Inhibition of Cell Migration by 24-kDa Fibroblast Growth Factor-2
Is Dependent upon the Estrogen Receptor*
Randolph S.
Piotrowicz
,
Lan
Ding
,
Pamela
Maher§, and
Eugene
G.
Levin
¶
From the Departments of § Cell Biology and
Molecular and Experimental Medicine, Scripps Research
Institute, La Jolla, California 92037
Received for publication, June 6, 2000, and in revised form, October 30, 2000
 |
ABSTRACT |
The single-copy gene for fibroblast growth
factor-2 (FGF-2) encodes for multiple forms of the protein with
molecular masses of 24, 22.5, 22, and 18 kDa. We reported previously
that the 24-22-kDa FGF-2 forms inhibit the migration of endothelial
and MCF-7 cells by 50% and 70%, respectively. Here we show that this
inhibition of migration is mediated by the estrogen receptor (ER). We
have found that depletion of the receptor in either cell line abrogates the inhibitory activity of 24-kDa FGF-2 while re-introduction of the ER
into deficient cells once again promotes the inhibitory response. To
determine whether exposure to 24-kDa FGF-2 resulted in the activation
of the estrogen receptor, 3T3 cells were cotransfected with estrogen
receptor cDNA and an estrogen regulatory element-luciferase gene
reporter construct and treated with 24- and 18-kDa FGF-2. The high
molecular weight form stimulated luciferase activity 5-fold while
18-kDa FGF-2 at the same concentration had no effect. Treatment of
ER-positive MCF-7 cells transfected with the reporter construct only
showed the same results. Inclusion of the pure estrogen antagonist ICI
182,780 blocked the increase in luciferase activity by 24-kDa FGF-2,
further indicating that the response was estrogen receptor dependent.
Expression of dominant negative FGF receptor 1 inhibited ER activation,
indicating that this was the cell surface receptor mediating the
effect. Although growth factor-dependent activation of the
ER was reported to require mitogen-activated protein kinase-induced
phosphorylation at Ser118 in COS and HeLa cells, this
mechanism is not involved with the activation by 24-kDa FGF-2. These
results suggest that the addition of 55 amino acids to the
amino-terminal end of 18-kDa FGF-2 by alternative translation alters
FGF-2 function and allows for the activation of a second signaling
pathway involving the estrogen receptor.
 |
INTRODUCTION |
Multiple forms of FGF-21
are produced from a single-copy gene as a result of alternative
translation initiation at an AUG codon or at three in-frame upstream
CUG codons. This results in the synthesis of 4 FGF-2 isoforms of 24, 22.5, 22, and 18 kDa (1, 2). The cellular localization and apparent
functions of 18 kDa and the three higher molecular weight forms of
FGF-2 (hmwFGF-2) differ. The 18-kDa FGF-2 is mostly cytoplasmic and is
exported to the cell surface, where it is localized to the basement
membrane or extracellular matrix in association with matrix heparins
and heparans (3, 4). In contrast, undetectable or extremely low levels
of hmwFGF-2 are present in the media of cultured cells, suggesting that
hmwFGF-2 is not released from cells. Instead, the majority of the
cellular hmwFGF-2 is directly translocated into the nucleus (5, 6). The
residues associated with nuclear translocation are RG repeats found at
several sites within the amino-terminal region of hmwFGF-2 (7) that is
absent from the 18-kDa FGF-2 form. Potential differences in function
have also been reported for the 18-kDa versus the hmwFGF-2
forms. Transfection and expression of 18-kDa FGF-2 in 3T3 cells results
in increased growth, motility, and levels of surface
1
integrins. In contrast, 3T3 cells transfected with the cDNA for
hmwFGF-2 show enhanced growth but no increase in migration nor integrin
expression (8, 9). Additionally, PC12 cells overexpressing 18-kDa FGF-2
were found to differentiate toward the neuronal phenotype whereas
overexpression of the higher molecular weight isoforms resulted in a
stabilization of the endocrine phenotype (10). Thus, the family of
FGF-2 growth factors demonstrate isoform-specific functions that differ
depending on the cell type studied. Further control over the effect of
the FGF-2 forms is provided by the extracelluar environment which regulates the ratio of their synthesis (11).
In a recently published article (12), we demonstrated that endothelial
cells could be stimulated to secrete hmwFGF-2 in a regulated manner to
levels capable of affecting cell behavior in an autocrine and paracrine
fashion. The effects were 2-fold: stimulation of cell proliferation and
inhibition of migration. The increase in proliferation was comparable
to that promoted by an equal amount of 18-kDa FGF-2, indicating that
the stimulation was independent of the additional amino-terminal
peptide. On the other hand, the effect on migration was opposite to
that of 18-kDa FGF-2. Although 18-kDa FGF-2 promotes cell motility, the
hmwFGF-2 forms inhibited migration of endothelial cells by 50% and
mammary carcinoma MCF-7 cells by greater than 70% even in the presence of unrelated mitogens that promote cell migration such as VEGF and
IGF-1. Thus, we showed that, in addition to cellular effects promoted
by hmwFGF-2 transported into the nucleus, these forms could also
dramatically alter cell function from outside the cells.
Among the various intracellular signaling pathways that are employed by
peptide growth hormones to transmit signals into the nucleus is one
that involves the activation of the ER. Although intuitively contrary
to the characterization of these receptors as steroid hormone
receptors, it has been shown repeatedly that they can be activated in a
ligand independent manner. Several peptide growth hormones including
EGF and IGF-1 activate the receptor in a variety of cell types and
in vivo (13-16). In this study, we demonstrate that the ER
mediates the inhibition of migration by 24-kDa FGF-2 in both
endothelial cells and MCF-7 cells and that these forms of the growth
factor can activate the ER leading to the transactivation of a reporter
gene. In addition, we show that the mechanism by which 24-kDa FGF-2
activates the ER is different from that of other peptide growth
hormones. This is the first report of an isoform-specific function of a
growth factor being linked to an alternate intracellular signaling pathway.
 |
MATERIALS AND METHODS |
Synthesis of 24-kDa FGF-2--
To generate pure recombinant
24-kDa FGF-2, full-length 24-kDa FGF-2 cDNA (obtained from Dr. M. Stachowiak, State University of New York, Buffalo, NY) was inserted in
frame into a pPIC9K yeast expression vector (Invitrogen) between the
SnaBI and AvrII restriction sites of the pPIC9K
vector directly downstream of the DNA encoding the X-factor secretion
signal region. The His4 mutant of P. pastoris GS 115, the
methanol utilization-positive phonetype (Mut+), was transformed by
electroporation with the pPIC9K construct vector linearized with
SacI. The His+ yeast transformants were grown on MD plates,
and those carrying the kanT gene further selected for
multiple integrated copies by replating on plates containing 4.0 mg/ml
G418 antibiotic. The multicopy transformant was grown in 50 ml of BMGY
medium with glycerol as the sole carbon source until the cultures
reached an A600 = 2-6 (~16-18 h). The cells
were collected by centrifugation at 1500-3000 × g for
5 min at room temperature and the cell pellet resuspended in BMMY medium to induce expression. The cells were grown for 4 days, and 100%
methanol was added every 24 h to a final concentration of 2% to
maintain protein expression. The medium was cleared of the yeast by
filtration and mixed with 5 ml of heparin- Sepharose for 2 h at
4 °C. The gel was washed with 50 ml of buffer A (20 mM
Tris- HC1, pH 7.4, 5 mM EDTA, 2 mM EGTA) and
then with buffer A containing 0.5 M NaCl. The recombinant
24-kDa basic fibroblast growth factor was eluted with buffer A
containing 3 M NaCl. The eluate was dialyzed against 4 liters of 20 mM Tris, pH 7.4, 145 mM NaCl.
Protein purity was assessed by 10% SDS-PAGE and Coomassie Blue
staining and Western blot analysis using antibodies generated against a
12-amino acid peptide found within the amino-terminal domain of 24-kDa
FGF-2 (17).
Cross-linking and
Immunoprecipitation--
125I-18-kDa FGF-2 (10 µCi/ml;
Amersham Pharmacia Biotech) was added to the cells cultured in 60-mm
dishes for 2 h at 4 °C. Cells were washed twice with
phosphate-buffered saline and disuccinimidyl suberate (0.37 mg/ml;
Pierce) added for 15 min at room temperature. The reaction was stopped
with 0.2 M ethanolamine; after rinsing in
phosphate-buffered saline, the cells were solubilized with 1% Triton
X-100 in 50 mM HEPES, pH 7.5, 50 mM NaCl, 5 mM EDTA. Insoluble material was removed by centrifugation
and the supernatant used for immunoprecipitation. For
immunoprecipitation of FGFR1-4, 200 µg of each cell lysate was
incubated with 2 µg of the polyclonal antibodies against FGFR1-4
(Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C, and
immune complexes were collected on protein A-Sepharose (Amersham
Pharmacia Biotech). Complexes were washed twice in a buffer containing
20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton
X-100, and 10% glycerol, then once in phosphate-buffered saline. Bound
proteins were eluted by boiling in 2× Laemmli sample buffer, separated
by SDS-polyacrylamide gel electrophoresis (PAGE), and placed on film
(Kodak BioMax).
Immunoblot Analysis--
Cells were washed twice in Dulbeco's
phosphate-buffered saline and then lysed with 0.5% Triton X-100 in 10 mM imidazole, pH 7.15, 40 mM KCl, 10 mM EGTA, 1 mM phenylymethylsulfonyl fluoride, 10 mM benzamidine, and 100 µg/ml leupeptin. The lysates
were spun for 2 min at 14,000 × g to pellet cell
debris and then subjected to the bicinchoninic acid protein assay
(Pierce). The Triton X-100-insoluble material remaining on the flask,
containing the cell nuclei, was washed twice with the lysis buffer and
then solubilized in SDS-PAGE reducing sample buffer (50 mM
Tris, pH 6.8, 5%
-mercaptoethanol, 2% SDS, and 10% glycerol).
Samples representing equivalent amounts of each fraction were subjected
to electrophoresis on 12% polyacrylamide gels. Upon completion of
electrophoresis, proteins were transferred to nitrocellulose membranes,
blocked for 1 h at room temperature in 5% nonfat milk in 20 mM Tris, pH 7.4, 145 mM NaCl (Tris-buffered saline) with 0.05% Tween 20 and then incubated with dilutions of
primary antibodies in Tris-buffered saline with Tween 20 containing 0.1% bovine serum albumin. Antibodies used in this study were anti-18-kDa FGF-2 monoclonal antibody (Sigma), anti-human ER-
monoclonal antibody (Neomarkers), anti-smooth muscle cell actin monoclonal antibody (Sigma), and anti-FGFR1 monoclonal antibody (Santa
Cruz Biotechnology). Bound antibody was detected using donkey
anti-mouse or rabbit IgG conjugated to horseradish peroxidase (Jackson
Laboratories) and the ECL chemiluminescence reagent. The stained
immunoblots were placed against x-ray film (BioMax MR, Kodak) to
generate chemiluminographs.
Culture of MCF-7, Endothelial, and NIH3T3 Cells--
All cells
were maintained at 37 °C under 5% CO2 in humidified
incubators and subcultured to cell densities of one third to one sixth
that of confluent cultures. MCF-7 cells were maintained in phenol
red-free minimal essential media supplemented with 1 mM
sodium pyruvate, 10% fetal calf serum, and 10 µg/ml insulin. For the
generation of estrogen receptor-negative populations, the cells were
subcultured in steroid-deficient medium (SDM) prepared with phenol
red-free minimal essential medium and 10% dextran/charcoal-stripped fetal calf serum. These MCF-7 cells were cultured in SDM for at least 6 months prior to use. The NIH3T3 cells were grown in Dulbecco's modified Eagle's medium containing 1 mM each penicillin
and streptomycin and 10% calf serum. Bovine aortic endothelial cells
were grown in Dulbecco's modified Eagle's medium containing 25 mM HEPES and 4.0 g/liter glucose supplemented with 1 mM sodium pyruvate, 1 mM nonessential amino
acids, 1 mM penicillin/streptomycin, and 10% fetal calf sera.
Migration and Growth Assays--
Endothelial or MCF-7 cells were
harvested with trypsin, counted, centrifuged, and resuspended at 1 × 105 cells in 0.5 ml of Dulbecco's modified Eagle's
medium, 0.5% bovine serum albumin. Cells were added to the upper well
of a Boyden chamber containing an 8.0-µm pore size polycarbonate
membrane separating the two chambers of a 6.5-mm Transwell (Costar).
The upper wells were placed into lower wells containing 0.75 ml of Dulbecco's modified Eagle's medium/0.5% bovine serum albumin to which 10 ng/ml IGF-1(Sigma) or 10 ng/ml VEGF (Sigma) was added as a
chemoattractant. Both chambers contained the 24-kDa FGF-2 at the
appropriate concentrations. After 4-6 h of incubation at 37 °C in
5% CO2, nonmigratory cells on the upper membrane surface were removed with a cotton swab, and the cells that traversed and
spread on the lower surface of the filter were fixed and stained with
Diff-Quik (Dade-Behring). The filter was mounted on a glass slide, and
four phase-contrast photomicrographs/membrane were taken
(magnification, ×100). The number of cells per field was counted from
contact sheets and the results compared with control chambers that had
no 24-kDa FGF-2 added. To measure growth rates, MCF-7 cells (6 × 103) were plated in growth medium for 48 h, the medium
changed to assay medium containing phenol red-free modified Eagle's
medium supplemented with 1 mM sodium pyruvate and 0.3%
lactalbumin hydrolysate plus or minus growth factors, and the cultures
were allowed to incubate another 24 h. Two hours prior to the
termination of the experiment, [3H]thymidine was added.
The cultures were washed with phosphate-buffered saline and then ice
cold methanol (2×), 5% trichloroacetic acid was added two times for
10 min each and discarded, and the DNA extracted with 0.3 N
NaOH. The number of counts/min were determined on a liquid
scintillation counter.
Plasmids and Transfection--
MCF-7 cells or NIH3T3 cells were
plated for transfection at a density of 1 × 105
cells/well in a six-well tissue culture dish for 24 h. ER-positive MCF-7 cells were transfected with 2 µg of pVitEREluc, a reporter construct containing three copies of the Xenopus
laevis vitellogenin estrogen regulatory element proximal to
the thymidine kinase promoter driving expression of the luciferase
cDNA (provided by Dr. C. Glass, University of California, San
Diego, CA). NIH 3T3 cells were cotransfected with 2 µg of the
reporter plasmid and 3 µg of pSG5 plasmid containing the full-length
wild type cDNA for the human ER (pSG5hER, HEGO) or one of two
truncated forms of the ER (HE15 containing amino acids 1-282 of the ER
or HEG19 containing amino acids 178-595) provided by Dr. P. Chambon
(Unite 84 de Biologie Moleculaire et de Genetique de I'NSERM,
Strasbourg Cedex, France) (18). A mutant of the estrogen receptor in
which serine 118 was changed to alanine was generated by the method of
Kunkel (19), and the mutation was confirmed by sequencing. The FGFR1
expression plasmid was constructed by cloning the entire coding
sequence of the three Ig-like domain form of human FGFR1 into
pcDNA3.1 (Invitrogen). The dominant negative mutant lacking the
tyrosine kinase domain was constructed by truncating the full-length
receptor at the KpnI site (between Gly-402 and Thr-403)
in the juxtamembrane domain. The DNA was mixed with
LipofectAMINE (Life Technologies, Inc.) in 1.8 ml of Opti-MEM I (Life
Technologies, Inc.), allowed to sit for 45 min at room temperature, and
the mixture was added to the cells that had been washed twice with
Opti-MEM I. Transfection was allowed to occur for 5.5 h at
37 °C. The transfected cells were cultured for 24 h in their
respective growth medium and the vehicle or agonists added at
concentrations described under "Results" for another 24 h.
When applicable, ICI 182,780 (Zeneca Pharmaceuticals) or the MAPKK
inhibitor PD98059 was added 30 min prior to the addition of agonist.
The cells were lysed with 0.1 M potassium phosphate, pH
7.8, 0.2% Triton X-100, 1 mM dithiothreitol, and the
insoluble material was removed by centrifugation at 10,000 × g. ER transactivation ability was determined by measuring
luciferase activity (20).
 |
RESULTS |
Depletion of the ER and Loss of Inhibition--
A requirement for
the presence of ER in 24-kDa FGF-2-mediated inhibition of migration was
tested in MCF-7 and endothelial cells by depleting the receptor content
and testing the effect on 24-kDa FGF-2-mediated migration. To reduce
the level of the ER in endothelial cells, cultures were maintained in
the presence of 500 nM ICI 182,780 which promotes changes
in ER conformation which target the receptor for degradation (21).
Immunoblot analysis for ER content demonstrates the reduction in the
level of both the cytosolic (cyt) and nuclear
(nuc) ER of the ICI-treated cells (Fig.
1A). ICI-treatment reduced the
amount of the 67-kDa ER by greater than 70%, in the cytoplasm and to
undetectable levels in the nucleus as compared with parallel cultures
of the same cells cultured in the absence of the anti-estrogen.
Equivalent loading of cellular material was demonstrated by reprobing
the blot with an anti-
actin peptide antibody. The loss of the ER had no significant effect on the base rate of endothelial cell migration (1.0 versus 0.86 ± 0.16, ER-positive
versus ER-negative) nor VEGF-induced enhanced migration
(1.8 ± 0.2 versus 2.0 ± 0.2, ER-positive
versus ER-negative), indicating that basal motility and
chemokinetic stimulation through the VEGF receptor were not impeded by
the loss of the ER (Fig. 1B). However, the reduction in ER
levels resulted in the loss of the inhibitory response to 24-kDa FGF-2.
The migration rate of receptor positive endothelial cells was reduced
to 48 ± 8% of control values but declined only 10% to 90 ± 16% with ER-negative cells. The same was true in the presence of
VEGF. The migration rate was reduced dramatically by 24-kDa FGF-2 in
VEGF-treated ER-positive cells (to 40 ± 12% of ER-positive
control cells), while the ER-negative cells showed no such decline.
Further evidence for the involvement of the ER in the inhibition of
migration in endothelial cells was obtained with short term exposure of
the cells to 10 nM ICI 182,780, which results in the
inactivation of ER function without the loss of ER antigen (21).
Simultaneous exposure of endothelial cells to ICI and 24-kDa FGF-2
resulted in a loss of the inhibitory potential of the 24-kDa FGF-2;
motility was inhibited only 25% in cultures treated with ICI
versus a 60% decline of migration in its absence (data not
shown). Thus, ER function as well as ER protein is required for the
inhibition of migration by the 24-kDa FGF-2.

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Fig. 1.
A, prolonged incubation in ICI 182,780 depletes endothelial cells of ER antigen. Endothelial cells were
incubated in complete medium containing 500 nM ICI 182,780 for 10 days and the cultures extracted with 0.5% Triton X-100. After
removing the soluble material, the nuclei were extracted with SDS. Both
samples were fractionated by SDS-PAGE and the ER detected by
immunoblotting with anti-ER antibodies. The blots were reprobed for
actin to confirm equal loading. B, depletion of the ER
abrogates the inhibitory effect of 24-kDa FGF-2. ICI-treated cells were
used to test the effect of 5 ng/ml 24-kDa FGF-2 on the migration rates
of the estrogen-negative versus estrogen-positive
endothelial cells. Cell motility was tested in Boyden Chamber assays in
the presence and absence of 10 ng/ml VEGF. The number of migrating
cells were counted and compared with the number migrating in the
absence of 24-kDa FGF-2. ER deficiency had no effect on the basal
migration of endothelial cells with or without VEGF but abrogated the
inhibitory effect of 24-kDa FGF-2. C, reduction of ER
antigen in MCF-7 cells by prolonged culture in steroid-deficient
medium. Replicate cultures of MCF-7 cells were maintained continuously
in either complete (RM) or SDM. With SDM the level of ER was
undetectable in the cytoplasm by Western blot analysis and
significantly reduced in the nucleus. D, the effect of 5 ng/ml 24-kDa FGF-2 on ER-negative and positive MCF-7 cell mobility was
tested in Boyden Chamber assays with 10 ng/ml IGF-1 as a
chemoattractant. The data show that the inhibition of migration is
dependent on the ER but basal motility is not. The results are
presented as a ratio of the number of 24-kDa FGF-2-treated cells
migrating onto the bottom of the membrane to that occurring in the
absence of 24-kDa FGF-2. The data are presented as the mean ± S.D. cyt, cyto, cytosolic; nuc,
nuclear; C, control.
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Depletion of the ER had the same effect on 24-kDa FGF-2-mediated
inhibition of MCF-7 migration (Fig. 1, C and D).
MCF-7 cells were cultured and maintained in SDM, which results in a
nonclonal population of cells that no longer exhibit a mitogenic
response to
-estradiol (12). This lack of estrogen responsiveness is accompanied by a decrease in ER levels as assessed by immunoblot analysis (Fig. 1C). Cytoplasmic ER was undetectable, while
nuclear ER levels were substantially reduced. 24-kDa FGF-2 inhibited
the migration of these ER-positive cells by 77 ± 18%, while in
ER-negative cells the migration rates actually increased to 1.3 ± 0.22 times those of control, ER-positive cells (Fig.
1D).
Reestablishing the 24-kDa FGF-2 inhibition of migration in ER-negative
MCF-7 cells could be achieved by re-introducing the estrogen receptor
to its normal levels. MCF-7 cells propagated in SDM (ER-negative; Fig.
2A, lane
2) were placed in serum-free Opti-MEM I for 4 h and
then returned to SDM for 24 h. This short term exposure to
Opti-MEMI resulted in the appearance of endogenous ER at levels
approximating those of the MCF-7 cells retained in normal medium (Fig.
2A, compare lanes 1 and 3).
Fig. 2B shows that the migration rate of the cells remaining
in SDM (corresponding to lane 2, panel
A) is not affected by 24-kDa FGF-2 but replicate cultures
stimulated to produce the ER (lane 3, panel
A) only migrated 37.1 ± 10.7% as fast as the control
cells. Although 24-kDa FGF-2 inhibits the migration of MCF-7 cells, it
acts as a stimulus for cell proliferation equally as well as 18-kDa
FGF-2 (17). Therefore, the effect of ER-depletion on the growth
promoting activities of 24-kDa FGF-2 was examined using MCF-7 cells
cultured in SDM or regular medium (Fig. 2C). Cells were
incubated with 24-kDa FGF-2 or 18-kDa FGF-2 and the rate of
proliferation determined by thymidine incorporation studies. No change
in the rate of cell proliferation was observed in either case
regardless of the presence or absence of the ER (3.7 ± 0.3 versus 3.3 ± 0.2, 18-kDa FGF-2; 3.4 ± 0.3 versus 3.7 ± 0.4, 24-kDa FGF-2). Thus, the involvement of the ER in 24-kDa FGF-2 function is specific to the inhibition of
migration and is not relevant to its effect on cell division.

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Fig. 2.
A, effect of introduction of the ER into
MCF-7 cells on migration. MCF-7 cells made ER negative by prolonged
growth in steroid-deficient medium were placed in Opti-MEM I in the
absence of serum for 4 h and then placed back into
steroid-deficient complete DMEM for 24 h. This treatment resulted
in the appearance of ER to levels equal that in control cells grown
continuously in normal medium as demonstrated by immunoblot analysis
with anti-ER . Lane 1, control cells maintained
in complete medium. Lane 2, cells maintained in
steroid-deficient medium. Lane 3, cells shown in
lane 2 treated with Opti-MEM I medium.
B, relative migration of ER-negative MCF-7 cells and
ER-negative MCF-7 cells stimulated to produce ER by treatment with
Opti-MEM I. Cells represented by lanes 2 and
3 in panel A were treated with 5 ng/ml
24-kDa FGF-2 and the rates of migration in response to 10 ng/ml IGF-1
measured. The migration rates are presented as a ratio of the number of
migrating cells in the presence of 24-kDa FGF-2 plus IGF-1 to those
treated with IGF-1 alone (open bar). The data are
presented as the mean ± S.D. C, the ER is not
necessary for growth stimulation. ER-positive or negative MCF-7 cells
(6 × 103) were plated in their respective growth
medium for 48 h and then incubated in serum-deficient medium
supplemented with 5 ng/ml 24-kDa FGF-2 or 18-kDa FGF-2 for 24 h.
Two hours prior to the termination of the experiment 100 µCi/ml
[3H]thymidine was added to the cultures. At the end of
the experiment, the DNA was extracted with 0.3 N NaOH. The
results are presented as the increase in the number of 3H
counts/min compared with cultures not treated with growth factor. The
data are presented as the mean ± S.D.
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Activation of the ER by 24-kDa FGF-2--
To assess the effect of
24-kDa FGF-2 on ER activity, we measured the ability of the growth
factor to modulate the transcriptional activity of ER in transient
transfection assays. 3T3 cells were cotransfected with a plasmid
containing a triple estrogen regulatory element (ERE) proximal to the
thymidine kinase promoter driving luciferase reporter gene expression
and a plasmid containing the cDNA for the full-length human ER-
(22). Fig. 3A shows the low
level of endogenous ER in the 3T3 cells and the increase in ER levels
24 h after transfection of the ER cDNA. Treatment with estradiol for 24 h had no effect on the intracellular content of
the recombinant ER (Fig. 3B). Treatment of these cells with 10 nM estradiol increased the luciferase activity in the
cell extracts 3.6 ± 0.1-fold. Dose-dependent
increases in luciferase activity also were observed with increasing
doses of 24-kDa FGF-2 from 5 to 20 ng/ml. As the concentration of the
growth factor was raised, there was a corresponding increase in
luciferase activity up to 2.5 times control levels. Higher doses (30 ng/ml) had no additional effect. When the cells were transfected with
the reporter plasmid only (no ER expression), no increase in luciferase
activity was observed, even at 20 ng/ml, demonstrating that the ER was necessary for the stimulation of luciferase gene transcription. In
contrast to the higher molecular weight FGF-2, 18-kDa FGF-2 at 20 ng/ml
concentration had little effect on luciferase activity, establishing
the specificity of ER activation for the higher molecular weight forms
of FGF-2. Mammalian cell-derived recombinant hmwFGF-2 produced in COS
cells (17) gave identical results (data not shown). The ability of
24-kDa FGF-2 to activate the ER also was tested in MCF-7 cells using
the natural endogenous ER and the luciferase reporter construct. Cells
were transfected with the reporter construct only and treated with
estradiol or 24-kDa FGF-2. Treatment with 10 nM estradiol
induced an increase in luciferase activity to 2.6 ± 0.4 times
control (Fig. 3C). Addition of 24-kDa FGF-2 created a
dose-dependent increase in luciferase activity to 2.3 ± 0.3 times control at 10 ng/ml. Further evidence for
ER-dependent 24-kDa FGF-2 activation of luciferase
expression was acquired with the pure anti-estrogen ICI 182,780 (Fig.
3D). This anti-estrogen inhibited the level of luciferase
activity achieved with 10 ng/ml 24-kDa FGF-2 in a
dose-dependent manner. Thus, consistent results were
observed in 3T3 cells expressing an exogenous source of ER and in MCF-7
cells using the endogenous receptor.

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Fig. 3.
24-kDa FGF-2 activates ER transcriptional
activity. A, Western blot analysis of extracts of 3T3
cells transfected with the ER gene. NIH 3T3 at 50% confluence in
six-well tissue culture dishes were transiently transfected with 3 µg
of plasmid pSG5hER containing the human ER cDNA and 24 h later
the cell extracts analyzed for ER by Western blot analysis. Replicate
cultures were treated with 10 nM estradiol
(E2) for 24 h to determine whether the
steroid had any effect on ER levels. B, transactivation of
the ER by 24-kDa FGF-2. NIH 3T3 cells were cotransfected with plasmid
pSG5hER containing the cDNA for human ER and 2 µg of 3xERE
-luciferase reporter construct and 24 h later treated with 10 nM estradiol, 5-30 ng/ml 24-kDa FGF-2, or 20 ng/ml 18-kDa
FGF-2. After an additional 24 h, the cultures were extracted and
the samples assayed for luciferase activity. Replicate cultures were
transfected with the reporter construct only and treated with 20 ng/ml
24-kDa FGF-2 (ERE only). The failure of the FGF-2 alone to promote
luciferase activity demonstrates that the activity observed in the
cotransfected cultures was ER-dependent. Data are presented
relative to those from untreated (control) cultures (mean ± S.D.). C, activation of endogenous ER by 24-kDa FGF-2 in
MCF-7 cells. MCF-7 cells maintained in normal media (ER-positive) were
transfected with the luciferase reporter construct for 24 h and 10 nM estradiol or 2-10 ng/ml 24-kDa FGF-2 added for an
additional 24 h. Cells were extracted and the samples assayed for
luciferase activity. Data are presented relative to those from
untreated cultures (mean ± S.D.). D, inhibition of
24-kDa FGF-2 transactivation of ER by the pure estrogen antagonist ICI
182,780. MCF-7 cells transfected with the ERE-luciferase reporter
plasmid and treated with 10 ng/ml 24-kDa FGF-2 and 0.1, 1, and 10 nM ICI for 24 h before luciferase activity was
measured. A dose-dependent reduction in ER transactivation
is observed.
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18-kDa FGF-2 binds to and activates four different high affinity
tyrosine kinase receptors, FGFR1/flg to FGFR4, although not all are
found in a single cell type (23-26). To determine which of the four
receptors were present in the MCF-7 cells, aortic endothelial cells,
and 3T3 cells employed in these studies, cells extracts were analyzed
by cross-linking 125I-18-kDa FGF-2 to the cells and
performing immunoprecipitation with antibodies against each of the four
receptors (Fig. 4A). FGFR1 was
the only receptor detected in each cell type and was the only one
present in the 3T3 and endothelial cells. The doublet observed in 3T3
cells represents the two and three IG-like domain isoforms of FGFR1
(data not shown). The possibility that FGFR1 mediates the activation of
the ER by 24-kDa FGF-2 was tested in 3T3 cells by expressing a dominant
negative FGFR1 in which the tyrosine kinase domain had been deleted.
Expression of this receptor and the wild type receptor following
transient transfection is shown in Fig. 4B (right
panel). In cells expressing the mutant FGFR1, no increase in
ER activation occurred with 10 ng/ml 24-kDa FGF-2 treatment while the
activation by estradiol was not affected. When the experiments were
repeated with cells transfected with a plasmid containing wild type
FGFR1 cDNA, no difference in ER activation was found when compared
with control cells.

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Fig. 4.
Activation of the ER by 24-kDa FGF-2 is
mediated by FGFR1. A, analysis of 3T3, MCF-7, and
endothelial cells for the presence of FGF receptors. Cross-linking
experiments were performed with 125I-18-kDa FGF-2 and cell
lysates immunoprecipitated with antibodies to each of the FGFRs.
Immunoprecipitates were fractionated by SDS-PAGE and the gels exposed
to x-ray film. T47D cell lysates were analyzed concurrently to
demonstrate positive staining for each receptor type. The doublet in
the 3T3 cells represents the two and three Ig-like domain isoforms of
FGFR1. The numbers underneath the panel identify the
receptor specifically identified by the antibody used. N,
nonimmune control. B, loss of activation with the expression
of a FGFR1 tyrosine kinase-negative receptor. 3T3 cells were
transfected with plasmids containing wild type FGFR1 or the tyrosine
kinase mutant and the expression analyzed by immunoblot analysis with
antibodies to the receptor (right panel). To test
the effect of the mutant receptor, cells were cotransfected with the
plasmids containing the tyrosine kinase negative FGFR1 cDNA,
estrogen receptor, and the reporter gene construct and treated with
either 10 ng/ml 24-kDa FGF-2 or 10 nM estradiol
(filled bars). Control cultures were transfected
in the same manner except the mutant plasmid was replaced with
pcDNA3 vector at the same concentration (open
bars). FGFR1, The experiments were repeated with cells
transfected with plasmid containing wild type FGFR1 cDNA
(hatched bar). Results are presented relative to
those from replicate cultures containing the appropriate plasmid but
left untreated. wt, wild type; mut, mutant.
|
|
Binding of FGF-2 to the extracellular domain of FGFR results in
activation of the intrinsic kinase activity causing tyrosine phosphorylation of a number of signaling intermediates including MAPKs
(27-33). In COS and HeLa cells, ER activation by EGF depends on the
phosphorylation of the ER (34). The intracellular pathway responsible
for this phosphorylation event is the MAPK signal transduction cascade
and results in the phosphorylation of Ser118 of the ER,
which is also the target of a phosphorylation event induced by
estradiol (35-37). To determine whether 24-kDa FGF-2 can activate the
MAPK signal transduction cascade, 3T3 cells were treated with 18- and
24-kDa FGF-2 and the activation of Erks 1 and 2 examined by Western
blot analysis with antibodies against the phospho-form of the enzymes
(Fig. 5). Both growth factors were added
to the cells at 10 ng/ml for increasing amounts of time, and the levels
of phospho and total protein analyzed. MAPK phosphorylation was
elevated by both 18- and 24-kDa FGF-2 within 10 min and remain elevated
for at least 1 h after treatment. The constant total levels of
Erk1/2 show that this increase reflected the phosphorylation of the
protein and not differences in loading. Addition of antibodies to the
18-kDa COOH-terminal domain or the amino-terminal end of 24-kDa FGF-2
neutralized the effect on MAPK phosphorylation (lower
panel). Direct evidence for the involvement of the MAPK
pathway in the activation of the ER was sought with the MAPKK inhibitor
PD98059. Dose titration analysis of the effect of this inhibitor on the
phosphorylation of ERK1/2 in 3T3 cells showed that 1 µM
or greater was capable of inhibiting the phosphorylation of MAPK
induced by 24-kDa FGF-2 (Fig. 6).
However, instead of reducing ER activation in parallel with the loss of
MAPK phosphorylation, 1 µM PD98059 treatment increased
the level of luciferase activity in response to 10 ng/ml 24-kDa FGF-2
by 1.5 times (2.1 ± 0.1 versus 3.6 ± 0.1) while
10 µM inhibitor stimulated the 24-kDa FGF-2 effect by
2.5-fold (to 4.9 ± 0.1; Fig. 6B). Addition of 1 and 10 µM PD98059 alone stimulated ER activation (~2-fold),
although not as much as when 24-kDa FGF-2 was present. The same pattern
of PD98059-enhanced FGF-2-dependent ER activation was
observed with 5 and 10 ng/ml 24-kDa FGF-2 treatment (data not
shown).

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Fig. 5.
Induction of MAPK phosphorylation by 24-kDa
FGF-2. 3T3 cells were treated with 10 ng/ml 24-kDa FGF-2 or 18-kDa
FGF-2 for increasing amounts of time, the cells were extracted, and the
protein lysates were immunoblotted with antibodies to either
phospho-MAPK (P-MAPK) or total MAPK. In some experiments,
antibodies to the 18-kDa domain or the amino-terminal end of 24-kDa
FGF-2 ( 18kD and 24kD,
respectively) were incubated for 30 min with the protein and the
mixture added to the cells for 10 min. Cells were then analyzed for
phospho-MAPK. C, control.
|
|

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Fig. 6.
Inhibition of MAPKK does not inhibit 24-kDa
FGF-2 activation of the ER. A, 3T3 cells were
pretreated with increasing concentrations of MAPKK inhibitor PD98059
for 15 min and the ability of 10 ng/ml 24-kDa FGF-2 to induce MAPK
phosphorylation determined. As little as 1 µM inhibitor
eliminated MAPK phosphorylation in these cells with no effect on the
level of total enzyme. P-MAPK, phospho-MAPK. B,
the effect of PD98059 on ER activation by 24-kDa FGF-2. 3T3 cells were
cotransfected with plasmid pSG5hER and the 3xERE-luciferase reporter
construct and 24 h later treated with 1 and 10 µM
PD98059 for 15 min and then 10 ng/ml 24-kDa FGF-2 (solid
bars) or vehicle (hatched bars). After
an additional 24 h, the cultures were extracted and the samples
assayed for luciferase activity. Data are presented relative to those
from transfected cells left untreated (control) (mean ± S.D.).
|
|
Specific activation factor domains of the ER have been related to the
activation of the receptor by estradiol or peptide growth factors. In
COS and HeLa cells, it is the AF-1 domain that mediates the growth
factor-induced transactivation while the AF-2 domain is required for
estradiol-induced activation (38-40). To determine whether 24-kDa
FGF-2 activation of the ER can be localized to a specific domain of the
receptor, 3T3 cells were transfected with truncated forms of the ER
cDNA containing either the AF1 or AF2 domains plus the DNA binding
domain (Fig. 7). In contrast to what has
been found with other growth factors, increases of the reporter
luciferase activity in response to 24-kDa FGF-2 occurred with the
truncated ER containing the AF2 domain (HEG19; 5.4 ± 2.0-fold)
but not the AF1 domain (HE15; 0.9 ± 0.1-fold). The increase with
HEG19 was comparable to that observed with estradiol, which also
occurred through the AF2 domain (7.4 ± 2.5-fold). Little difference was observed between 24-kDa FGF-2 activation of the truncated HEG19 ER and the wild type (HEGO; 4.4 ± 2.0-fold). In addition to the domain specificity of EGF-induced ER activation, phosphorylation of the receptor at Ser118 is required in
COS1 and HeLa cells (39). Conversion of this serine to alanine
eliminated activation of the receptor. To determine whether
Ser118 phosphorylation was involved with 24-kDa FGF-2
activation of the ER, this residue was substituted with alanine by site
directed mutagenesis. Comparison of the effect of 24-kDa FGF-2 on cells containing the mutant ER versus the wild type receptor
showed a 5.6 ± 0.22-fold increase in luciferase activity in cells
containing the mutant ER and 6.0 ± 0.29-fold with the wild type
receptor.

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Fig. 7.
Activation of the ER does not depend on the
AF-1 domain nor phosphorylation of serine 118. A,
schematic presentation of the ER deletion mutants. The domains and the
corresponding AF-1 and AF-2 regions are illustrated. B, 3T3
cells were transfected with either the full-length wild type ER
(HEGO), one of the two truncated ER forms missing the AF-1
or AF-2 domains (HEG19 or HE15, respectively), or
ER in which serine 118 had been changed to alanine (s118a).
The cultures were treated with 10 ng/ml 24-kDa FGF-2 (open
bars) or 10 nM estradiol (filled
bars) for 24 h and the cells extracted and assayed for
luciferase activity. Data are presented relative to those extracts from
transfected cells not treated with growth factor (mean ± S.D.).
|
|
 |
DISCUSSION |
The synthesis of various molecular weight isoforms of FGF depends
upon the mechanism of alternative translation initiation (1, 2). This
process employs three CUG initiation sites upstream from a traditional
AUG start site. This results in three higher molecular weight forms
containing an amino-terminal peptide of increasing length coupled to
the 18-kDa FGF-2. The presence of this peptide alters the cellular
localization of FGF-2 from the cytoplasm (18 kDa) to the nucleus due to
the presence of nuclear localizing RG repeats within the amino-terminal
extension (3-6). In addition to differences in localization, the
hmwFGF-2 forms also have been found to modify cell behavior differently
from the 18-kDa form. This difference was first suggested by
experiments in which 18-kDa or hmwFGF-2 forms were overexpressed in 3T3
cells (8, 9). All forms stimulated proliferation, but, whereas the
smaller form stimulated migration, the hmwFGF-2 had no effect. Overexpression of the different forms also had distinct effects on PC12
cell phenotype with the higher molecular weight FGF-2-expressing cells
displaying neuronal properties and 18-kDa FGF-2-expressing cells being
more endocrine-like (10). Thus, not only is localization of the
proteins altered by the amino-terminal extension, so is their function.
Further control of cell behavior by this mechanism can occur since the
different isoforms are expressed in response to changing environmental
conditions (41), e.g. untransformed endothelial, epithelial,
and fibroblastic cells express primarily the 18-kDa FGF-2 while stress
conditions stimulate the synthesis of the CUG forms (11).
The difference in the function of 18-kDa and hmwFGF-2 forms was further
demonstrated by studies in which recombinant hmwFGF-2 was added to
endothelial cells and MCF-7 cells and their effects on cell migration
evaluated (12). Cell migration was inhibited to greater than 50% by
hmwFGF-2. This occurred even in the presence of saturating amounts of
mitogens that stimulate cell motility (VEGF, IGF-1, 18-kDa FGF-2),
suggesting that the effect of hmwFGF-2 can override the signals
generated by these other factors. In this study we provide evidence
that this effect on migration results from a change in the
intracellular signaling pathway that the cell employs to transmit
FGF-2-activated signals. With the synthesis of the amino-terminal end,
FGF-2 can activate pathways that lead to the transactivation of the ER.
An association between inhibition of migration and the activation of
the ER is demonstrated by the parallel changes between the two with
reduction in ER accompanied by loss of the inhibitory effect. This link
was found in two different cell types (endothelial and MCF-7), which
had been depleted of their ER by two different methods (maintenance in
SDM and ICI 182, 780 treatment, respectively). In addition, short term
treatment with ICI 182,780 blocked the inhibition of migration
indicating that the ER must not only be available but also needs to be
functional. The latter premise is consistent with our observations that
24-kDa FGF-2 has the capacity to transactivate the ER as demonstrated by the expression of an estrogen regulatory element controlled gene.
Again, this occurred in two different cell types regardless of whether
the ER was expressed from a transfected plasmid (3T3 cells) or was the
natural product of the endogenous gene (MCF-7 cells).
The activation of the ER by a peptide growth hormone is not unusual;
direct evidence for peptide growth factor activation of the ER has been
obtained in multiple cell types (13-16) and in ER knockout animals
(42). Transforming growth factor-
or EGF treatment of HeLa, Ishikawa
(endometrial), and BG-1 (ovarian) cells transiently transfected with
ER-responsive target genes results in the dose-dependent
activation of theER in an estrogen-independent manner (38). In primary
rat uterine cell cultures (43), GH3 pituitary cells (16), and the
neuroblastoma cell line, SK-ER3 (44), ER-dependent gene
expression is stimulated by IGF-1. Growth factors also mimic estrogen
in their ability to increase the expression of ER target genes, such as
the progesterone receptor and the iron-binding glycoprotein,
lactoferrin (45). EGF and IGF-1 also promote cell growth through the ER
both in vitro and in vivo (38, 43). What
distinguishes hmwFGF-2 from these other growth factors is the fact that
it affects motility, which has not yet been associated with peptide
activation of ER. The mechanism by which EGF activates the ER has been
shown to occur through the EGF plasma membrane receptor (38) and
activation of specific MAPKs resulting in the phosphorylation of serine
118 of the ER. This occurs, however, in only certain cell types
(e.g. COS and HeLa cells). Studies employing vascular smooth
muscle cells failed to show involvement of either MAPKs or
Ser118 phosphorylation, indicating that multiple pathways
are available for ER activation by these peptide hormones (20). In
fact, overexpression of the dominant positive MAPKK in endothelial
cells inhibited basal ER activity, suggesting that in these cells there
is a reverse relationship between MAPKK activity and ER activity.
Our results are more consistent with those reported for the smooth
muscle cells than the studies performed with COS or HeLa cells.
Although 24-kDa FGF-2 clearly stimulates the phosphorylation of MAPK,
inhibition of that phosphorylation with the MAPKK inhibitor PD98059 did
not reduce the level of ER activation. In fact, we observed an increase
in the basal level of ER activity and an increase in the 24-kDa FGF-2
induced activation of the ERwith the addition of the MAPKK inhibitor.
Despite the independence from the MAPK pathway, it is apparent that a
fully functional FGFR1 receptor must be present for 24-kDa FGF-2 to
activate the ER. It is also likely that the tyrosine kinase activity of
FGFR1is responsible for the transduction of the 24-kDa FGF-2 generated signals for ER activation. The diversity of the different transducer proteins associating with FGFR1 and the number of signaling
intermediates that are activated may reflect the capacity of the
different forms of FGF-2 to exert the complex array of biological
responses in the same cell, a characteristic shared by other tyrosine
kinase receptor-binding growth factors (46-48).
In addition, the mechanism of ER activation by FGFR1 appears to involve
distinct domains of the ER. Previously, the AF-1 domain was shown to be
required for peptide growth factor ER activation while the AF-2 domain
mediated the estradiol effect. The use of the same truncated forms of
the ER transfected into 3T3 cells demonstrated the opposite effect with
24-kDa FGF-2. Thus, activation of the ER by 24-kDa FGF-2 requires the
AF-2 and not the AF-1 domain. Consistent with these results is our
observation that the elimination of the putative MAPK phosphorylation
site Ser118 by conversion to alanine has no effect on the
activation of the receptor, a result consistent with the effect of
growth factors on the ER in smooth muscle cells but not in COS or HeLa cells.
Alternative translation site usage was first characterized as occurring
during viral replication, particularly among the picornaviruses. It is
a mechanism that allows for the expansion of gene diversity within a
highly restricted genome by changing the localization and the functions
of a single gene. Although much less common, alternative translation
initiation using noncanonical codons also exists for mammalian proteins
other than FGF-2. Among these are several whose function changes with
the synthesis of their alternate forms. For example, there are two
forms of GATA-1 that differ in their transactivation potential and
developmental expression (49), the cAMP-responsive element modulator is
converted from a transcriptional activator to a repressor (50), and
Egr3 transcription factors differ in their ability to activate
transcription (51). As transcription factors, the changes in these
three proteins most likely involve how they interact directly with DNA
or other DNA-binding proteins. In contrast, changes in 24-kDa FGF-2
function involve a more complex process involving multiple steps
between cell surface receptor activation, ER activation, and the
inhibition of migration. This represents the novelty of our
observations on FGF-2 that, in addition to the functional change that
occurs with the additional amino-terminal peptide, there is also an
activation of a separate signaling pathway that can be directly
linked to the change in function.
 |
ACKNOWLEDGEMENT |
We are very grateful for the expert technical
assistance of Jeanine Witkowski.
 |
FOOTNOTES |
*
This work was supported by Grant R01 CA81209 from the
National Institutes of Health.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.
¶
To whom correspondence should be addressed: La Jolla Inst. for
Molecular Medicine, 4570 Executive Dr., San Diego, CA 92121. Tel.:
858-587-8788 (ext. 120); Fax: 858-587-6742; E-mail:
glevin@ljimm.org.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M004868200
 |
ABBREVIATIONS |
The abbreviations used are:
FGF-2, basic
fibroblast growth factor;
VEGF, vascular endothelial growth factor;
hmwFGF-2, high molecular weight FGF-2;
IGF-1, insulin-like growth
factor-1;
EGF, epidermal growth factor;
ER, estrogen receptor;
SDM, steroid-deficient medium;
MAPK, mitogen-activated protein kinase;
MAPKK, mitogen-activated protein kinase kinase, FGFR, fibroblast growth
factor receptor;
PAGE, polyarylamide gel electrophoresis;
ERE, estrogen
regulatory element.
 |
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