The Inhibition of Fibroblast Growth Factor-2 Export by Cardenolides Implies a Novel Function for the Catalytic Subunit of Na+,K+-ATPase*

Robert Z. FlorkiewiczDagger , Jerry Anchin, and Andrew Baird

From PRIZM Pharmaceuticals, San Diego, California 92121

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-alpha .

Because cardenolides are known to inhibit ion transport activity mediated by Na+,K+-ATPase, we investigated whether there are functional interactions between FGF-2 and their only known molecular target: the alpha -subunit of Na+,K+-ATPase. Export of FGF-2 from COS-1 cells is selectively inhibited when co-transfected with expression vectors encoding the alpha -subunit and FGF-2. Moreover, antibodies to the alpha -subunit specifically co-immunoprecipitate FGF-2 along with the alpha -subunit while conversely, antibodies to FGF-2 specifically co-immunoprecipitate the alpha -subunit along with FGF-2. Finally, the ion transporting activities of the Na+,K+-ATPase can be uncoupled from protein export. Varying the external concentration of K+ has little effect on export of FGF-2.

Taken together, these data: 1) identify a novel activity for cardenolides; 2) suggest a previously unknown role for the alpha -subunit of Na+, K+-ATPase in FGF-2 export; and 3) raise the possibility that the alpha -subunit itself may be an integral component of this alternate exocytic pathway mediating translocation of cytosolic FGF-2 to the cell surface.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -subunit of human chorionic gonadotropin (hCGalpha ) (12) or the alpha 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-hCGalpha immune serum (Biodesign Inc.), or a mouse monoclonal antibody raised against the alpha 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-alpha 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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.

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 alpha -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.

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 hCGalpha from transfected COS-1 cells was used to determine if ouabain specifically blocks FGF-2 export. Secretion of hCGalpha 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 hCGalpha in conditioned media was completely unaffected by the presence of as much as 20 mM ouabain, approximately 80% of hCGalpha 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 hCGalpha . 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 hCGalpha (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 hCGalpha secretion. COS-1 cells were transfected with an SV-40 based expression vector encoding hCGalpha , 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-hCGalpha 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.

The alpha -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 alpha -subunit (25). Since ouabain also inhibits FGF-2 export, we designed experiments to investigate whether the alpha -subunit might also play a role in FGF-2 export. To gain insight into how the alpha -subunit might be involved in protein export, COS-1 cells were co-transfected with plasmid expression vectors encoding FGF-2 and the rat alpha 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 alpha 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 alpha 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 hCGalpha which traffics through the ER/Golgi also has no effect on FGF-2 export. Moreover, when co-transfection experiments are performed with hCGalpha and alpha 1-subunit, the rate and extent of hCGalpha secretion remains unchanged (not shown).


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Fig. 3.   Co-transfection and overexpression of the alpha 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 alpha 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.

The alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 1-subunit are only with the 100-kDa form.


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Fig. 4.   Immunoblot analysis of transfected COS-1 cells overexpressing the alpha 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 alpha 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-alpha 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.

Further evidence supporting the notion that specific interactions can occur between FGF-2 and the alpha 1-subunit was obtained when immune complexes from co-transfected COS-1 cells were prepared using anti-alpha 1-subunit antibodies rather than anti-FGF-2 antibodies (Fig. 5). Just as anti-FGF-2 antibodies can co-immunoprecipitate the 100-kDa alpha 1-subunit, antibodies to the alpha 1-subunit co-immunoprecipitate 18-kDa FGF-2. The 100-kDa band representing the alpha 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 hCGalpha and the alpha 1-subunit, no co-precipitated signal is detected (Fig. 6). Immune complexes prepared with anti-alpha 1-subunit antibodies do not co-precipitate hCGalpha and immune complexes prepared with anti-hCGalpha antibodies do not co-precipitate the alpha 1-subunit of Na+,K+-ATPase. Thus, even though both hCGalpha and the alpha 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 alpha 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-alpha 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 alpha 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 alpha 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.   HCGalpha does not co-immunoprecipitate with the alpha 1-subunit of Na+,K+-ATPase. COS-1 cells were mock transfected, transfected with an SV-40 based expression vector encoding hCGalpha or the alpha 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 alpha 1-subunit or polyclonal rabbit anti-hCGalpha antibody as described under "Materials and Methods." Immune complexes were separated by 12% SDS-PAGE and the signal visualized by fluorography. The location of hCGalpha (open arrow) and the alpha 1-subunit (closed arrow) are shown. The location of 14C-labeled molecular size standards are indicated at the right.

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.

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 alpha 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 alpha -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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 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 alpha 1-subunit co-immunoprecipitate with anti-alpha 1-subunit antibodies and anti-FGF-2 immune serum, respectively. In addition, co-expression of the alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha 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 alpha beta 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 alpha -subunit of Na+,K+-ATPase. However, despite extensive searches (28), the only characterized binding site for cardenolides at the cell surface is the catalytic alpha -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 alpha -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 alpha beta 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 alpha -subunit could be regulated and/or in balance between a functional sodium pump (alpha beta heterodimer) and a functional PMTA that may or may not include the beta -subunit. Indeed, Blanco et al. (32) have shown that the alpha -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 gamma -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -subunit of Na+,K+-ATPase implicates the alpha -subunit as a structural and/or regulatory component in this pathway. The function of the alpha -subunit as it is involved in export of FGF-2 may or may not include a beta -subunit and can be uncoupled from the ion transporting activity of Na+,K+-ATPase.

    ACKNOWLEDGEMENTS

We especially thank Elin Florkiewicz for comprehensive and expert technical assistance, Marie Ryder for preparing ELISA data, and Carolyn Machamer for the plasmid encoding hCGalpha . We also thank Jerry Lingrel, Jack Kyte, and Richard Majack for many helpful discussions.

    FOOTNOTES

* This work was originally funded by National Institutes of Health Grant DK11811. Some of the experiments presented in this paper were performed when the authors (R. Z. F. and A. B.) were at the Department of Cell Biology, The Scripps Research Institute. This is paper 9387-CB from the Scripps Research Institute.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.

Dagger To whom correspondence should be addressed: 11035 Roselle St., San Diego, CA 92121. Tel.: 619-625-0100; Fax: 619-625-0050; E-mail: rzflork{at}prizmpharm.com.

1 The abbreviations used are: ER, endoplasmic reticulum; FGF, fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; Me2SO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; hCGalpha , human chorionic gonadotropin-alpha ; PAGE, polyacrylamide gel electrophoresis; PMTA, plasma membrane translocation apparatus.

2 R. Z. Florkiewicz and A. Baird, unpublished observations.

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Results
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