Basic fibroblast growth factor (FGF-2) is one of
a select group of proteins that can exit cells through an alternate,
endoplasmic reticulum/Golgi apparatus independent exocytic pathway.
This alternate pathway has been termed protein export. In an attempt to
better understand this process, we have identified a family of related compounds, "cardenolides," that inhibit FGF-2 export. The
cardenolides inhibit FGF-2 export in a time and concentration dependent
fashion. Inhibition of FGF-2 export is specific in that the
cardenolides have no effect on conventional protein secretion as
measured by their inability to block release of the secreted protein
human chorionic gonadotropin-
.
Taken together, these data: 1) identify a novel activity for
cardenolides; 2) suggest a previously unknown role for the
-subunit of Na+, K+-ATPase in FGF-2 export; and 3) raise
the possibility that the
-subunit itself may be an integral
component of this alternate exocytic pathway mediating translocation of
cytosolic FGF-2 to the cell surface.
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INTRODUCTION |
Growth factors act to modify cell growth and differentiation
through interactions with the extracellular ligand-binding domains of
their cognate plasma membrane receptors. Accordingly, it is generally
well accepted that growth factors must be secreted from cells to elicit
their biological effects. Over the last 20 years, a number of
laboratories have designed a series of elegant experiments to show that
protein secretion first requires targeting of the nascent polypeptide
chain to the endoplasmic reticulum
(ER)1 via a hydrophobic
signal peptide sequence (1-3). Co-translational translocation of the
secretory protein across the lipid bilayer of the ER involves passage
through an aqueous channel that includes the Sec61p complex (4-6).
Once in the ER lumen, secretory proteins are transported through the
Golgi apparatus and vesicular intermediates that ultimately fuse with
the plasma membrane, thereby releasing their protein cargo into the
extracellular milieu.
The primary translation product(s) of all secretory proteins include a
hydrophobic signal peptide (leader) sequence. However, recently, a
small number of extracellular proteins have been described that do not
contain this leader peptide sequence (7). Moreover, the selective
release (export) of these "leaderless" proteins can be
unequivocally distinguished from conventional ER/Golgi-mediated protein
secretion, in a manner that is not a consequence of impaired plasma
membrane integrity or cell death (8-12).
Two prototypic members of the fibroblast growth factor (FGF) family of
polypeptide growth factors, FGF-1 and FGF-2, do not contain a
hydrophobic signal peptide sequence (13-15). Yet, data from a variety
of experiments indicate that both proteins are detected
extracellularly, they interact with one or more integral plasma
membrane receptors (16) and are exported from transfected cells in an
ATP-dependent, brefeldin A-resistant, ER/Golgi-independent manner (9, 10, 12). However, even though their multifunctional extracellular activities have been well documented, the precise molecular mechanisms mediating their export are unknown.
In an attempt to better understand the process responsible for FGF-2
export, we reasoned that small molecules causing specific perturbations
of the plasma membrane might interfere with FGF-2 translocation and
release. In this paper, we describe how this strategy led to the
identification of a class of molecules known as cardenolides that alter
the export of FGF-2 from transfected COS cells.
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MATERIALS AND METHODS |
Cell Culture, DNA Transfections, and Reagents--
COS-1 cells
were obtained from the American Tissue Type Culture Collection and
cultured in Dulbecco's modified Eagle's medium (DMEM, Life
Technologies, Inc.) supplemented with 10% fetal bovine serum (Sigma),
2 mM L-glutamine (Life Technologies, Inc.), 1 mM sodium pyruvate (Life Technologies, Inc.), 0.1 mM non-essential amino acids (Life Technologies, Inc.), 100 units/ml penicillin (Life Technologies, Inc.), and 100 units/ml
streptomycin (Life Technologies, Inc.). Media containing varying
concentrations of Na+ or K+ were prepared
following the DMEM recipe (Life Technologies, Inc.) with the specific
modifications as described below. When necessary, choline chloride was
used to replace NaCl or KCl and to keep the final osmolarity constant.
Except for essential and nonessential amino acids (× 50 and 100 stock
solutions, respectively), cell culture grade media components were
purchased from Life Technologies, Inc., Sigma, or were of the highest
quality available, prepared as separate stock solutions in
dH2O and filter sterilized. To vary K+
concentrations, the Na+ concentration was kept constant at
84.91 mM (44 mM NaHCO3, 0.91 mM NaH2PO4, 10 mM
sodium pyruvate (Life Technologies, Inc.), 15 mM from the
glutamine stock solution (Life Technologies, Inc.), 15 mM
from the penicillin and streptomycin stock solution (Life Technologies,
Inc.)). Choline chloride was substituted for NaCl and adjusted along
with a KCl stock solution to achieve the desired final concentrations
of K+. To vary Na+ concentrations, equimolar
KHCO3 and KH2PO4 were substituted
for NaHCO3 and NaH2PO4,
respectively, KOH was used instead of NaOH to adjust pH, the penicillin
and streptomycin solution was omitted, pyruvic acid was substituted for
sodium pyruvate, a glutamine stock solution in dH2O was
made from powder, the KCl concentration was maintained at 30 mM and choline chloride replaced NaCl to achieve the
desired final Na+ concentrations.
COS-1 cells were transfected as described previously with SV-40 based
plasmid cDNA expression vectors encoding the 18-kDa isoform of
human FGF-2, referred to as p18dx in a previous publication (12), the
-subunit of human chorionic gonadotropin (hCG
) (12) or the
1-subunit of rat Na+,K+-ATPase (pCMVOuabain,
PharMingen). Briefly, COS-1 cells were transfected for 30 min with 10 µg of CsCl-purified plasmid DNA mixed with 1.0 ml of transfection
(TX) buffer (140 mM NaCl, 3 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2,
0.9 mM Na2HPO4, 25 mM
Tris, pH 7.4). The TX buffer was removed and the cells were cultured in
complete media supplemented with 100 µM chloroquine
(Sigma) for 90 min. Media containing chloroquine was removed, cells
were washed two times with complete media and cultured in complete
media until the next step in the experiment was performed. In
co-transfection experiments we used 5 µg of each CsCl-purified
plasmid DNA.
Ouabagenin, digoxin, digoxigenin, digitoxin, and digitoxigenin were
purchased from Sigma. Ouabain was purchased from Calbiochem. In
experiments designed to determine IC50 values, 1 mM digitoxose (Sigma) served as a negative control. Unless
otherwise stated, media containing cardenolides and digitoxose were
prepared as follows. An initial 500 mM stock solution was
prepared in 100% Me2SO. A dilution series of working
stocks were prepared in chase media so that the final concentration of
Me2SO was 2%. To determine IC50 values,
working stocks were then added to cell culture media to give final
cardenolide concentrations of 10
4 through
10
11 and a final Me2SO concentration of
0.2%. In the metabolic labeling experiments shown in Figs. 1 and 2,
the initial ouabain stock solutions were prepared directly in chase
media and dissolved by heating briefly at 65 °C. Serial dilutions
were prepared in chase media and used for the dose-response curve shown
in Fig. 1, panel C.
Over the time course of the experiments described in Figs. 1 and 2, a
number of criteria were used to determine the viability of COS-1 cells
in the presence of varying concentrations of ouabain. COS-1 cells do
appear to swell and would eventually die in the presence of ouabain,
however, over the time course of these experiments no difference in
viability could be detected between treated and control cells by the
following assays: lactic dehydrogenase activity in conditioned media
(Sigma), uptake and enzymatic conversion of
3-(4,5-dimethylthiazol-2-yl)-5-(3-lacboxymethoxyphenyl)-2H-tetrazolium (Progmega Corp.), and trypan blue dye exclusion (Life Technologies, Inc.). In addition, we have previously described the use of neomycin phosphotransferase as a marker for expression of a cytosolic protein in
transfected COS-1 cells (12). Therefore, we also conducted control
metabolic pulse-chase and immunoprecipitation experiments in the
presence of 10 mM ouabain with transfected COS-1 cells overexpressing the cytosolic protein neomycin phosphotransferase. No
neomycin phosphotransferase was detected in conditioned media and the
cell-associated signal remained the same. If ouabain was destroying
COS-1 cells we would have expected metabolically labeled neomycin
phosphotransferase in the media fraction and reduced signal in the cell
fraction.
Stock solutions were prepared to test compounds at the highest possible
concentration without toxicity: thapsigargin (Calbiochem) an inhibitor
of Ca2+-ATPase (17), 10 mM stock in 100%
Me2SO, the highest final concentration tested was 1 µM; bafilomycin (Calbiochem), an inhibitor of vacuolar H+-ATPase (18), 16 µM stock in 100%
Me2SO, the highest final concentration tested was 50 nM; amiloride (Sigma), an inhibitor of Na+
channels (19, 20), 200 mM stock in 100% Me2SO,
the highest final concentration tested was 1 mM;
glibenclamide (Research Biochemicals International), an inhibitor of
K+ channels (21, 22), 100 mM stock in 100%
Me2SO, the highest final concentration tested was 100 µM; verapamil (Sigma), an inhibitor of the multidrug
resistance gene product p-glycoprotein (23), 40 mM stock solution in 100% ethanol, the highest final
concentration tested was 100 µM; and trifluoroperazine
(Sigma), also an inhibitor of p-glycoprotein (23), the
highest final concentration tested was 10 µM. These
compounds were shown to have no effect on FGF-2 export as determined by
metabolic pulse-labeling, chase, and immunoprecipitation experiments as
well as by using a standard sandwich ELISA.
Metabolic Labeling, Immunoprecipitations, Western Transfer, and
Immunoblot Analysis--
Forty to 48 h after DNA transfections,
COS-1 cells were metabolically pulse-labeled for 15 min with 100 µCi
of [35S]methionine and [35S]cysteine
(Trans35S-label, ICN Biomedicals, Inc.) in 1.0 ml of
methionine- and cysteine-free DMEM as described (12). After pulse
labeling, cell monolayers were washed once with chase media composed of
DMEM supplemented with 10 mM unlabeled methionine (Sigma),
10 mM unlabeled cysteine (Sigma), 20 µg/ml heparin
(Sigma), 0.5% dialyzed fetal bovine serum (Life Technologies, Inc.).
Transfected cells were then cultured in 2.0 ml of chase medium for the
indicated lengths of time. Where noted, the chase medium was made from
individual stocks to achieve the different final concentrations of
K+ or Na+ as described above.
Cell lysates and conditioned media fractions were processed for
immunoprecipitations or Western transfer and immunoblotting as
described previously (12). Briefly, the media fraction was removed
divided into two 1-ml samples and clarified by centrifugation, 13,000 RPM for 10 min at 4 °C. To each 1 ml of clarified media fraction 400 µl of ice-cold lysis buffer was added. Cells were removed from the
plate using 1 ml of ice-cold lysis buffer and clarified by
centrifugation, 13,000 rpm for 10 min at 4 °C. The lysis buffer used
in these experiments contains 1% Nonidet P-40 (Calbiochem), 0.5%
deoxycholate (Sigma), 20 mM Tris pH 7.5, 5 mM
EDTA, 2 mM EGTA, 0.01 mM phenylmethylsulfonyl
fluoride (Calbiochem), 10 ng/ml aprotinin (Sigma), 10 ng/ml leupeptin,
and 10 ng/ml pepstatin (ICN Biomedicals, Inc.). Both cell and medium
fractions were then incubated at 21 °C for 45 min with guinea pig
anti-FGF-2 immune serum (12) (1:200), rabbit anti-hCG
immune serum
(Biodesign Inc.), or a mouse monoclonal antibody raised against the
1-subunit of Na+,K+-ATPase (Upstate
Biotechnology Inc.). GammaBind G-Sepharose (Pharmacia Biotech Inc.) was
added for an additional 30-min incubation at 21 °C.
G-Sepharose-bound immune complexes were pelleted, washed three times
with ice-cold lysis buffer, and four times with ice-cold immunoprecipitation wash buffer (IP-wash buffer) containing the same
protease inhibitors as lysis buffer. IP-wash buffer is 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2, 1%
deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (Bio-Rad).
Immune complexes were eluted directly into Laemmli SDS gel sample
buffer and separated by 12% SDS-polyacrylamide gel electrophoresis
(12% SDS-PAGE). The gel was processed for fluorography, dried, and
exposed to x-ray film at
70 °C. To determine the IC50
of each cardenolide or the effect of the other compounds listed in
Table I, we used a standard sandwich ELISA technique. Briefly,
quantitation of FGF-2 exported into conditioned media was measured
using monoclonal antibody F155C prepared in our laboratories for
capture followed by biotinylated rabbit polyclonal anti-FGF-2 antibody
(R & D Systems Inc.). For ELISA, 48 h post-transfection, cells
were washed for 75 s with 0.1 M NaCO3
buffer which removes the majority of cell surface bound FGF-2 as
previously shown (12). Cells were then cultured in media containing
dilutions of the specific compound being tested, 0.5% fetal bovine
serum and 20 µg/ml heparin until the media fractions were removed,
clarified, and the amount of FGF-2 measured.
For Western blot analysis, total proteins from 3 × 105 transfected or control COS-1 cells were prepared. Cells
were removed from the tissue culture plate by incubation in Dulbecco's
phosphate-buffered saline without MgCl2 and without
CaCl2 (Life Technologies, Inc.) containing 10 mM EDTA and 5 mM EGTA. Cells were pelleted,
lysed in 100 µl of lysis buffer, and clarified by centrifugation as described above. Supernatants from clarified cell extracts were mixed
with 4 × Laemmli gel sample buffer, heated at 65 °C for 20 min, separated by 12% SDS-polyacrylamide gel and transferred to
nitrocellulose membrane support (0.45 µm pore size, Schleicher and
Schuell) in cold buffer containing 25 mM
3-[dimethyl(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (Research Organics, Inc.), pH 9.5, 20% methanol for 90 min at
constant amperage (0.4 amps). Western transfers were blocked at room
temperature for 1 h in buffer containing 10 mM Tris pH 7.5, 150 mM NaCl, 5 mM NaN3, 0.35%
Tween 20, and 5% nonfat dry milk (Carnation Co., Los Angeles, CA).
Transfers were incubated with primary monoclonal
anti-
1-Na+,K+-ATPase antibody diluted 1:300
in blocking buffer at 4 °C for 16 h. After incubation with
primary antibody, transfers were washed at room temperature with 1 liter (10 changes) of wash buffer containing 150 mM NaCl,
500 mM sodium phosphate, pH 7.4 (Sigma), 5 mM
NaN3, and 0.05% Tween 20. Transfers were then incubated
for 30 min at room temperature in blocking buffer containing 1 µg/ml
rabbit anti-mouse IgG (H+L, affinipure, Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA), washed in 1 liter of wash buffer,
incubated for 1 h in 100 ml of blocking buffer containing 15 µCi
of 125I-protein A (ICN Biochemicals, Costa Mesa, CA) and
washed with 1 liter of buffer as described above. Signal was visualized
by autoradiography using RX Fuji Medical x-ray film.
 |
RESULTS |
Cardenolides Inhibit Export of FGF-2 from COS-1 Cells--
In a
previous publication (12), we provided evidence for the existence of an
ATP-dependent plasma membrane translocation apparatus
(PMTA) as a component of the FGF-2 export pathway. On this basis, it
was proposed that specific perturbations of the plasma membrane might,
as a consequence, interfere with FGF-2 export. For example, disrupting
the movement of ions across the plasma membrane is known to interfere
with the function of other proteins involved in the active transport of
amino acids and glucose into cells (24). Therefore, in an attempt to
identify small molecules that could alter the export of FGF-2, we
evaluated those that were already known to modulate the activity of
plasma membrane proteins involved in ion transport. One such molecule
is the cardioglycoside ouabain. Ouabain belongs to a class of compounds
called the cardenolides (cardioglycosides and their aglycone
derivatives) that interact with the catalytic
-subunit of
Na+,K+-ATPase and thereby inhibit its ability
to (i) exchange K+ for Na+, (ii) hydrolyze ATP,
and (iii) maintain an electrochemical gradient across the plasma
membrane.
When COS-1 cells are transfected with an expression vector encoding the
18-kDa isoform of FGF-2, they export this growth factor into the
culture media in a time-dependent fashion (Fig.
1, panel A). After an 8-h
chase, approximately 60% of the 35S-metabolically labeled
FGF-2 is detected in the conditioned media. When the identical
experiment is performed with chase media supplemented with 100 µM of the cardioglycoside ouabain, the amount of
metabolically labeled FGF-2 detected in the conditioned medium is
reduced by over 75% (Fig. 1, panel B). Because the amounts
of radiolabeled FGF-2 immunoprecipitated from the corresponding cell
extracts remain relatively constant during the time course of these
experiments, ouabain appears to block plasma membrane translocation of
FGF-2 and not its intracellular stability. The inhibition of FGF-2
export is concentration-dependent with a half-maximal
activity (IC50) of ~0.1 µM ouabain (Fig. 1,
panel C). The high initial concentration tested in the
experiments shown in Fig. 1, panels B and C,
necessitated that ouabain be dissolved directly in chase media by
heating briefly at 65 °C. As a consequence of the well characterized
effect of ouabain on Na+,K+-ATPase activity,
COS-1 cells do increase in volume during these experiments. However,
over the time course of these experiments, there is no difference
between treated and control cell viability and/or plasma membrane
integrity as measured by trypan blue dye exclusion and levels of lactic
dehydrogenase activity in conditioned media. Therefore, the presence of
ouabain in an 8-h pulse-chase experiment appears to block FGF-2 export
from transfected COS-1 cells in the absence of detectable cytotoxicity
or cell death.

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Fig. 1.
FGF-2 export: time and concentration
sensitivity to ouabain. COS-1 cells were transfected with an SV-40
based expression vector encoding the 18-kDa isoform of human FGF-2.
Forty-eight hours thereafter, transfected COS-1 cells were
metabolically pulse-labeled for 15 min, washed, and incubated in chase
media in the absence (panel A) or presence of 100 µM ouabain (panel B). Ouabain was dissolved
directly in chase media by heating briefly at 65 °C. At the times
indicated, cell (C) and media (M) fractions were prepared and incubated with polyclonal guinea pig anti-FGF-2 immune serum as described under "Materials and Methods." Immune complexes were fractionated by 12% SDS-PAGE, and the signal detected by fluorography. The location of 14C-labeled molecular size
standards are indicated at the right. In other experiments
(panel C), transfected COS-1 cells were metabolically pulse-labeled and incubated in chase media containing various concentrations of ouabain for 10 h and then processed as described above. In this case, signal intensity was quantified by densitometry and graphed as percent of the total (cell plus media) FGF-2 detected in
the media fraction. By 10 h after metabolic pulse labeling, 80%
of the radiolabeled FGF-2 has been chased into the conditioned media of
control untreated cells. Results are representative of two to four
experiments.
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A number of ouabain-related compounds were also tested to determine if
the ability to inhibit export of FGF-2 was shared by other members of
the cardenolide family, which include the aglycone derivatives of the
cardioglycoside. The cardenolides tested include ouabain, ouabagenin,
digoxin, digoxigenin, digitoxin, and digitoxigenin (Table
I). In these experiments we determined
the IC50 value for each cardenolide. Dilutions of working
stock solutions were prepared in chase media so that the final
concentration of Me2SO was 0.2%. All cardenolides capable
of inhibiting the catalytic activity of the
-subunit of
Na+,K+-ATPase were also found to inhibit FGF-2
protein export.
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Table I
Compounds tested as potential inhibitors of FGF-2 export from COS-1
cells
In all cases, COS-1 cells were transfected with an SV-40 based
expression vector encoding the 18-kDa isoform of human FGF-2. Forty-eight hours later, transfected cells were washed with 0.1 M NaCO3 buffer (pH 11) and incubated in media
containing serial dilutions of cardenolides or other compounds until
the media fractions were processed and the amount of FGF-2 measured by
ELISA as described under "Materials and Methods." The IC50
values were calculated for compounds that had an inhibitory effect on
FGF-2 export. If no effect was detected, the highest final
concentration that was tested is indicated. NKA is used as an
abbreviation for Na+,K+-ATPase.
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Control metabolic pulse-labeling experiments were performed in the
presence of inhibitors of other ion transporters or channels to
determine if the cardenolide effect on FGF-2 export was a general consequence of altering any ion transport process (Table I). Thapsigargin was used as an inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase, bafilomycin of vacuolar
H+-ATPase, glibenclamide inhibits K+ channels,
amiloride inhibits Na+ channels while verapamil and
trifluoperazine were used to block transport activity of the multidrug
resistance/P-glycoprotein. All inhibitors were tested at the
concentrations listed in Table I. When treated for 8 h in chase
media, the concentrations listed are the highest that were not
cytotoxic to COS-1 cells. None of these compounds blocked, reduced, or
elevated the rate or extent of FGF-2 export from transfected COS-1
cells. Identical results were also obtained by sandwich ELISA.
Together, these data imply that the only ion transporter or ion channel
involved in regulating FGF-2 export appears to be
Na+,K+-ATPase and that the cardenolides, as a
class of molecules, specifically interfere with this process in COS-1
cells.
Cardenolides Selectively Block Export and not Protein Secretion
from COS-1 Cells--
The ER/Golgi-dependent secretion of
hCG
from transfected COS-1 cells was used to determine if ouabain
specifically blocks FGF-2 export. Secretion of hCG
from transfected
COS-1 cells was examined in the absence (Fig.
2, panel A) or presence (Fig.
2, panel B) of ouabain. The appearance of
35S-metabolically labeled hCG
in conditioned media was
completely unaffected by the presence of as much as 20 mM
ouabain, approximately 80% of hCG
is still released into the
conditioned media. In these experiments, we used the highest
concentration of ouabain that can be easily dissolved in chase media
(with brief heating at 65 °C) to emphasize that there is no effect
on conventional ER/Golgi trafficking and secretion of hCG
. Likewise,
lower concentrations of ouabain have no effect. In metabolic
pulse-labeling or ELISA-based experiments, the ouabain-related
cardioglycosides as well as their aglycone derivatives listed in Table
I were also tested and shown to have no effect on secretion of hCG
(not shown). We conclude that cardenolides selectively interfere with
export of FGF-2 from the cytosol into the extracellular milieu while
having no obvious effect on conventional ER/Golgi-dependent
protein trafficking and secretion from COS-1 cells.

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Fig. 2.
Ouabain has no effect on hCG
secretion. COS-1 cells were transfected with an SV-40 based
expression vector encoding hCG , metabolically pulse-labeled 48 h thereafter, washed, and incubated in chase media in the absence
(panel A) or presence of 20 mM ouabain
(panel B). Ouabain was dissolved directly in chase media by
heating briefly at 65 °C. At the times indicated, the cell
(C) and corresponding media (M) fractions were
processed, immune complexes prepared using rabbit polyclonal
anti-hCG antibody, separated by 12% SDS-PAGE and the signal
visualized by fluorography. The location of 14C-labeled
molecular size standards are indicated at the right.
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The
-Subunit of Na+,K+-ATPase Modulates
FGF-2 Export--
Ouabain inhibits the ion transport activity of
Na+,K+-ATPase by binding to sites in the
catalytic
-subunit (25). Since ouabain also inhibits FGF-2 export,
we designed experiments to investigate whether the
-subunit might
also play a role in FGF-2 export. To gain insight into how the
-subunit might be involved in protein export, COS-1 cells were
co-transfected with plasmid expression vectors encoding FGF-2 and the
rat
1-subunit of Na+,K+-ATPase. The cell and
media fractions of metabolically pulse-labeled COS-1 cells were
immunoprecipitated with anti-FGF-2 antibodies. As shown in Fig.
3, coexpression of the
1-subunit with
18-kDa FGF-2 dramatically slows the rate of FGF-2 protein export into the conditioned media (panel A), compared with control cells
expressing FGF-2 only (panel B). When measured by
densitometry, the FGF-2 signal is stable and less than 20% of the
total FGF-2 signal is detected in the media 8 h after labeling.
Yet, within the same time frame, over 50% of the radiolabeled FGF-2 is
chased into the conditioned media when COS-1 cells are transfected with
the FGF-2 expression vector alone. These data suggest that the
1-subunit is in some way involved in FGF-2 export and that when
overexpressed, it competes for FGF-2 or in some other way specifically
interferes with maintenance of a functional FGF-2 export pathway in
COS-1 cells. In control experiments, this is not the consequence of co-overexpression. The co-overexpression of the cytosolic protein neomycin phosphotransferase with FGF-2 has no effect on FGF-2 export
(12). In addition, co-overexpression of hCG
which traffics through
the ER/Golgi also has no effect on FGF-2 export. Moreover, when
co-transfection experiments are performed with hCG
and
1-subunit, the rate and extent of hCG
secretion remains unchanged (not
shown).

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Fig. 3.
Co-transfection and overexpression of the
1-subunit of Na+,K+-ATPase interferes with
FGF-2 export. COS-1 cells were co-transfected with an SV-40 based
expression vector encoding the 18-kDa isoform of human FGF-2 plus an
SV-40 based expression vector encoding the 1-subunit of rat
Na+,K+-ATPase (panel A) or the
expression vector encoding human 18-kDa FGF-2 alone (panel
B). Forty-eight hours thereafter, transfected COS-1 cells were
metabolically pulse-labeled for 15 min, washed, and incubated in chase
media as described under "Materials and Methods." The cell
(C) and corresponding media (M) fractions were immunoprecipitated with polyclonal guinea pig anti-FGF-2 immune serum
at the indicated times. Immune complexes were separated by 12%
SDS-PAGE and the signal visualized by fluorography. The location of a
100-kDa band that co-immunoprecipitates with FGF-2 is marked by a
double-headed arrow, FGF-2 by a single-headed
arrow. The location of 14C-labeled molecular size
standards are indicated at the right.
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The
1-Subunit of Na+,K+-ATPase Interacts
with 18-kDa FGF-2--
In addition to blocking FGF-2 export,
immunoprecipitation of co-transfected COS-1 cell extracts revealed a
previously unobserved metabolically labeled protein of approximately
100-kDa (see double headed arrow in Fig. 3). This protein
band is reproducibly detected throughout the time course of the
experiment, although the intensity of the radiolabeled
co-immunoprecipitated signal decreases at the longer time points. The
100-kDa band is not detected in conditioned media and its estimated
size is consistent with it being the rat
1-subunit encoded by the
expression vector co-transfected with the plasmid encoding 18-kDa
FGF-2. In control experiments, cell extracts prepared from transfected
COS-1 cells expressing the rat
1-subunit alone or FGF-2 alone were
mixed and then immunoprecipitated with anti-FGF-2 immune serum (not
shown). Under these conditions, the 100-kDa band was not detected,
suggesting that interactions between FGF-2 and the
1-subunit may
occur de novo in co-transfected cells but do not form as a
result of nonspecific associations during sample preparation.
The hypothesis that the 100-kDa band is the
1-subunit was confirmed
by Western transfer and immunoblot analysis. Total cell extracts were
prepared from mock-transfected COS-1 cells and from COS-1 cells
transfected with the expression vector encoding the rat
1-subunit.
The samples were mixed with 4 × Laemmli gel sample buffer,
separated by 12% SDS-PAGE, transferred to nitrocellulose support, and
probed with monoclonal antibody to the
1-subunit (Fig.
4). Under these conditions, the same
overexpressed 100-kDa band noted in Fig. 3 is detected in extracts
prepared from COS-1 cells transfected with the vector encoding the rat
1-subunit of Na+,K+-ATPase. The 100-kDa band
is also present in mock transfected COS-1 cells, although at lower
endogenous levels of expression as would be expected of a kidney
derived cell line. As a consequence of transfection efficiency, the
increase in
1-subunit signal accounts for the elevated levels of
expression in the transfected cell population, approximately 15% of
the total cell number. The band observed at approximately 140-kDa has
not been identified. However, it does not co-immunoprecipitate with
FGF-2 (Fig. 3), indicating that any interactions occurring between
FGF-2 and the
1-subunit are only with the 100-kDa form.

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Fig. 4.
Immunoblot analysis of transfected COS-1
cells overexpressing the 1-subunit of
Na+,K+-ATPase. Total cell extracts were
prepared from 3 × 105 control transfected COS-1 cells
(lane 1) and from cells transfected with an SV-40 based
expression vector encoding the 1-subunit of rat
Na+,K+-ATPase (lane 2). Extracts
were fractionated by 12% SDS-PAGE, transferred to nitrocellulose
support, incubated with a monoclonal anti- 1-subunit antibody,
washed, incubated with rabbit anti-mouse polyclonal antibody, and then
with 125I-protein G as described under "Materials and
Methods." Signal is visualized by autoradiography. The location of
prestained molecular size standards are indicated at the
right.
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Further evidence supporting the notion that specific interactions can
occur between FGF-2 and the
1-subunit was obtained when immune
complexes from co-transfected COS-1 cells were prepared using
anti-
1-subunit antibodies rather than anti-FGF-2 antibodies (Fig.
5). Just as anti-FGF-2 antibodies can
co-immunoprecipitate the 100-kDa
1-subunit, antibodies to the
1-subunit co-immunoprecipitate 18-kDa FGF-2. The 100-kDa band
representing the
1-subunit only co-immunoprecipitates from cells
co-transfected with the expression vector encoding FGF-2. When similar
co-transfection experiments are carried out with expression vectors
encoding hCG
and the
1-subunit, no co-precipitated signal is
detected (Fig. 6). Immune complexes
prepared with anti-
1-subunit antibodies do not co-precipitate hCG
and immune complexes prepared with anti-hCG
antibodies do not
co-precipitate the
1-subunit of
Na+,K+-ATPase. Thus, even though both hCG
and the
1-subunit traffic through the ER and Golgi, they do not form
nonspecific complexes by co-immunoprecipitation from co-transfected
COS-1 cell extracts. Similar experiments performed with cDNA
expression vectors encoding cytoplasmic proteins (i.e.
neomycin phosphotransferase) also fail to show an interaction (12).
Taken together, these data suggest that the
1-subunit specifically
interacts with FGF-2 either directly or indirectly through the
formation of stable complexes involving other proteins.

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Fig. 5.
Co-immunoprecipitation of 18-kDa FGF-2 using
an anti- 1-subunit antibody from co-transfected COS-1 cells.
COS-1 cells were mock transfected or co-transfected with an SV-40 based
expression vector encoding the 18-kDa isoform of FGF-2 plus an SV-40
based expression vector encoding the 1-subunit of rat
Na+,K+-ATPase. Forty-eight hours thereafter,
transfected COS-1 cells were metabolically pulse-labeled for 15 min,
washed, and cell extracts processed as described under "Materials
and Methods." Immune complexes were prepared using a monoclonal
antibody to the 1-subunit, separated by 12% SDS-PAGE and the signal
visualized by fluorography. The location of 14C-labeled
molecular size standards are indicated at the right.
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Fig. 6.
HCG does not co-immunoprecipitate with the
1-subunit of Na+,K+-ATPase. COS-1 cells
were mock transfected, transfected with an SV-40 based expression
vector encoding hCG or the 1-subunit of rat
Na+,K+-ATPase, or co-transfected as indicated.
Forty-eight hours after transfection, cells were metabolically
pulse-labeled for 15 min, cell extracts prepared and immunoprecipitated
with a monoclonal antibody to the 1-subunit or polyclonal rabbit
anti-hCG antibody as described under "Materials and Methods."
Immune complexes were separated by 12% SDS-PAGE and the signal
visualized by fluorography. The location of hCG (open
arrow) and the 1-subunit (closed arrow) are shown.
The location of 14C-labeled molecular size standards are
indicated at the right.
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Uncoupling Ion Transport from 18-kDa FGF-2 Export--
The
K+ and Na+ electrochemical gradient across the
plasma membrane is maintained by Na+,K+-ATPase
and is known to be an important driving force for the transport of
various amino acids and glucose into cells (24). In intact cells, the
ion transport activity of Na+,K+-ATPase can be
inhibited in the presence of low external K+ concentration.
Therefore, we also tested whether FGF-2 export is functionally linked
to the electrochemical gradient maintained by
Na+,K+-ATPase in COS-1 cells.
To determine if a functional electrochemical gradient was required for
FGF-2 export, transfected COS-1 cells were metabolically pulse-labeled
and then incubated in chase media containing varying K+
concentrations. Chase media containing varying concentrations of
K+ were prepared from individual stocks of DMEM components
as described under "Materials and Methods." The Na+
concentration was kept constant at 84.91 mM. Choline
chloride was substituted for NaCl and replaced KCl to achieve the
indicated final concentrations of K+, 0.5% dialyzed fetal
bovine serum was also used to reduce the possibility of introducing
small amounts of K+ from this source. After metabolic
labeling, all cell and media fractions were processed by
immunoprecipitation 8 h later. No significant difference in the
media or cell associated 35S-labeled FGF-2 signals can be
detected by varying external K+ (Fig.
7).

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Fig. 7.
Varying the external K+
concentration does not alter FGF-2 protein export. COS-1 cells
were transfected with an SV-40 based expression vector encoding the
18-kDa isoform of FGF-2. Forty-eight hours later, transfected cells
were metabolically pulse-labeled for 15 min, washed, and incubated for
8 h in chase media containing the indicated concentration of
K+, as described under "Materials and Methods"
(panel A, B, and C). Chase media was made from
individual stock components, the Na+ concentration was kept
constant at 84.91 mM, choline chloride was substituted for
NaCl and replaced KCl to achieve the indicated final concentrations of
K+. The cell (C) and corresponding media
(M) fractions were processed, immune complexes prepared,
separated by 12% SDS-PAGE, and the signal visualized by fluorography.
The entire gel is shown in panel A, while only the bands
corresponding to 18-kDa FGF-2 are shown in panels B and
C. The location of 14C-labeled molecular size
standards are indicated at the right.
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In parallel, experiments were performed to determine if the external
Na+ concentration might have an unexpected effect on the
rate and/or extent of FGF-2 export. In these experiments, chase media
containing varying concentrations of Na+ were prepared as
described under "Materials and Methods." Chase media with varying
Na+ concentrations included equimolar KHCO3 and
KH2PO4 substituted for NaHCO3 and
NaH2PO4, respectively, choline chloride was
used to substitute for NaCl and the KCl concentration was kept constant at 30 mM. As shown in Fig. 8,
there is no detectable effect of varying external Na+
concentration on 18-kDa FGF-2 export from transfected COS-1 cells. In
addition, there was no obvious cytotoxic affect on COS-1 cells cultured
for 8 h using media with varying Na+ or K+
concentrations. Thus, while there is a functional (Fig. 3) and physical
(Fig. 5) association between FGF-2 and the
1-subunit, protein export
appears to be independent of the electrochemical gradient established
by Na+,K+-ATPase (Fig. 7) and there is no
effect of varying the external Na+ concentration (Fig. 8).
Although these data implicate the involvement of the
-subunit of
Na+,K+-ATPase in 18-kDa protein export, they
also suggest that it does so in a manner that is mechanistically
distinct from its previously characterized activities in mediating
K+ and Na+ transport.

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Fig. 8.
Varying the external Na+
concentration does not alter FGF-2 protein export. COS-1 cells
were transfected with an expression vector encoding the 18-kDa isoform
of FGF-2, metabolically pulse-labeled, washed, and incubated in chase
media for 8 h containing the indicated concentrations of
Na+, as described under "Materials and Methods"
(panels A and B). Media with varying
Na+ concentrations were prepared from individual stocks
such that equimolar KHCO3 and
KH2PO4 were substituted for NaHCO3
and NaH2PO4, respectively, KOH was used instead
of NaOH to adjust pH, the KCl concentration was 30 mM and
choline chloride replaced NaCl to achieve the indicated final
Na+ concentrations. Eight hours after metabolic labeling,
the cell (C) and corresponding media (M)
fractions were processed, immune complexes prepared, separated by 12%
SDS-PAGE, and the signal visualized by fluorography. The entire gel is
shown in panel A, while only the bands corresponding to
18-kDa FGF-2 are shown in panel B. The location of
14C-labeled molecular size standards are shown at the
right.
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DISCUSSION |
We have demonstrated that export of FGF-2 from COS-1 cells can be
specifically inhibited by the class of molecules known as cardenolides,
the cardioglycosides and their aglycone derivatives. We also present
evidence that there is a functional interaction between FGF-2 and the
1-subunit of Na+,K+-ATPase that appears to
be required for export of FGF-2 from COS-1 cells. Both FGF-2 and the
1-subunit co-immunoprecipitate with anti-
1-subunit antibodies and
anti-FGF-2 immune serum, respectively. In addition, co-expression of
the
1-subunit of Na+,K+-ATPase inhibits
FGF-2 export from cells, but export is not dependent upon an
electrochemical gradient maintained by heterodimeric
Na+,K+-ATPase. The effects of co-overexpression
of the
1-subunit on FGF-2 export are specific, in that
co-overexpression of other proteins have no effect on FGF-2 export. It
is likely that co-overexpression of the
1-subunit interferes with
export by preferentially competing for 18-kDa FGF-2 or other components
of the pathway, preventing access to a fully functional PMTA. These
data suggest that FGF-2 associates with the
1-subunit directly or,
perhaps indirectly, following formation of complexes with other
proteins that remain to be identified. Taken together, these data
strongly implicate the
1-subunit of
Na+,K+-ATPase as a structural and/or regulatory
component of the FGF-2 export pathway.
Several possible models can serve to link, at the molecular level, the
known actions of cardenolides, the activity of their target (the
1-subunit of Na+,K+-ATPase) and their
ability to inhibit FGF-2 export. For example, Na+,K+-ATPase itself could be a component of
the plasma membrane translocation apparatus (PMTA) through which FGF-2
is "exported." This possibility, however, seems rather unlikely in
view of (i) the physical nature of the
Na+,K+-ATPase 
heterodimer that exchanges
2 extracellular K+ for 3 intracellular Na+ (26,
27), (ii) the size differences between these ions and 154 amino acid
FGF-2, and (iii) our ability to dissociate ion pump activity from
protein export (Figs. 7 and 8).
Alternatively, the PMTA exporting FGF-2 from cells may be regulated via
the electrochemical gradient maintained by
Na+,K+-ATPase. But, this possibility is not
compatible with the dissociation of FGF-2 export from ion transport as
described in Figs. 7 and 8. Instead, it is possible that there is a
novel cardenolide-sensitive protein target that is distinct from the
catalytic
-subunit of Na+,K+-ATPase.
However, despite extensive searches (28), the only characterized
binding site for cardenolides at the cell surface is the catalytic
-subunit of Na+,K+-ATPase. While there have
been isolated reports describing 10- and 31.5-kDa ouabain-binding
proteins, no molecules have been structurally characterized
(29-31).
The data currently available fit best with the hypothesis that the PMTA
mediating translocation of FGF-2 exists as a higher ordered protein
complex, most likely made up at least in part by the
-subunit itself
plus an as yet unidentified protein or proteins that together form a
functional PMTA. Although the minimal functional
Na+,K+-ATPase ion transporter (sodium pump)
consists of a single 
heterodimer, higher order multimeric
structures have been described (26, 27). It is conceivable that these
higher order structures may function in FGF-2 export and not ion
transport. Involvement of the
-subunit could be regulated and/or in
balance between a functional sodium pump (
heterodimer) and a
functional PMTA that may or may not include the
-subunit. Indeed,
Blanco et al. (32) have shown that the
-subunit itself
can organize into stable oligomeric structures that localize on the
cell surface but that have no known function. In addition, there have
also been reports describing a putative
-subunit of
Na+,K+-ATPase (31, 33) and another ouabain
receptor protein (29, 30) that could be involved in formation of the
PMTA mediating FGF-2 protein export. Structural, functional, and/or
regulatory similarities may exist between the PMTA mediating protein
export and the translocon mediating protein translocation through the ER (34). The ER translocation complex (35), as well as the mammalian
multidrug resistance gene product (P-glycoprotein) have been reported
to function as ion channels in addition to peptide translocators (36,
37).
The observations described here are also compatible with a model in
which the catalytic
-subunit functions in a chaperone-like fashion
(via a vesicular intermediate) rather than as a component of the PMTA
per se. In this instance, the
-subunit would play a role
in delivering FGF-2 to the cytoplasmic face of the plasma membrane
where it is then transferred to the PMTA or is extravesciculated through a basolateral release mechanism (38) and into the extracellular matrix, a known proteolytically protected storage site for FGF-2 (39-42). A similar explanation has been proposed for FGF-1 export involving synaptotagmin following heat shock (43) as well as for the
release of the exported proteins "VDP" (44) and galectin (45).
Although we cannot formally rule out this chaperone model, the
existence of a vesicular intermediate is weakened by experiments showing that nocodazole, NH4Cl, chloroquine, monensin,
cytochalasin, and colchicine have no effect on FGF-2 export in the
COS-1 cell system.2
Furthermore, if an intracellular vesicular intermediate were involved,
the cardenolide would need to cross multiple lipid bilayers before it
could interact with binding sites on the
-subunit localized in the
lumen of the vesicle. It is also noteworthy to mention that the results
presented here do not preclude the possibility that cytoplasmic
interactions between FGF-2 and the
-subunit of
Na+,K+-ATPase reflects a heretofore
unrecognized role for FGF-2 in modulating Na+,K+-ATPase activity. The catalytic subunit
of Na+,K+-ATPase is ubiquitous in cells and
tissues (28), much like FGF-2 (14) and it is often found at the
basolateral membrane (33) where FGF-2 is deposited into the
extracellular matrix.
The data presented here suggest that the known activities of
cardenolides should be re-evaluated in light of the role they appear to
play as inhibitors of FGF-2 protein export. In this context, several
investigators (46, 47) have described the existence of endogenous
cardenolides that may function in regulation of blood pressure.
However, it is conceivable that their normal physiological function may
also be as endogenous regulators of FGF-2 export. Moreover, differences
in breast cancer outcome, including reduced tumor mass and lower growth
potential (48, 49) have been described in patients on digoxin therapy,
perhaps due to the anti-proliferative effects of cardenolides (50, 51). Whether these observations can be correlated to levels of circulating FGF-2 measured in biological fluids (52, 53) or the elevated levels
reported in certain cancer models (54-56) is not known but currently
under investigation.
In conclusion, the data shown indicate that the FGF-2 export pathway in
COS-1 cells is cardenolide sensitive. Co-immunoprecipitation of FGF-2
with the
-subunit of Na+,K+-ATPase
implicates the
-subunit as a structural and/or regulatory component
in this pathway. The function of the
-subunit as it is involved in
export of FGF-2 may or may not include a
-subunit and can be
uncoupled from the ion transporting activity of
Na+,K+-ATPase.
We especially thank Elin Florkiewicz for
comprehensive and expert technical assistance, Marie Ryder for
preparing ELISA data, and Carolyn Machamer for the plasmid encoding
hCG
. We also thank Jerry Lingrel, Jack Kyte, and Richard Majack for
many helpful discussions.