,3 (Genzyme Corp.,
Cambridge, MA) for 2 h at which time
[35S]cysteine/methionine was added to 50 µCi/ml (1180 Ci/mmol; Trans-35S-label; ICN Biochemicals Inc., Costa
Mesa, CA) for an additional 2 h. Extracellular matrix was prepared
as described (31). Briefly, labeled cell monolayers were rinsed with
PBS, and the cytosolic and nuclear proteins were extracted by
subsequent washes with hypotonic buffer and sodium deoxycholate. The
remaining labeled extracellular matrix proteins were recovered by
addition of electrophoresis buffer to the washed wells following by
scraping. The samples were subjected to 10% SDS-PAGE, and the gels
were treated with Fluoro-HancerTM autoradiography enhancer
(Research Products International Corp., Mt. Prospect, IL) for 30 min
followed by drying and autoradiography.
For the FN assay, 2 × 105 cells were plated in 6-well
tissue culture plates. The cells were treated overnight with or without 10 ng/ml TGF-
1 or 30 µg/ml monoclonal mouse
anti-TGF-
1,2,3 in cysteine/methionine-free media. The
next day, [35S]cysteine/methionine was added to 50 µCi/ml for 2 h. The media were collected and subjected to
adsorption on gelatin-Sepharose beads (Amersham Pharmacia Biotech) in
the presence of 0.5% Triton X-100 as described (34). The samples were
resolved by 7% SDS-PAGE, and the gels were treated with
Fluoro-HancerTM for 30 min followed by drying and autoradiography.
Antisense TGF-
1 Oligodeoxynucleotides and Cell
Transfection--
Antisense 14-base phosphorothioate
oligodeoxynucleotides corresponding to the human TGF-
1
mRNA and the corresponding scrambled sequence control were
synthesized by Trilink Biotec. Inc. (San Diego, CA) and consisted of
the antisense sequence of 5'-CGA TAG TCT TGC AG-3' and scrambled
control sequence of 5'-GTC CCT ATA CGA AC-3' previously shown to
completely and specifically eliminate TGF-
1 expression
(64). To introduce the oligonucleotides into fibrosarcoma HT1080
subclone H4 cells or the egr-1-transfected clones, a
cationic liposome-mediated transfection method was used (65). Briefly,
oligonucleotides dissolved in 1 volume of antibiotic-free medium were
mixed with LipofectinTM reagent (Life Technologies, Inc.)
dissolved in same volume of antibiotic-free medium and incubated for 15 min at room temperature. Thereafter, the oligonucleotides-liposome
complexes were diluted with 4 volumes of antibiotic-free medium and
then added to cells that had been grown to 60% confluence and washed
twice with antibiotic-free medium. The concentration of
oligonucleotides and Lipofectin in the transfection medium was 1 mM and 1%, respectively. After 4 h, fresh normal
growth medium containing 5% fetal bovine serum was added. Forty-eight
hours later the cells were analyzed for the expression of PAI-1 or FN.
Oligonucleotides and Electrophoretic Mobility Shift
Assay--
Nuclear extracts were prepared from the clone of maximum
EGR-1 expression (H4E9) and from non-expressing cells (H4, H4N) as described (35). The protein concentrations in the nuclear extracts were
determined by protein assay reagent (Bio-Rad). Synthetic double-stranded oligonucleotides bearing sequences corresponding to
either
75 to
52 base pairs or
4 to +18 base pairs of the human
FN promoter, termed sites A and B, respectively (36), were
selected based on an analysis of the sequence of the human FN promoter region for the presence of GCEs (Transcription
Element Search Software). The DNA sequences for the two
oligonucleotides are for site A,
5'-GATCTCTCTCCTCCCCCGCGCCCCGGGG-3'; and for site B,
5'-GATCTCCGACGCCCGCGCCGGCTGTG-3'. The prototypic
EGR-1-binding sites are underlined. The oligonucleotides of sites A and
B were end-radiolabeled with [
-32P]ATP by use of T4
polynucleotide kinase according to supplier specification (Amersham
Pharmacia Biotech) and used as "probes" A and B. Gel shift assays
were performed as follows: nuclear extracts (20 µg) were incubated
with radiolabeled DNA probe (1 × 105 cpm) for 20 min
at 4 °C in a 20-µl reaction containing 25 mM HEPES, pH
7.9, 60 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 100 µg/ml
spermidine, 10% glycerol, and 100 µg/ml bovine serum albumin.
Protein-DNA complexes were separated from free DNA probe by
electrophoresis through 6% nondenaturing acrylamide gels in 0.5× Tris
borate/EDTA. The gels were dried and exposed to x-ray film (Kodak
X-OMAT, Kodak) for autoradiography. For the competition experiments,
excess unlabeled oligonucleotides for sites A and B, or
oligonucleotides containing two consensus EGR-1-binding sequences (GCE)
and mutated EGR-1-binding sequences (mGCE), were incubated with the
reaction mixture for 15 min at 4 °C before the addition of the
radiolabeled probes A and B. Similarly, in the antibody supershift
experiments, the specific antibodies against Sp1 (Santa Cruz
Biotechnology, Santa Cruz, CA) or rabbit polyclonal Egr-1 antiserum
(15) were added to the binding reactions and incubated before the
appropriate radiolabeled probe was added. Recombinant GST-EGR-1 fusion
protein or wild-type FLAG-tagged-EGR-1 and
FLAG-tagged-EGR-1
S348A/S350A mutant fusion proteins were used as
controls. Mutations were introduced at the DNA-binding sites by the
Quick Change method using polymerase chain reaction primers containing
the S348A/S350A mutations (Stratagene, La Jolla, CA).
Cell Adhesion Assay--
ELISA plates (Sarstedt Inc., Newton,
NC) or 35-mm polystyrene Petri dishes (Falcon, Becton Dickinson,
Bedford, MA) were used for adhesion assays. For the cell adhesion assay
in Petri dishes, the cells were added to polystyrene Petri dishes at
3 × 104 cells/cm2. After incubation for 4 or 7 h, non-attached cells were washed off by using PBS at
37 °C, and adherent cells were harvested by adding trypsin and
counted with a Coulter counter (Coulter Electronics Inc., Hialaeh, FL).
In some cases, 96-well ELISA plates were coated with 5 µg/ml or 0.25 µg/ml human plasma FN (Boehringer Mannheim) or 5 µg/ml recombinant
active human PAI-1 (American Diagnostica Inc, Greenwich, CT) for 1 h at 37 °C. Non-specific sites were blocked by addition of 0.1%
bovine serum albumin in PBS, and cells were plated at 4 × 104 cells/well. After incubation for varying times, any
non-adherent cells were removed with gentle washing with warm PBS.
Adherent cells were stained with 1% crystal violet in 20% methanol
for 15 min and then washed with distilled water and solubilized with 2% SDS. The absorbance of the solution at 590 nm was quantified using
a microtiter plate reader. For PAI-1 neutralization, monoclonal antibody against human PAI-1 (American Diagnostica Inc., Greenwich, CT)
was added to plates at 10 µg/ml together with cells, and its effects
on adhesion were observed. For FN inhibition, GRGDSP and GRGESP
peptides, provided by Dr. R. Pasqualini (The Burnham Institute, La
Jolla, CA), were added at 10-50 µg/ml at the time of cell addition to the FN-coated plates.
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RESULTS |
EGR-1 Increases the Expression of PAI-1 in Fibrosarcoma HT1080
Cells--
Fibrosarcoma HT1080 subclone H4 cells have been stably
transfected with an expression vector for wild-type EGR-1 (14). A series of transfectants that express graded amounts of EGR-1 were used
to determine whether PAI-1 or FN was synthesized and secreted in
proportion to the amount of EGR-1 expressed by these cells. The
relative expression of EGR-1 was reconfirmed here by Western blot
analysis (Fig. 1A). The results
confirmed that H4E9 clone expresses the largest amount of EGR-1, taken
as 100%, whereas the remaining clones express graded levels of EGR-1:
H4E2 (21%) and H4E3 (8.7%). H4E4 and H4E6 do not express detectable
levels of EGR-1 similar to the results for the parental H4 cells and empty vector control cells H4N (Fig. 1A).

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Fig. 1.
The secretion of PAI-1 is increased in
EGR-1-expressing clones of fibrosarcoma HT1080 cells.
A, Western blot analysis of EGR-1 expression in the series
of EGR-1 stably transfected of H4 cells, a subclone of HT1080. Protein
was extracted from parental H4 cells, empty vector transfectant (H4N),
and from stable EGR-1 transfectants (H4E2, H4E3, H4E4, H4E6, and H4E9).
100 µg of protein of each extract was analyzed by 7% SDS-PAGE,
transferred to polyvinylidene difluoride membranes, and detected by
using a polyclonal EGR-1 antibody as described under "Materials and
Methods." The band intensity for EGR-1 in H4E9 clone was considered
as 100%, and the relative band intensities of the other samples were
represented relative to that of H4E9. B, assay for the
production of PAI-1. Cells were incubated with cysteine/methionine-free
DMEM in the presence or absence of 10 ng/ml rhTGF- 1 for 2 h and
labeled with [35S]methionine/cysteine for additional
2 h. After the cells were lysed by hypotonic buffer and sodium
deoxycholate, ECM proteins were harvested by adding SDS-sample buffer
and scraping the culture wells. PAI-1 is observed as 48-kDa bands after
10% SDS-PAGE and autoradiography. C, densitometric analyses
of PAI-1 expression in the absence (white bars) or presence
(black bars) of TGF- 1. The values plotted represent the
average of five independent experiments. Inset, the
expression of PAI-1 as a function of EGR-1 is shown.
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The PAI-1 expression was examined in these graded EGR-1-expressing
clones. In metabolically labeled EGR-1-expressing H4 clones, H4E9 cells
expressed the greatest amount of PAI-1, whereas H4E2 also expressed
more PAI-1 than parental cells or negative control clones (Fig.
1B). Quantitative analysis of the average of five independent experiments showed >5-fold elevation of PAI-1 in H4E9 clone (Fig. 1C). In contrast, The pCMV empty
vector-transfected clones or EGR-1-negative clones, H4E6 and H4E4
(G418-resistant clones), expressed low levels of PAI-1 similar to the
parental H4 cells (Fig. 1, B and C). Indeed, the
expression of PAI-1 and EGR-1 is highly correlated with the expression
of EGR-1 (RPEARSON = 0.971, p < 0.0003) (Fig. 1C, inset).
We previously showed that expression and secretion of TGF-
1 was in
direct proportion to the levels of expression of EGR-1 in these graded
clonal series (RPEARSON = 0.96) and functions in
an autocrine loop to regulate growth of these cells (18). Moreover,
TGF-
1 can stimulate the expression of PAI-1 (37-38), suggesting
that EGR-1-induced expression and secretion of TGF-
1 may account for
the increased expression of PAI-1 by the EGR-1-expressing cells. To
test whether TGF-
1 could in fact stimulate the expression of PAI-1
in our clones, the cells were labeled with
[35S]cysteine/methionine in the presence or absence of 10 ng/ml of recombinant human TGF-
1 (rhTGF-
1) for 4 h. The
expression of PAI-1 was monitored by electrophoresis and
autoradiography (Fig. 1B). All clones exhibited a
significant (p < 0.05) increase in the secretion of
PAI-1 following treatment with rhTGF-
1 (Fig. 1C). Thus,
the secretion of PAI-1 in the clones of our series all responded to
rhTGF-
1 treatment. Moreover, the total PAI-1 observed for each clone
correlated with basal PAI-1 levels observed in the absence of
stimulation (Fig. 1C) with exogenous rhTGF-
1, suggesting
that the effect of addition of rhTGF-
1 was additive with stimulating
effects of endogenous EGR-1/TGF-
1 system. These data demonstrated
that TGF-
1 can regulate the induction of PAI-1 in H4-transfected
clones, indicating that the correlation between EGR-1 and PAI-1 may be
mediated by TGF-
1 induction.
EGR-1 Increases the Expression of Fibronectin in Fibrosarcoma
HT1080 Cells--
To determine the levels of FN secreted by the
EGR-1-expressing or non-expressing clones, the cells were metabolically
labeled for 2 h, and the labeled FN was adsorbed from the
conditioned medium with gelatin-Sepharose beads that are known to
specifically bind FN (42). The characteristic band of FN at 220 kDa was
observed at maximum intensity in the medium from H4E9 cells, whereas a similar protein occurred in the medium from H4E2 and H4E3 clones with a
band intensity intermediate between H4E9 cells, parental cells, and
EGR-1-non-expressing clones (Fig.
2A). The quantitative analysis of
the average of three independent experiments showed a 3.5-fold average
induction of FN in the H4E2 clone and 38-fold average increase of FN in
the H4E9 clone compared with the negative control cell clones (Fig.
2B). Similar to the analysis for PAI-1, we determined the
correlation between EGR-1 expression and FN expression and found they
are very highly correlated (RPEARSON = 0.985, p < 0.00005) (Fig. 2B, inset). These
results suggest that at least one factor that is important for FN
secretion by the graded clonal series is EGR-1.

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Fig. 2.
The secretion of FN is increased in
EGR-1-expressing clones of HT1080 cells. A, the
secretion of FN in HT1080 clones. H4 parental cells, empty vector
transfectant (H4N), or egr-1 transfectants (H4E series) were
incubated with or without 10 ng/ml rhTGF- 1 for 12 h. After
metabolic labeling with [35S]cysteine/methionine for
2 h, the FN secreted into the media was purified by adsorption to
gelatin-Sepharose. The protein was analyzed by SDS-PAGE and visualized
by autoradiography. The relevant portion of the fluorogram is shown.
FN, 220 kDa is indicated (arrow). B,
densitometric analysis of fibronectin secretion in the absence
(white bars) or presence (black bars) of
rhTGF- 1. The bars represent the mean values from three
independent experiments. Inset, the expression of
fibronectin secretion as a function of EGR-1 is shown.
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TGF-
1 Regulates the Expression of PAI-1 but not FN in
EGR-1-expressing Cells--
Previous studies indicated that the
expression of FN was stimulated by TGF-
1 in prostatic carcinoma
cells (26), colon cancer cell Moser (24), and lung mink Mv1Lu (CCL64;
American Type Culture Collection) (38). In order to determine whether
EGR-1-induced TGF-
1 might regulate the expression of FN in HT1080
fibrosarcoma cells, rhTGF-
1 was added to all clones at the same
concentration that stimulates a high level of expression of PAI-1. In
contrast to the effects on PAI-1, in parallel experiments we observed
only a weak induction of FN (<1.5-fold) for all clones (Fig. 2,
A and B), suggesting the regulation of FN is
distinct from PAI-1 regulation.
To determine further the effect of TGF-
1 on the secretion of PAI-1
and FN in fibrosarcoma HT1080 cells, the expressions of PAI-1 and FN
were examined as a function of the concentration of rhTGF-
1 (0.001 to 100 ng/ml) on parental H4 cells (Fig.
3A). The induction of PAI-1
expression by rhTGF-
1 is dose-dependent in H4 cells.
When the concentration of rhTGF-
1 is increased, the expression of
PAI-1 dramatically increased (Fig. 3, A and B),
up to 9-fold compared with basal level of PAI-1 in H4 cells. The
half-maximal stimulation was achieved at a rhTGF-
1 concentration
0.05 ng/ml (EC50 = 2 × 10
12
M), with the near-maximal effect being observed at
100
ng/ml (4 × 10
9 M) (Fig. 3B),
indicating that H4 cells are very sensitive to TGF-
1. In contrast,
the induction of FN by rhTGF-
1 did not exhibit a detectable response
at 0.05 ng/ml (Fig. 3A). A weak response of 2.4-fold was
seen at a 200-fold higher concentration of 10 ng/ml (4 × 10
10 M) (Fig. 3B).

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Fig. 3.
Effects of rhTGF- 1
on the secretion of PAI-1 and FN in HT1080 subclone H4 cells.
A, the secretion of PAI-1 (upper panel) and FN
(lower panel) by H4 parental cells was measured after
incubation with the indicated concentrations of rhTGF- 1 as described
under "Materials and Methods." B, the relative
expression level of PAI-1 and FN following stimulation with rhTGF- 1
was based on the densitometric analysis of A as described
under "Materials and Methods."
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These data suggest that PAI-1 but not FN is preferentially regulated by
TGF-
1. The high correlation between EGR-1 and FN (Fig. 2B,
inset) without the role of TGF-
1 suggests, as one possibility, that EGR-1 directly regulates the FN gene. Therefore, we
next examined whether the TGF-
1 is required for the secretion of
both PAI-1 and FN in egr-1-regulated HT1080 cells.
Neutralizing TGF-
antibody was added to block the TGF-
1 effect in
EGR-1-expressing clones. The results are shown in Fig.
4. The basal level of PAI-1 was reduced
1.4-fold in control cells (H4 and H4N), but there was 2.7-fold
reduction of PAI-1 secretion in EGR-1-expressing cells (H4E2 and H4E9)
in the presence of TGF-
antibodies, supporting the observation that
the increased secretion of PAI-1 by EGR-1 may require the expression of
TGF-
1. In contrast, the expression of FN was only
slightly altered by the addition of TGF-
antibody (<1.4-fold) in
the EGR-1-expressing cells (H4E2 and H4E9) (Fig. 4). Thus, consistent
with the dose-response studies, the experiments using TGF-
neutralizing antibody suggest that the expression of PAI-1 by EGR-1 but
not FN is mediated by TGF-
1.

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Fig. 4.
TGF- -neutralizing
antibodies inhibit the induction of PAI-1 but not FN in
EGR-1-expressing cells. Cells were grown in the absence or
presence 30 µg/ml monoclonal antibody against
TGF- 1,2,3 overnight, and labeled with
[35S]cysteine/methionine, and the secretion of PAI-1
(upper panel) or FN (lower panel) was
measured as described under "Materials and Methods." The relative
expression of PAI-1 or FN was normalized to the basal level of PAI-1 or
FN in parental H4 cells and indicated as fold increase of PAI-1 or
FN.
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Similar results were also obtained in experiments in which antisense
TGF-
1 oligonucleotides were utilized to block TGF-
1 function. In
previous studies, using a TGF-
1 antisense oligodeoxynucleotide complementary to the mRNA of human TGF-
1, both
transcript levels and protein production were completely and
specifically eliminated (55, 64). Therefore, we examined the expression
of PAI-1 after lipofection with the same TGF-
1 antisense
or the scrambled sequence control oligonucleotides (Fig.
5). The level of PAI-1 in EGR-1-expressing cells (H4E9) was reduced by over 75% in both EGR-1-expressing clonal
cell lines to near basal levels similar to those of control cells (H4
and H4N), whereas the levels of PAI-1 were not influenced by
transfection with the scrambled sequence oligonucleotide or by use of
the lipofection reagent alone (Fig. 5, upper panel). In
contrast, the expression of FN was not inhibited in
EGR-1-expressing cells by transfection of either antisense
TGF-
1 or scrambled sequence oligonucleotide (Fig. 5,
lower panel). These experiments strongly support the results
based on the TGF-
1 antibody blocking experiments, showing TGF-
1
is required for expression of PAI-1 but not FN by
egr-1-transfected cells. In addition, the lack of effect of
antisense TGF-
1 on FN secretion supports the observations on the dose-response studies (Fig. 3) that TGF-
1 is not involved in
mediating expression of FN in the egr-1-transfected
cells.

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Fig. 5.
Antisense TGF- 1
oligonucleotides inhibit the induction of PAI-1 but not FN in
EGR-1-expressing cells. Parental H4 cells, empty vector
transfectant (H4N), and EGR-1-expressing cells (H4E9) were transiently
transfected with TGF- 1 antisense oligonucleotides or
scrambled control oligonucleotides or were treated with Lipofectin
reagent alone as a control. After 48 h, cells were analyzed for
expression of PAI-1 or FN as described in Figs. 1-4. The relative
protein level was normalized to that of parental H4 cells treated with
the Lipofectin reagent alone as indicated at the bottom of
each lane as fold secretion of PAI-1 or FN.
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Nuclear and Recombinant EGR-1 Bind to the Proximal Region of the
Human Fibronectin Promoter--
The high correlation of FN secretion
with EGR-1 expression independently of TGF-
1 suggests that EGR-1 may
directly regulate FN gene. We examined the promoter region
of the human FN gene (exon 1) consisting of 742 base pairs
in 5'-flanking region (36), and we observed two potential EGR-1-binding
sites by using Transcription Element Search Software. Two sequences
containing potential EGR-1 consensus sites termed A and B were
identified, and double strand probes containing these sequences were
synthesized separately (Fig. 6A).
In order to test whether EGR-1 binds to either of these motifs,
electrophoretic mobility shift assays were carried out. A recombinant
wild-type FLAG-tagged-EGR-1 fusion protein and control mutant
FLAG-tagged-EGR-1
S348A/S350A fusion protein were used for these
assays. The FLAG-tagged-EGR-1
S348A/S350A fusion protein is a serine
alanine mutant at positions 348 and 350 in the EGR-1 zinc finger
domain, thereby reducing DNA binding activity (79-80). Fig.
6B shows that a specific DNA-protein complex occurred when wild-type protein was combined with either of the DNA probes and that
the complexes were absent when the EGR-1 mutant protein was used in the
reaction in place of the wild-type protein (compare lanes 1 and 2 and lanes 6 and 7, arrow). When
EGR-1 antibody was added to the reaction, this complex disappeared
(lanes 5 and 8), consistent with previous studies
showing that anti-EGR-1 did not promote a supershift but in fact caused
a dissociation of the complex (67, 68). In contrast, addition of an
anti-Sp1 antibody did not dissociate the complex or produce a
supershift. In fact, the use of anti-Sp1 antibodies with the
FLAG-tagged-EGR-1 fusion protein preparation is associated with a
slight but reproducible increase in band intensity (Fig. 6B,
lane 1 versus 3 and lane 2 versus 4). Moreover, experiments with FLAG-tagged-EGR-1
fusion protein preparation and probe B often lead to less intense bands than experiments with probe A. In order to clarify these observations, similar experiments were carried with a different EGR-1-containing preparation, GST-EGR-1 fusion protein (Fig. 6C). As before
(Fig. 6B), specific DNA-protein complexes occurred when
GST-EGR-1 protein was combined with either of the DNA probes (Fig.
6C, lanes 2 and 6).
Furthermore, for both sites the addition of the unlabeled oligonucleotides with the consensus GCE sequence completely
"competes-off" probe binding (Fig. 6C, lanes
3 and 7). Similarly, the addition of the
unlabeled oligonucleotides with a mutated EGR-1-binding sequence (mGCE)
has no effect (Fig. 6C, lanes 4 and
8). These results confirm the results of experiments with
recombinant FLAG-tagged-EGR-1 fusion protein. Moreover, the experiments
show that the complexes formed with the GST-EGR-1 preparation under the
same conditions as with the FLAG-tagged-EGR-1 fusion protein are of
similar intensity, suggesting similar binding affinities, whereas the
intensity changes observed with the FLAG-tagged fusion protein are not
representative of quantitative properties. In support of this, direct
titration experiments with unlabeled probes demonstrated very similar
binding for each site (Fig. 6C, lanes 10-13 and
lanes 15-18). The sum of results indicates that both site A
and B form specific complexes with recombinant protein.

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Fig. 6.
EGR-1 protein specifically binds to the human
FN promoter. A, schematic
representation of human FN promoter. The location of two GCE
sites and transcription start sites on the human FN promoter
(36) is indicated by the shaded boxes. B,
recombinant wild-type FLAG-tagged-EGR-1 (wt) or mutant
FLAG-tagged-EGR-1 S348/350A (mut) protein was incubated
with the indicated 32P-labeled oligonucleotide probes
(A or B) and processed for EMSA as described
under "Materials and Methods." The specific antibodies
(anti-Egr-1 and anti-Sp1) were preincubated with
protein for 15 min at room temperature where indicated. The specific
protein-DNA binding bands are indicated by the arrows.
C, recombinant GST-EGR-1 fusion protein. D, the
nuclear protein extracted from egr-1-transfected H4 cells
(H4E9), parental H4 cells, or empty vector-transfected cells were
incubated with human FN 32P-labeled
oligonucleotide probes A and B and analyzed by EMSA as described under
"Materials and Methods." The mobility shift competition assay with
GST-EGR-1 or nuclear extracts from H4E9 cells in the absence or
presence of 10-, 20-, 100-, and 200-fold excess of unlabeled
oligonucleotide probes A and B or unlabeled oligonucleotides containing
two consensus EGR-1-binding sequences (GCE) and mutated EGR-1-binding
sequences (mGCE) were indicated at the top of the panel. For
antibodies supershift experiments, the specified antibodies against
Egr-1 or Sp1 were preincubated with the extracts for 15 min at 4 °C
where indicated. The specific DNA-EGR-1 complexes are indicated by the
arrows.
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Similar results were obtained when nuclear extracts from the
EGR-1-expressing clone (H4E9) and EGR-1-lacking cells (H4 and H4N) were
used in place of the recombinant proteins. A prominent complex was
observed when nuclear extracts from H4E9 cells were incubated with
oligonucleotide probes A and B (Fig. 6D, lanes 3 and 13, arrow). This complex was not detected for the
nuclear extracts from control cells H4 and H4N (Fig. 6D,
lanes 1 and 2 and lanes 11 and 12). Specificity of binding was determined by titration
experiments using unlabeled oligonucleotides. The complex formation by
the H4E9 nuclear extracts were inhibited in dose-dependent manner by addition of the unlabeled oligonucleotides A or B (Fig. 6D, lanes 6-8 and lanes 16 and
17). Also, competition with oligonucleotides containing two
consensus EGR-1-binding sequences (GCE), but not mutated
EGR-1-binding sequences (mGCE), resulted in dissociation of complex
(Fig. 6D, lanes 4 and 5 and
lanes 14 and 15). Again, the addition
of EGR-1 antibody dissociated the complex that was formed with H4E9
nuclear extracts (Fig. 6D, lanes 10 and 19), but addition of the Sp1 antibody had no effect
(Fig. 6D, lanes 9 and 18).
These results argue against significant binding of any Sp1 that may be
in these extracts to either sites (66). Thus, factors in nuclear
extracts of EGR-1-expressing cells but not control cells recognized
both A and B sites of human FN promoter in a
sequence-specific manner. We conclude from these results that EGR-1
binds specifically to the promoter region of human FN gene
consistent with a direct role in the regulation of FN expression.
Enhanced Expression of Fibronectin and PAI-1 in EGR-1-expressing
Cells Increases Adherence to a Substratum--
We have previously
found that expression of EGR-1 in HT1080 slows proliferation, restores
density-dependent growth arrest, and promotes a flattened
and non-refractive phenotype (14, 15). Conversely, elimination of
endogenous EGR-1 tends to promote a more transformed phenotype (15).
Both FN and PAI-1 have been shown to interact with FN receptors and to
modulate cell adhesion and behavior in a variety of cell types
including HT1080 cells (27, 74-78). To determine whether
endogenous PAI-1 or FN influences the adhesion of HT1080
cells, we compared the attachment efficiency of the various cells to
untreated polystyrene "Petri" dishes. Polystyrene plastic plates
have high hydrophobic nature, inhibit cell adhesion, and are commonly
used for growing cells in suspension culture. Many types of adherent
cells fail to attach on this substratum (70), but EGR-1-expressing
cells (H4E9 clone) can attach this kind of substratum significantly
better than the parental cells (H4) or empty vector cells (H4N) (Fig.
7). For example, we observed that 22% of
H4E9 cells adhere to polystyrene Petri dishes 4 h after plating,
compared with
8% for the controls (H4 9% and H4N 7%)
(p < 0.006) (Fig. 7). At 7 h after plating, 88%
of H4E9 cells had attached to polystyrene Petri dishes, whereas only
58% of the control H4 or H4N cells adhered to Petri dishes
(p < 0.0001) (Fig. 7), consistent with functional
roles for endogenous PAI-1 and/or FN.

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Fig. 7.
Enhancement of cell adhesion in
EGR-1-expressing cells to substratum. A, the parental
H4 cells, empty vector control cells, and egr-1-transfected
clone H4E9 were plated in 35-mm plastic polystyrene Petri dishes or
tissue culture dishes treated for the indicated times. The percentage
of adhesion was calculated based on the numbers of starting cells.
B, cells were plated in 96-well ELISA plates that were
precoated with FN or PAI-1 as described under "Materials and
Methods." After incubation for the indicated times, the cells
were stained with crystal violet and the O.D. at 590 nm was read using
microtiter plate reader. The percentage of adherent cells was
calculated based on the number of starting cells. In this
representative experiment the values plotted are means ± S.D.
(n = four dishes). Similar results were obtained in
three other independent experiments.
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To test whether authentic FN or PAI-1 in fact facilitates attachment,
we carried out adhesion assays in the presence and absence of rhPAI-1
and human plasma FN (Fig. 7B). When the various cells were
exposed to 96-well polystyrene "ELISA" plates precoated with 5 µg/ml rhPAI-1 for 4 h, they exhibited adhesion efficiencies similar to untreated plates (cf. Fig. 7, A and
B). However, by 7 h about 70% of the cells attached to
the PAI-1-treated plates although no consistent differences were
observed between EGR-1-expressing cells and EGR-1-lacking control cells
(Fig. 7B). Addition of neutralizing anti-PAI-1 antibodies
(Fig. 8) had little effect on the adhesion of
control cells but partially inhibited the attachment of the FN-secreting H4E9 cells by about 20%. The inhibition is reproducible and significant (p < 0.03). These results suggest, as
one possibility, that any role of PAI-1 on the attachment of H4
cells and derivatives depends on other ECM components such as FN (see
below).

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Fig. 8.
Cell adhesion on PAI-1-coated plates.
The 96-well ELISA plates were precoated with recombinant active human
PAI-1 in PBS for 1 h. Monoclonal antibodies against PAI-1 or
GRGDSP and GRGESP peptides were added as indicated at the time of cell
plating. Seven hours later, the adhesive cells were stained with
crystal violet, and the O.D. at 590 nm was read using a microtiter
plate reader. The percentage of adherent cells was calculated based on
the number of starting cells. In this representative experiment the
values plotted are mean ± S.D. (n = five wells). Similar results were obtained in three other independent
experiments.
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To test whether authentic FN facilitates attachment, we tested the
adhesion of cells on 96-well polystyrene plates precoated with 5 µg/ml FN for 4 h. We found that 88% of H4E9 cells attached to
the plates and ~70% of H4 and H4N control cells attached (Fig. 7B). Thus, we found that FN promoted about 20 times greater
adhesion than for uncoated or PAI-1-coated plates similar to the
results reported by Planus et al. (27). Virtually all cells
attached to FN-coated plates by 7 h (Fig. 7B). To
determine whether the increased adhesion depended on specific FN
interactions, we added Arg-Gly-Asp (RGD)-containing peptides which
block FN binding to its receptors, such as
5
1 or
v
3
integrin (61, 69). The addition of moderate amounts of GRGDSP peptide
(10 µg/ml) reduced the adhesion of H4 or H4N to <20% but had no
effect on the adhesion of H4E9 cells (Fig.
9A). However, when the GRGDSP
concentration was increased to 50 µg/ml, the adhesion of H4E9 cells
was completely eliminated (Fig. 9A). The control peptide
(GRGESP) had negligible effects even at 50 µg/ml (Fig.
9A). These results indicate that H4 cells and derivative
cells interact with FN in an RGD-dependent manner.
Moreover, the greater amount of GRGDSP required for the inhibition of
attachment of the FN-secreting H4E9 cells supports a functional role of
endogenous FN.

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Fig. 9.
Cell adhesion on FN-coated plates.
A, the 96-well ELISA plates were precoated with 5 µg/ml FN
in PBS for 1 h. Monoclonal antibodies against PAI-1, GRGDSP
peptide, GRGESP peptide control, or combination of anti-PAI-1 antibody
and GRGDSP peptide were added at the time of cell plating ("Materials
and Methods"). Four hours after plating, the adhesive cells were
analyzed with crystal violet as described in Fig. 8. In this
representative experiment the values plotted are mean ± S.D.
(n = five wells). Similar results were obtained in
three other independent experiments. B, the 96-well ELISA
plates were precoated with 0.25 µg/ml FN in PBS for 1 h and
treated and analyzed as for A. * indicates the comparison of
adhesion of EGR-1-expressing H4E9 cell in the presence or absence of
PAI-1 antibody by t test.
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Since the attachment of FN-secreting H4E9 cells is partially inhibited
by the addition of anti-PAI-1 antibodies (Fig. 9A), we asked
whether anti-PAI-1 antibodies inhibit attachment to FN-coated plates.
The addition of anti-PAI-1 antibodies to cells exposed to FN-coated
plates (Fig. 9A) had the opposite results compared with the
effect of the antibodies on cell adhesion to PAI-1-coated plates
(cf. Figs. 8 and 9A). The antibody blocked the
attachment of the control cells by 50-60% but had no effect on
EGR-1-expressing H4E9 cells (Fig. 9A). Increased amounts of
anti-PAI-1 had weak inhibitory effect on the adhesion of H4E9 cells
(data not shown). However, addition of anti-PAI-1 antibodies to these
cells when attached to plates coated with less FN (20× less)
substantially and significantly reduced adhesion to levels
indistinguishable from control cells (Fig. 9B). Consistent
with this, the combination of small (10 µg/ml) amounts of GRGDSP
peptide together with anti-PAI-1 treatment reduced attachment of all
three cell types by approximately 50% compared with the effects of low
dose GRGDSP alone (Fig. 9A). These results, together with
the effects of anti-PAI-1 antibodies on cells adherent to PAI-1-coated
surfaces (Fig. 8), show that PAI-1 facilitates attachment only in the
context of FN-coated surfaces or FN-secreting cells.
In summary, the observations support the conclusions that FN and, to a
lesser extent, PAI-1 facilitate attachment of H4 and derivative cells
and that endogenous FN is also functional in promoting increased and
RGD-dependent cell adhesion interactions. Endogenous FN
further functions to facilitate the attachment role of endogenous
PAI-1.
 |
DISCUSSION |
Regulation of Fibronectin and PAI-1 by EGR-1 Are
Distinct--
HT1080 and related clonal lines studied here express
little or no EGR-1, a feature that is similar to a variety of human
tumor cell lines including breast carcinoma and glioblastoma cells
(13). Stable expression of EGR-1 in several tumor cell lines, such as U251 glioblastoma cell line, ZR-75 breast carcinoma, Saos2 osteogenic sarcoma cells that lack EGR-1 expression, leads to more normal cell
morphology especially in HT1080 fibrosarcoma cells. The expression of
EGR-1 in HT1080 stimulates the expression and secretion of TGF-
1 in
direct proportion to the amount of EGR-1 expressed in a series of five
clonal lines and inhibits cell growth. The addition of neutralizing
anti-TGF-
1 antibody to the EGR-1 expression cells actually causes a
near doubling of growth thereby completely reversing the growth
inhibitory effect in EGR-1 in HT1080 cells (18). Moreover,
EGR-1-expressing HT1080 cells but not control cells strongly activate
reporter constructs containing the EGR-1-binding sequences of the
TGF-
1 promoter, and this effect is blocked by the
TGF-
1 transcription suppressor, WT-1 (18). Thus, EGR-1 suppresses the growth and transformation of HT1080 cells by induction of a TGF-
1-dependent autocrine loop.
In this study, we provide evidence that the suppression of
transformation by TGF-
1 involves the coordinated effects for the secretion of TGF-
1, FN, and PAI-1. Both the FN (23, 24,
26) and PAI-1 (25, 26, 72) genes have been reported to be
regulated by the TGF-
1 signal transduction mechanism in several cell
types. We reasoned that the secretion of FN and PAI-1 may be a
consequence of the TGF-
1 autocrine loop known to be functional in
the EGR-1-expressing cells (18). Indeed, addition of rhTGF-
1 to
control HT1080 cells mimicked the effect of EGR-1 in that considerable
increased secretion of PAI-1 is observed with an EC50
10
12 M consistent with the presence of
high affinity TGF-
1 receptors (41) that functionally regulate PAI-1
transcription and secretion. Conversely, the addition of neutralizing
anti-TGF-
antibody to EGR-1-expressing cells but not control cells
blocks the secretion of PAI-1. Moreover, the use of previously
characterized antisense TGF-
1 oligonucleotides confirms this result
thereby strongly supporting the presence of a functional TGF-
1
autocrine loop as responsible for the regulation of PAI-1 secretion.
Thus, in HT1080 cells, the expression of EGR-1 appears to mediate the
expression of PAI-1 by direct induction of TGF-
1, which in turn
regulates the secretion of PAI-1 via a highly effective
TGF-
1-autocrine loop. These relationships are summarized in Fig.
10.

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Fig. 10.
Model for mechanism of the suppression of
transformation by EGR-1 in fibrosarcoma HT1080. Dashed
lines indicate that the expression of FN receptor is regulated by
FN and TGF- 1 but not demonstrated in this study (see text). EGR-1
directly transactivates both TGF- 1 and FN
genes leading to increased steady state synthesis and secretion of
TGF- 1 and FN. Secreted TGF- 1 becomes activated and induces
increased steady state PAI-1 expression via TGF- 1 receptor-mediated
signal transduction. Both FN and PAI-1 augment ECM formation and
density-dependent growth control.
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However, similar regulation of the secretion of FN was not observed.
Thus, the addition of rhTGF-
1 did not stimulate the secretion of FN
to an appreciable extent, and the secretion that was observed occurred
at over 2 orders of magnitude higher concentration of rhTGF-
1 than
that known to mediate TGF-
1-dependent PAI-1 secretion.
Conversely, specific anti-TGF-
antibodies that effectively blocked
PAI-1 secretion had no effect on FN secretion. These results were
obtained in parallel with the results for PAI-1 that, therefore, provided a convenient positive control. The sum of observations strongly argues against regulation of FN secretion by TGF-
1 in HT1080 cells.
The human FN gene contains at least four GC-rich sequences
at least two of which consistent with the consensus sequence for EGR-1-binding site (36). Interestingly, the site termed A here has been
observed to positively regulate transcription of FN by Sp-1
in embryonal carcinoma cells (66). SP-1 binds GC-rich sequences and
commonly interacts with EGR-1 in the regulation of a variety of genes
(19). In direct binding studies of either recombinant EGR-1 or nuclear
extracts of EGR-1-expressing cells, we observed complex formation with
both sites. These complexes were disrupted by anti-EGR-1 antibodies, a
known characteristic of EGR-1 antibodies on specific EGR-1-DNA
complexes (67-68). Anti-Sp-1 sera, on the other hand, had no effect on
the complex indicating that SP-1 was not likely associated with either
GCE in HT1080 cells. These observations indicate that EGR-1 interacts
with known positive transcription activation sites of the FN
promoter and strongly supports the view that EGR-1 directly induces the
transcription of FN. Since we have not examined the
transcription properties of these sites, it remains an hypothesis that
EGR-1 directs the expression of FN via sites A and/or B. However, this hypothesis provides an explanation for the strong
correlation of FN secretion and EGR-1 expression observed here.
These relationships are summarized in the model of Fig. 10.
Phenotypic Consequences of Coordinated Gene Expression by
EGR-1--
Several studies have shown that one of the most important
growth regulatory mechanisms of TGF-
1 is its stimulatory effects on
the accumulation of ECM proteins such as FN (23, 24, 26). The
functional effects of FN on oncogenic transformation have been studied
in detail. The expression level of FN in fibrosarcoma cell lines is
commonly very low. For instance, in JEG-3 cells it is 0.01% of total
protein, in TE671 is 0.002%, in HeLa Bu25 is 0.008%, and in HT1080 is
0.004% (43). In contrast, normal fibroblasts exhibit high basal levels
of FN (0.3% of total protein) (43). FN expression is decreased also in
a variety of other transformed cells (44-45). Both FN and its receptor
are down-modulated during the chemical transformation of murine
fibroblasts (46-47). Ha-ras transformation of fibroblasts
significantly reduced the expression of FN and its receptor (48).
Absence or reduced expression of FN and its receptor is thought to play
a major role in determining the malignant phenotype (49-50).
Down-modulation of FN in melanocytes promotes malignant behavior (51).
Conversely, the addition of FN peptides has been shown to inhibit
experimental metastasis of melanoma cells (52). Similarly, the level of
expression of FN is significantly correlated with low metastatic
potential of breast carcinoma (53). Overexpression of recombinant FN
suppresses the transformed phenotype in fibrosarcoma HT1080 (33). The
HT1080 cells that re-express FN adopt a more flattened morphology, have reduced proliferation and growth in soft agar, suppressed
tumorigenicity in vivo, and reduced cell migration (33).
It is likely that the EGR-1-induced expression of TGF-
1, PAI-1, and
FN have functional roles in the suppression of transformation of HT1080
cells. Here we used adhesion assays to provide an indication of the
functional role of the secreted products. Anti-PAI-1 antibodies preferentially inhibited the attachment of control cells but not EGR-1-expressing cells. Moreover, RGD-containing peptides that are
known to specifically disrupt the association of FN with FN receptors
completely inhibited the adhesion of EGR-1-expressing cells in a
dose-related manner. Strong inhibition also could be achieved by using
RGD-containing peptides at lower doses in association with anti-PAI-1
antibodies. Thus, it appears that the actual products of the regulation
proposed here (Fig. 10) are both secreted and participate in the
formation of specific ECM-cell association to effect enhanced
cell-substratum attachment.
PAI-1 participates in the stabilization of the extracellular matrix and
enhanced adhesion by at least two broad mechanisms (27, 54). First, it
interacts with an organized structure consisting of the urokinase
plasminogen activator (uPA)-urokinase plasminogen activator receptor
(uPAR) and inhibits the serine protease activity of uPA, thereby
blocking the conversion of serum plasminogen to plasmin, a broad
specificity protease that activates collagenases and metalloproteases
(54). These and related proteases destabilize the ECM thereby
facilitating mobility and the metastatic phenotype (54, 72-73). The
organization of the PAI-1·uPA·uPAR complex on HT1080 cells has been
examined in detail and is thought to preferentially associate with the
1 integrin subunit for cells adherent to FN, laminin, or
vitronectin-coated surfaces (74). PAI-1 directly influences the nature
of FN and vitronectin cell surface associations (27, 75-78). For
example, the addition of antibodies against PAI-1 to primary human
muscle satellite cells, which secrete PAI-1, is reported to cause near
complete inhibition of adhesion (27). Similarly, the role of PAI-1 in
the adhesion of the cells studied here was only apparent in the
presence of FN. On uncoated surfaces cells that secrete ample amounts
of both FN and PAI-1, such as H4E9, are at least partially susceptible to the blocking of adhesion by anti-PAI-1, whereas the control cells,
which express considerably less of each factor, establish attachments
to ELISA plates that are not susceptible to inhibition by anti-PAI-1
(Fig. 8). Conversely, when FN is supplied as precoated surfaces, the
interactions formed by the small amounts of PAI-1 secreted by control
cells (H4 and H4N, Fig. 1) are readily inhibited by anti-PAI-1, whereas the increased amounts of PAI-1 and FN secreted by H4E9 cells appear too extensive to be neutralized readily (Fig. 8).
Finally, the use of increased anti-PAI-1 antibody or decreased FN at
the same antibody concentration (Fig. 9B) demonstrated the role of endogenous PAI-1 on the adhesion of EGR-1-regulated H4E9 cells.
The combined use of RGD-containing peptides and anti-PAI-1 lead to
complete inhibition of adhesion by the minimal PAI-1/FN-expressing control cells and over 60% inhibition of adhesion to FN-coated surfaces. These results argue that PAI-1 expressed by EGR-1-regulated cells is indeed a functional product that facilitates attachment and
that this effect is at least partially dependent upon the presence of
FN as described previously (27, 74-78). Similarly, the complete
inhibition of attachment in a dose-dependent manner by
RGD-containing peptides but not RGE control peptides strongly argues
that the EGR-1-induced FN is a fully functional product. Of the two
factors, FN has by far the major effect on adhesion of H4 cells.
Control of Transformation by EGR-1 (Fig. 10)--
The enhanced
PAI-1- and FN-mediated attachments observed here likely have important
consequences in suppressing the transformation of HT1080 cells. There
is considerable evidence that FN receptors, especially the
5
1 integrin, play a role in modulating
cellular transformation and metastasis (56-57) and that HT1080 cells
express
5
1 integrin (33, 74). Conversely,
reduced expression of integrin
5
1 in
Chinese hamster ovary cell variants leads to increased tumorigenicity
(58), whereas overexpression of integrin
5
1 inhibits anchorage-independent
growth and tumorigenicity (59-60). In our system, integrin
5
1 might be regulated in two ways, by
TGF-
1 stimulation (24, 62, 71) and/or by FN itself (63) (Fig. 10).
Consistent with this, our adhesion assays showed that GRGDSP peptides
disrupted adhesion in a dose-related manner. Thus, the
EGR-1-dependent secretion of TGF-
1 and FN, in addition to directly facilitating adhesion, may control increased expression of
FN receptors to enhance further attachment (Fig. 10).
These features argue that EGR-1 functions to promote enhanced cell-cell
and cell-substratum interactions that are known to facilitate
density-dependent growth control. An early observation of
the effects of stable expression of EGR-1 on HT1080 cells was the
3-fold decrease in the saturation density in a manner strictly correlated with the level of EGR-1 expression (14). We conclude that
the mechanism of the restored density arrest is due to the coordinated
expression of TGF-
1, FN, and secondarily regulated genes such as
PAI-1. A diagrammatic model representing this relationship is
summarized in Fig. 10. It is very likely that this mechanism is
significant in a variety of cells including glioblastoma cells (data
not shown).
EGR-1 as "Oikis" Factor--
Loss of integrin-mediated
cell-matrix contact by normal cells has profound consequences such as
loss of normal growth and induction of apoptosis, a phenomenon termed
"homelessness" or anoikis (81). Transformed cells circumvent this
process by developing means of anchorage-independent growth involving
growth-promoting oncogenes, such as activated ras, and
subvert apoptosis by deletion or mutation of p53 or overexpression of
Bcl-2. It is likely that the coordinated expression of TGF-
1, FN,
and PAI-1 by Egr-1 plays an important role in maintaining the
anchorage-dependent growth. Recent observation show that
the focal adhesion kinase is activated in EGR-1-expressing HT1080 cells
and that apoptosis is suppressed even in the presence of overexpressed
wild-type p53 (82). Thus, we propose that EGR-1 functions as a true
oikis factor, which works to establish normal contact and cell
attachment growth control. Now, it is important to examine the
generality of this mechanism in normal cells and tissues.