Differential regulation of fibroblast growth factor (FGF) receptor-1 mRNA and protein by two molecular forms of basic FGF. Modulation of FGFR-1 mRNA stability.

To evaluate possible functional differences between basic fibroblast growth factor (FGF) 2 isoforms we analyzed the effects of the 18-kDa FGF-2 which mainly localizes in the cytosol and that of the nuclear-targeted 22.5-kDa form on FGF receptors (FGFR) expression. These peptides were expressed at low amounts through a retroviral-infection system. Point mutated FGF-2 cDNAs under the control of the beta-actin promoter were used to infect a pancreatic cell line (AR4 2J) which does not produce FGF-2. Saturation and competition binding studies with 125I-FGF-2 revealed a 3-fold increase in both high and low affinity receptors in cells expressing the 22.5-kDa form and a 2-fold increase only in the high affinity receptors in cells producing the 18-kDa form. Kd values and molecular weights of the high affinity receptors were unaffected. Increasing cell densities or cell treatment with exogenous FGF-2 resulted in FGFR down-regulation as in control cells. Neutralizing anti-FGF-2 antibodies and suramin did not affect receptor density in control and in cells producing the 22.5-kDa form but further increased by 60 and 80%, respectively, the receptor level in cells synthesizing the 18-kDa form. These data suggest the involvement of the intracellular stored FGF-2 in FGFR up-regulation. Although all cells expressed FGFR-1, -2, and -3 mRNA only the FGFR-1 transcript was found increased, 6-fold in 22.5-kDa expressing cells and 3-fold in cell producing the shortest secreted isoform. The increase in FGFR-1 mRNA levels in the 22.5-kDa expressing cells was due to enhanced stability of the transcript. Confocal microscopy detected the presence of FGFR-1 at the cell surface whereas secretory isoforms of the receptor were not observed. Reverse transcriptase-polymerase chain reaction did not reveal significant differences in the expression of FGFR-1 variants. In the 22.5-kDa expressing cells exogenous FGF-2 evoked a stronger translocation of the calcium-phospholipid-dependent PKC. These results indicate that the transfected FGF-2 isoforms up-regulated FGFR-1 mRNA and protein. The 22.5-kDa form acted by increasing FGFR-1 mRNA stability enhancing cell responses to exogenous FGF-2.

tors (1)(2)(3)(4). The biology of FGF-2 is complex. Multiple isoforms of the growth factor (from 18 to 24 kDa) can be produced by the same cell from a single mRNA species, as a result of initiation of translation either at the AUG or at different CUG codons. FGF-2, like FGF-1, lacks the hydrophobic signal peptide and is therefore concentrated within its cell of origin. The shorter 18-kDa FGF-2 isoform initiated at the AUG codon is predominantly localized in the cytosol and is also found at the cell surface after secretion via a cellular pathway distinct from classical secretion (5)(6)(7)(8)(9). In contrast, polypeptides initiated at CUG codons are preferentially localized in the nucleus. They are larger than the 18-kDa form, possessing an additional amino-terminal sequence particularly rich in arginine residues which allows for their nuclear targeting (10,11). Furthermore, when the 18-kDa form is provided to cells exogenously it is internalized and specifically translocated to nucleoli probably through a putative nuclear localization signal located between residues 27 and 32 (10).
Production of multiple FGF-2 isoforms with different cellular localizations raises the possibility that each form may exert a specialized function. The internalized 18-kDa form has been shown to increase RNA polymerase I transcriptional activity (12). Furthermore, the intracellularly stored forms may participate in intracrine regulations leading to some of the broad biological effects exerted by FGF-2 (2,5,13,14). For instance, FGF-2-transfected cells expressing only the nuclear-translocated high molecular weight forms grow in the absence of serum and display the properties of immortalized cells through still unknown mechanisms (5,13). The cytosolic 18-kDa form appears also to exert some control at the intracellular level (1,13). However, the specific biological functions of the multiple FGF-2 isoforms and their mechanism of action still remain to be defined.
The biological effects of exogenous FGF-2 are mediated by high and low affinity receptors (3,4). Four FGFR cDNAs, encoding high affinity receptors possessing tyrosine kinase ac-tivity, have been identified and cloned. Different variants of FGFR-1 to 3 arising from alternative splicing of the primary transcript have also been described (3,4). There are also low affinity receptors (K d in the nM range) corresponding mainly to heparan sulfate proteoglycans (15)(16)(17). Cooperation between high and low affinity receptors plays an essential role in the induction of the biological effects of the extracellular FGF-2 (3,4,16).
High affinity FGFRs are down-regulated by exogenously added FGF-2 (18 -20), and cells transfected with FGF-2 cDNA show decreased levels of high affinity FGFR. This may be due to down-regulation induced by secretion of the 18-kDa form. Transfected cells treated with suramin, an inhibitor of FGF binding to the cell surface receptors, exhibit higher binding capacities than untreated cells, further suggesting that secreted FGF-2 may down-regulate FGFR (19,20). However, it is not known whether high molecular weight intracellular forms of FGF-2 are involved in FGFR modulation. Accordingly, in the present study we examined the respective roles of the 18-and 22.5-kDa FGF-2 forms on the expression of the type I high affinity FGF receptors (FGFR-1). We used a retroviral infection system previously shown to induce the production of low amounts of FGF-2 in the endothelial cell lines ABAE (13) and in the pancreatic acinar cell line AR4-2J (21). AR4-2J cells do not produce FGF-2 (21) and possess low levels of FGFR (22). These cells were induced to stably express the two molecular forms of FGF-2 under the control of the ␤-actin promoter. We now report that both FGF-2 molecular forms up-regulate the high affinity receptors and the levels of the FGFR-1 mRNA.

EXPERIMENTAL PROCEDURES
Cell Culture-Parental and transfected AR4-2J cells were maintained in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose (Life Technologies, Inc., Eragny, France) and supplemented with 10% fetal calf serum (Life Technologies, Inc.). Trypsin (0.05%), EDTA (0.02%) (Life Technologies, Inc.) was used to dissociate the cells at successive passages. Medium was replaced every 2 days. The cultures were regularly checked for the absence of contamination by mycoplasma. All the experiments were carried out at different passages after transfection, between the 6th and 30th and no changes in the different parameters were found. Cell number was determined with a Coulter counter (Coultronics, Margeny, France).
Transfections and Infections-The retroviral infection of AR4-2J cells was described previously (21). Briefly, the retrovirus packaging cells CRIP (23) were transfected with point mutated FGF-2 cDNAs and the replication-defective virus produced were used to infect AR4-2J cells. All Geneticin-selected clones issued from the same infection were shown to possess identical morphology by light and electron microscopy analysis, and to synthesize similar amounts of pancreatic secretory enzymes (21). Several clones issued from each transfection were used. Control CAT clones were obtained after infecting AR4-2J cells with the retroviral vector containing the chloramphenicol acetyltransferase reporter gene. They did not produce detectable amounts of any FGF-2 isoform. A3 clones were transfected with the FGF-2 cDNA point mutated at the level of a CUG initiation codon mutated to AUG. They synthesized only the 22.5-kDa FGF-2 form, at a concentration of about 2 ng/10 6 cells. A5 clones were transfected with the FGF-2 cDNA containing a stop codon upstream to the AUG initiation codon. They synthesized only the 18-kDa form, at a concentration of about 0.5 ng/10 6 cells.
Binding of 125 I-FGF-2-Cells were seeded in triplicate at a density of 60,000 cells/cm 2 in 35-mm dishes and washed three times with PBS the next day prior to initiating binding studies (15). Cells were treated 2 min with PBS containing 20 mM sodium acetate, pH 4. After three new washes with PBS, cells were incubated 30 min at 37°C in serum-free Dulbecco's modified Eagle's medium buffered at pH 7.4 with 25 mM Hepes, containing 0.2% gelatin. Cells were then washed with cold PBS and incubated at 4°C in 1 ml of Krebs-Hepes buffer, pH 7.4, containing 0.2% gelatin, 0.3 mg/ml trypsin inhibitor (Sigma), 0.5 mg/ml bacitracin (Sigma), and 125 I-FGF-2 (specific activity in the range of 2200 cpm/fmol, DuPont NEN, Les Ulis, France) at the desired concentrations. Cells were incubated 4 h at 4°C on an orbital shaker, washed three times with the incubation buffer, and then washed with 2 M NaCl in 25 mM Hepes, pH 7.4, at 4°C. NaCl fractions (corresponding to the low affinity binding sites) and cell lysates prepared with 1 M NaOH (corresponding to the high affinity binding sites) were counted for radioactivity. Nonspecific binding was determined in the presence of unlabeled recombinant FGF-2 (500 nM). Cells were counted in parallel dishes. Ligand saturation and competition binding data were analyzed with the LI-GAND program.
Cross-linking of 125 I-FGF-2-Following incubation with 125 I-FGF-2 (30 pM) in the presence or absence of cold FGF-2 (500 nM) as described above, cells were washed at 4°C, and then incubated at 23°C, in PBS containing 0.5 mM bis(sulfosuccinimidyl)suberate (Sigma). After 30 min, the cells were lysed in 2 ϫ electrophoresis sample buffer and subjected to SDS-PAGE analysis on a 6.5% resolving gel. The gels were dried and exposed to Kodak X-Omat AR films (Rochester, NY), at Ϫ70°C.
Effect of Cell Density, Exogenous FGF-2, and Suramin on 125 I-FGF-2 Binding-Cells were seeded in 35-mm dishes at increasing densities from 60,000 to 160,000 cells/cm 2 , in the presence of 10% fetal calf serum. Binding experiments were performed 24 h later as described above. To assess the effect of exogenous FGF-2 on 125 I-FGF-2 binding, cells were incubated for 3 h at 37°C in medium containing 10% fetal calf serum in the presence of increasing concentrations of the recombinant 18-kDa FGF-2. Cells were then washed extensively at pH 4, to remove bound FGF-2, prior to use in binding studies.
To assess the effect of neutralizing anti-FGF-2 antibodies or suramin on 125 I-FGF-2 binding, 24 h after plating cells were incubated in fresh serum-free medium containing 0.3 g/ml of a monoclonal anti-FGF-2 antibody (UBI, Lake Placid, NY) or in 10% fetal calf serum containing 40 M suramin (gift of Bayer Pharma, Sens, France). Based on cell detachment, this suramin concentration was found to be the maximal nontoxic concentration. Binding studies were then carried out 24 h later.
RNA Extraction and mRNA Determination-Total RNA was prepared by the guanidinium isothiocyanate method and the poly(A) ϩ fraction was separated by using oligo(dT) columns. Quantification of RNA was made spectrophotometrically at 260 nm. Northern blot analysis was performed as described previously (24). The FGFR-1 probe pCD115 (25) was a human cDNA encoding the extracellular domain (gift from Dr. M. Jaye, Rorer Central Research, Inc., King of Prussia, PA). FGFR-2 and -3 probes were provided by Dr. F. Bayard (INSERM U397, Toulouse, France) and FGFR-4 probe was provided by Dr. K. Alitalo (Cancer Biology Laboratory, University of Helsinki, Finland). Nylon sheets were prehybridized for 6 h at 42°C in 50% formamide, 1% SDS, 5 ϫ Denhardt's, 6 ϫ SSC, 5 mM EDTA, 100 g/ml salmon sperm DNA. Hybridizations were carried out at 42°C in the same medium in the presence of the 32 P-labeled probe. Blots were washed 2 ϫ 30 min at 55°C in 0.1 ϫ SSC and 0.5% SDS, then dried and exposed to Kodak X-Omat AR films at Ϫ70°C. Bands were quantified by an image analyzer (Biocom, Les Ulis, France).
To determine FGFR mRNA half-life, cells were grown for the indicated times in the presence of 5 g/ml actinomycin D (26). Poly(A) ϩ RNA fractions were then extracted, electrophoresed under denaturing conditions, and hybridized as described above. The amount of radioactivity of each band was quantified by using a PhosphorImager (Molecular Dynamics, Sunnyvale, Ca). The glyceraldehyde-3-phosphate dehydrogenase mRNA was analyzed in parallel.
Confocal Analysis of FGFR-1-FGFR-1 was visualized by an indirect immunofluorescence method on cells cultured on coverslips and fixed in situ. Cells were fixed with freshly prepared paraformaldehyde (3% w/v) in 100 mM cacodylate buffer at room temperature and then permeabilized 5 min in 50 mM Tris-HCl buffer, pH 7.6, containing 0.25% Triton X-100. A polyclonal antibody raised against a peptide sequence present in the acidic box of the chicken FGFR-1 (UBI) was used. This synthetic peptide of 26 amino acids began at position 120 from the starting methionine, in the region where FGFR-1 share many differences in the amino acid composition with respect to the other FGFR (4). Cells were incubated overnight at 4°C with the rabbit polyclonal antibody (1/250) in 50 mM Tris-HCl, pH 7.6. After 4 washes, the antigen-antibody complexes were revealed with fluorescein isothiocyanate-conjugated antirabbit ␥-globulins (1/80) (Institut Pasteur Production, France) at room temperature for 30 min. Rinsing, media and immunoserum dilutions contained 0.1% bovine serum albumin.
For control experiments, cells were incubated: (i) with anti-FGFR-1 antibodies preadsorbed with the synthetic peptide used for immunization, (ii) with fluorescein isothiocyanate-conjugated anti-rabbit ␥-globulins (1/80) in the absence of the primary antibody. The specificity of the antibody was checked by immunoprecipitating cell extracts in the presence of increasing concentrations of the immunizing peptide and pro-tein A-Sepharose. The cross-linking of the immunoprecipitates with 125 I-FGF-2 was followed by SDS-PAGE separation and autoradiography. As expected, one single band of about 165 kDa was observed corresponding to the receptor and the intensity of the band decreased with increasing peptide concentrations.
To evaluate the FGFR-1 distribution in the different cell compartments, confocal laser scanning microscopic studies were performed using a LSM10 Carl Zeiss microscope equipped with a 63 ϫ objective. Fluorescein-stained cells were illuminated with the 488 nm line of the Argon laser. Optical sections of 0.1 m were collected and analyzed. Each section was scanned eight times. Color pictures from screen images were taken on Ektachrome Kodak iso/100 films. 10 -15 cells from three independent experiments were analyzed.
RT-PCR of the FGFR-1 Receptor-Total RNA, previously heated 5 min at 90°C, was reverse transcribed at 42°C in the presence of 1 mM each dNTP, 1 unit/l RNasin (Promega, Charbonnières, France), 0.25 g/l oligo(dT), and 2 units/l Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The RT buffer contained: 50 mM KCl, 20 mM Tris-HCl, pH 8.4, 2 mM MgCl 2 , and 1 mg/ml nuclease-free bovine serum albumin. After a 1-h incubation the reaction was stopped at 95°C, 10 min. Amplification was then carried out in a final volume of 100 l, with 1.5 g of cDNA 2.5 units/l Taq polymerase (Perkin Elmer) in the same buffer and 100 pmol of each oligonucleotide primer. All reactions were performed for 30 cycles with cycling times of 4 min at 94°C, 1 min at 69°C, and 2 min at 72°C. Primers were synthesized by Eurogentec (Liège, Belgium). The sequences of the primers were chosen on the basis of the cDNA sequence of the rat FGFR-1 (27). Two sets of primers were used. The first one (SAS1) was 5Ј-CCTCTTCTGGGCT-GTGCTGGTCA-3Ј and 5Ј-TCCAGGTACAGAGGTGAGGTCATC-3Ј, covering the extracellular domain of the FGFR-1 from nucleotide 21 to 1127. The second one (SAS2) used for the amplification of the membrane-spanning and the intracellular domains from nucleotide 1075 to 2028, was 5Ј-CTGGAAGCCCTGGAAGAGAGACC-3Ј and 5Ј-GATCCG-GTCAAACAATGCCTCAGG-3Ј. The products of PCR amplifications were electrophoresed on a 5% polyacrylamide gel and stained with ethidium bromide (1 mg/ml).
Sizes of amplified products corresponded to those expected according to the position of the primers in the FGFR-1 cDNA sequence. The specificity of the PCR products was also checked by using restriction enzymes, selected according to the sequence of the rat FGFR-1 cDNA using computer analysis (PC/GENE, IntelliGenetics, Mountain View, CA). After gel electrophoresis, bands were extracted with the Gene-Clean II kit (BIO 101 Inc., Vista, CA), digested with restriction enzymes. BstEII was used for the SAS1 and XhoI for the SAS2 amplified products. Both enzymes were selected for their ability to digest each sequence at only one selected restriction point. The two enzymes gave rise to two fragments at the expected sizes. As negative controls we used H 2 O to replace RNA during RT-PCR and the restriction enzyme EcoRV which was unable to digest the PCR products according to the cDNA sequence.
Calcium-and Phospholipid-dependent Protein Kinases C Activity-The calcium and phospholipid-dependent PKC activity was assayed as described previously (28), by measuring the incorporation of 32 P from [␥-32 P]ATP into histone H1. A control assay was performed in the absence of diacylglycerol (diolein) and phosphatidylserine. The soluble and particulate cell fractions containing the PKC activity were obtained after a 100,000 ϫ g centrifugation followed by chromatography on a ion exchange DE52-cellulose column and the elution by 100 mM NaCl (28). PKC activity is determined by subtracting the activity measured in the absence of diacylglycerol and phosphatidylserine from that measured in the presence of both cofactors and expressed as picomoles of 32 P incorporated into histone H1 per min and per mg of protein.

Up-regulation of FGFR in FGF-2-expressing Cells-
The time course of 125 I-FGF-2 binding at 4°C was analyzed first. Association kinetics were similar in all the cell lines used and nonspecific binding was usually in the range of 20% of total binding. Fig. 1 illustrates the time course of 125 I-FGF-2 binding association and its dissociation following addition of unlabeled FGF-2 (500 nM), in control CAT cells not synthesizing FGF-2, and in A3 cells producing the 22.5-kDa FGF-2 form. Specific binding was found to be higher in A3 than in CAT (Fig. 1) and A5 cells.
Equilibrium saturation binding experiments were conducted in order to analyze the high affinity receptors. Typical ligand saturation isotherms and Scatchard plots are shown in Fig. 2. Nonspecific binding increased linearly with increasing concentrations of unlabeled FGF-2. A 3-fold increase in maximal 125 I-FGF-2 binding capacity was observed in A3 cells and a 2-fold increase in A5 cells, compared to those of parental AR4-2J and CAT cells (Table I). Maximal number of high affinity binding sites per cell were around 1200 in mock cells, comparable to values reported for other cell types expressing low levels of FGFR (15). They increased to about 3700 in A3 cells. No significant modifications were detected in the K d values for the high affinity receptors (about 50 pM) among the different cell lines (Table I).
Competition binding experiments were carried out to analyze the low affinity sites, by using 125 I-FGF-2 and unlabeled FGF-2 or FGF-1. Typical competition curves are reported in Fig. 3. In all cell lines, FGF-2 inhibited 125 I-FGF-2 binding with a slight higher potency than that displayed by FGF-1. Scatchard analysis of the inhibition data ( Fig. 3 and Table I) revealed a greater binding capacity for FGF-2 only in A3 cells. K d values (about 2 nM) were comparable among the different cell lines and similar to the data of the literature (15). Experiments performed with three different clones gave identical results (not shown).
Hill plot analysis of the experimental data obtained for both high and low affinity sites indicated that Hill coefficients were not far from 1, suggesting that there was no cooperativity between these two classes of receptors in any cell line.

FGF-2 Expression Did Not Modify the Apparent Molecular Weight of the High Affinity Receptors-
The binding data indicated that the FGFR levels were increased in FGF-2-expressing cells. We next performed cross-linking experiments with 125 I-FGF-2 in order to evaluate the size of the up-regulated FGFR. A single band with an apparent molecular mass of 165 kDa was observed in all cell lines (Fig. 4). Taking into account the presence of the recombinant 18-kDa 125 I-FGF-2 in the receptor-FGF-2 complex, the molecular mass of the receptor could be estimated at around 145 kDa. Unlabeled FGF-2 completely inhibited receptor labeling (Fig. 4, lane NS). Densitometry of the autoradiograms revealed a 1.5-fold increase in FGF-2 receptors in A5 cells and a 3-fold increase in A3 cells, in agreement with the data obtained by binding studies. These results confirm the up-regulation of the cell surface receptor number and demonstrate that the overexpressed receptors dis- play the same molecular weight than those present in CAT cells.
The High Affinity Receptors Are Still Down-regulated-Since increased cell density and extracellular FGF-2 are both known to induce down-regulation of the FGFR (20, 29 -30), we investigated whether cells transfected with FGF-2 exhibited an altered capacity to down-regulate FGF binding. For these studies A3 cells were utilized as they expressed the highest receptor level. CAT and A3 cells were plated at increasing densities and binding studies were performed 24 h later in order to avoid the influence of cell aging. Maximal binding capacity was found to be decreased by 46 and by 48% in A3 and mock cells, respectively, at the highest cell density (not shown).
The effect of exogenous FGF-2 was analyzed by incubating CAT and A3 cells at 37°C for 3 h with increasing concentra-tions of FGF-2 (19). Ligand binding was carried out after extensive washings with 2 M NaCl at pH 4.0 to remove the FGF-2 bound to the cells. Cell morphology after these washings was found unmodified. A dose-dependent decrease in specific binding was observed with IC 50 of 0.1 nM for A3 and 0.7 nM for CAT cells (not shown). Thus, endogenous FGF-2 expression did not modify the process of receptor down-regulation.
To analyze the effects on FGF receptor levels of the FGF-2 eventually secreted by transfected AR4 -2J cells, cells were grown in the presence of 30 g/ml of a neutralizing anti-FGF-2 antibody or 40 M suramin, which is known to reduce the FGFR internalization by interacting with the extracellular FGF-2 (19,20). Both suramin (Fig. 5) and anti-FGF-2 (not shown) did not alter binding in CAT and A3 cells and caused a further 80 and 60% increase, respectively, in A5 cells. Thus, the 22.5-kDa peptide remains in the intracellular compartments whereas as   FGF-2 Expression Increases the FGFR-1 mRNA Levels-In order to determine whether receptor up-regulation was related to an increase in levels of FGFR transcripts, we first identified the high affinity FGFR subtypes expressed in AR4-2J and transfected cells. FGFR-1 has been found in human exocrine pancreas (31). Northern blot analysis of the poly(A) ϩ fraction with specific probes revealed the presence of FGFR-1, FGFR-2, and FGFR-3 mRNAs of about 4.5, 4.4, and 4.5 kilobases, respectively (Fig. 6). The expression of FGFR-4 gene was undetectable. Only the FGFR-1 transcript was found increased, by 3-fold in A5 and 6-fold in A3 cells after correction of the poly(A) ϩ loaded, compared to control CAT cells (Fig. 7).
The High Molecular Weight FGF-2 Increases the FGFR-1 mRNA Stability-Since the results observed for FGFR-1 mRNA could be the consequence of a control exerted by FGF-2 on the stability of the transcript, we analyzed the half-life of the FGFR-1 in A3 cells expressing the higher level of the transcript. Cells were treated with actinomycin D for different times and the levels of the FGFR-1 poly(A) ϩ mRNA were determined. As shown in Fig. 8, Northern blots followed by the quantification of the radioactivity present in the different bands, revealed that the mRNA half-life was about 135 min for CAT cells and increased to about 375 min in A3 cells. These data suggest that at least the 22.5-kDa isoform increased the messenger level by acting on its stability.
To determine whether the FGFR-1 mRNA regulation occurred also at the transcriptional level, some nuclear run-on transcription assays were performed in CAT and A3 cells. While lipase transcription (performed as control) was clearly detectable in both cell lines, that of the FGFR-1 was too low to be quantified.
Confocal Analysis of the FGF Receptor Type 1-In order to localize the receptor protein, indirect immunofluorescence analysis by confocal microscopy was performed in the rat parental AR4-2J and transfected cells with anti-FGFR-1 antibodies. Immunofluorescence was found at the plasma membrane level in AR4-2J cells (Fig. 9) as in all transfected cells. The intracellular compartments appeared negative in the different planes, some of them crossing the nucleus. Similarly, no fluorescence could be observed in the extracellular spaces, suggesting the absence of secretory receptor isoforms.
RT-PCR Analysis of the FGFR-1 Variants Expressed by the FGF-2-producing Cells-Only one spliced form of FGFR-1 mRNA was detected by Northern blot. In order to see if FGF-2 also induced the expression of some other FGFR-1 variants, the more sensitive RT-PCR technique was used. The specificity of the amplification products was checked by using restriction enzymes able to digest the rat FGFR-1 cDNA at only one site and on the opposite, with restriction enzymes unable to digest the cDNA. Amplification of the cDNA encoding for the extracellular region of the receptor gave rise to two fragments of about 1100 and 800 bp in all cell lines (Fig. 10, left), corresponding to the isoforms possessing, respectively, 3 and 2 Ig-like domains (4). The 1100-bp cDNA was approximately 10 times more represented than the shortest form.
To study the intracellular domain of FGFR-1, the nucleotide sequence 1075-2028 containing the transmembrane region was amplified (Fig. 10, right). An amplification product of about 950 bp was obtained in all cell lines, corresponding to the expected size of the membrane-spanning and the intracellular domain. Some partially spliced forms of the primary transcript were observed in the extracellular domain of the receptor in A3 cells and also in the intracellular domain, in all cell lines (at 2100 bp). In AR4-2J cells, immature forms of some other mRNA have already been observed. 2 Thus, PCR amplifications revealed a FGFR-1 variant containing 2 Ig-like domains. According to the whole data the transfected cells did not appear to display major modifications in the expression of the FGFR-1 spliced variants.
Translocation of the Ca 2ϩ -Phospholipid-dependent PKC by Exogenous FGF-2 in FGF-2-expressing Cells-We subsequently examined whether the overexpressed receptors were functionally coupled to intracellular signaling pathways. For that purpose we followed the translocation of the PKC, one of the enzymes involved in some effects of this growth factor (3,4). Basal PKC activities were: 58 Ϯ 9.5 pmol of 32 P incorporated/ min/mg of protein in the cytosolic fraction for CAT cells (n ϭ 7), 41.3 Ϯ 2.3 for A3 cells (n ϭ 5), and 47 Ϯ 4.59 for A5 cells (n ϭ 8). These differences were not statistically significant. PKC activities in the particulate fractions were also similar in CAT and FGF-2-transfected cells. After FGF-2 addition to serumfree culture media at the concentration of 1 nM, PKC activity increased by 60% in the particulate fraction in A3 cells within 30 min (Fig. 11). PKC translocation was only increased by 20% in CAT and A5 cells (which overexpressed only the high affinity receptors). Thus, PKC activation is only significant in A3 cells which overexpress both high and low affinity receptors. DISCUSSION FGF-2 is synthesized as different molecular weight isoforms lacking the signal peptide sequence for secretion. The high molecular weight forms possess a nuclear-targeting sequence. All these peptides are concentrated in the cells. The multiple biological functions of FGF-2 have been suggested to result from the cooperation of these isoforms acting at specific cell compartments. The 18-kDa form can be secreted and the subsequent activation of the FGF receptors-tyrosine kinase elicits the cascade of intracellular events (3,4). The functions of the intracellular isoforms, including the non-secreted 18-kDa peptide, are still unknown. Previous studies reported that cells transfected by plasmidic vectors containing the FGF-2 cDNA expressed high amounts of FGF-2 and exhibited low levels of high affinity FGF receptors (19,20). Secretion of the 18-kDa form in the extracellular space has been suggested to be responsible for receptor internalization (18 -20). By contrast, in human malignant tissues, such as pancreatic cancers, glioma, and other brain tumors, up-regulation of high affinity FGFR concomitantly with an increased production of FGF-2 has been reported (31)(32)(33).
Transfected cells often express very high levels of FGF-2 (for instance in the range of 75-600 ng of FGF-2/million cells) (19,20), compared to normal and tumor cells (34). In these transfected cells, the FGFR down-regulation induced by secreted FGF-2 might mask the increased receptor biosynthesis by the FGF-2 forms localized in the intracellular compartments. Therefore, we used a retroviral infection system to obtain the production of lower levels of FGF-2 (about 0.5-2 ng/million cells) (13,34) and we chose a pancreatic acinar cell line (AR4-2J) in which cell differentiation can be easily determined by morphological analysis and by measuring the biosynthesis rate of the secretory enzymes (24). Inasmuch as AR4-2J cells do not produce FGF-2 (21), the biological effects resulting from the induction of low level expression of each isoform can be more easily analyzed. In contrast to cells overexpressing FGF-2, 2 M. Svoboda, personal communication. The RT-PCR was performed as described under "Experimental Procedures" with two sets of primers. The amplified products were separated by electrophoresis in a 5% polyacrylamide gel and visualized by staining with ethidium bromide. Left, the amplification products of the extracellular region obtained with the primer pair SAS1. Two bands of about 1100 (the major band) and 800 bp were obtained. Right, the cytosolic sequence containing the membrane-spanning domain was amplified with the primer pair SAS2. Two PCR products evaluated at about 2100 and 950 bp were obtained. 1, CAT; 2, A5; 3, A3. 1.5 g of the cDNA obtained by the reverse transcriptase were used for PCR amplifications. retroviral-infected A3 and A5 cells did not show any modification in cell morphology or differentiation state, biosynthesis and secretion of digestive enzymes, and cell regulation by glucocorticoids (21). On the other hand, by using this infection system identical properties are often observed in the different cell clones (35) as we indeed observed among the different clones isolated for each FGF-2 infection (21).
The present study provides evidence that the low level expression of either the cytosolic 18-kDa or the nuclear-targeted 22.5-kDa FGF-2 isoform increased the cell surface high affinity receptors, by 2-and 3-fold, respectively. By contrast, exogenously added FGF-2 exerted opposite effects resulting in FGFR down-regulation. Only the 22.5-kDa peptide was found to up-regulate the low affinity binding sites. Binding experiments performed on other clones expressing the FGF-2 peptides confirmed the above results. In control CAT cells, maximal binding capacities of high and low affinity receptors were identical to those of parental cells, indicating that the vector did not contribute to the modifications observed. Thus, the increase in the high affinity receptors reported in the present study on cultured cells resemble that which was published on tumor cells in vivo (31)(32)(33). In all cell lines the affinities of both classes of receptors remained unchanged as well as the greater affinity for FGF-2 compared to FGF-1.
Altering the cell density and adding exogenous 18-kDa FGF-2 down-regulated the high affinity receptors as in control cells. Cell treatment by neutralizing anti-FGF-2 antibodies and by suramin did not increase the high affinity binding sites on control and A3 cells expressing the nuclear-translocated isoform. A5 cells which synthesize the secretory 18-kDa FGF-2 molecular form (36), displayed as expected, a further increase (about 60 -80%) in the cell surface binding sites. On the other hand, confocal analysis of A3 cells with anti-FGF-2 antibodies confirmed that the 22.5-kDa form was localized in the nucleus and was absent at the cell surface and in the extracellular space (data not shown). Taken together these data suggest that the transfected FGF-2 up-regulated the high affinity receptors by acting at a level other than the down-regulation process.
FGF-2-producing cells and mock cells shared comparable cell diameters and doubling times (21), hence these parameters should do not play a role in receptor up-regulation. On the other hand, modifications of high affinity binding sites during cell differentiation have been reported both in vivo (37) and in vitro (38) in the absence of significant modulations of FGFR-1 mRNA levels (20,38). By contrast, morphological and biochemical data showed that the differentiation state of A3 and A5 cells was strictly comparable to that of control cells (21) and as reported below, FGFR-1 mRNA levels were found increased in the present study. These data suggest that cell differentiation is not responsible for the receptor modulation.
Although mRNAs encoding for FGFR-1, -2, and -3 were observed in all the cell lines, only the level of the FGFR-1 transcript was found increased, suggesting that FGF-2 exerts a positive control on the expression of this receptor. Cells producing the secretory 18-kDa form expressed lower levels of FGFR mRNA compared to A3 cells (a 3-fold increase in A5 cells against a 6-fold increase in A3 cells). This finding was the opposite of what was expected if the mRNA induction occurred via the activation of cell surface receptors. Therefore, these data further suggest that the receptor up-regulation did not occur through the activation of the cell surface receptors but rather through an intracrine mechanism. Similar conclusions were recently reached concerning proliferation of cells expressing the high molecular weight isoforms. Indeed, by using dominant negative FGF receptors it has been shown that proliferation was independent from the activation of the cell surface receptors (39).
We chose A3 cells to analyze the FGFR-1 mRNA stability since the 22.5-kDa form is not secreted leading therefore to an easier interpretation of the results obtained because of the absence of any receptor occupancy. A 3-fold increase in the FGFR-1 mRNA half-life was found compared to control cells. These data show first that the 22.5-kDa form is able to exert a control on the stability of the mRNA transcript(s), second that the increase in FGFR-1 mRNA level occurred at least in part, through a modulation of its half-life. The 22.5-kDa FGF-2 although chiefly concentrated in the nucleus, is also localized in the cytoplasm close to the endoplasmic reticulum (14), suggesting some possible function at the cytoplasmic level. However, whether the control exerted on the mRNA occurs at the cytoplasmic level remains to be confirmed. The existence of a control on mRNA stability by the 22.5-kDa isoform which has not previously been reported, opens new avenues for investigating the molecular mechanisms whereby this FGF peptide exerts its effects.
Immunofluorescent staining and confocal analysis revealed the presence of the FGFR-1 subtype at the cell surface of AR4-2J and transfected cells and did not detect a secretory FGFR-1 isoform in the extracellular space. PCR analysis revealed in all cell lines the FGFR-1 isoform containing the 3 Ig-like domains. However, it also revealed in all cell lines a mRNA species encoding the variant with 2 Ig-like regions, undetected by Northern blots. The corresponding protein was not observed by cross-linking studies, raising the possibility that this mRNA species was probably untranslated. Thus, the present data did not show any important modification of the FGFR-1 variant expressed in FGF-2-transfected cells. The higher affinity of the FGFR for FGF-2 than for FGF-1 in all cell lines, agrees with the presence of the IIIa sequence in the mRNA region encoding the third Ig-like domain (4), irrespective of the FGF-2 isoform expressed. The in vivo relevance of the up-regulation of the FGFR-1 could be suggested taking into account that the different FGF-2 isoforms are indeed produced by the same cell. The biosynthesis of each of them is under specific controls inducing preferentially the initiation of translation at the AUG or CUG codons (10). According to the predominant expression of the 18-kDa or the 22.5-kDa isoform, either the high affinity receptor or both high and low affinity receptors will be up-regulated. Thus, the endogenous isoforms appear to cooperate with the regulations evoked by exogenous FGF-2 and to amplify cell responses. The increase of the PKC translocation in A3 cells after exogenous FGF-2 stimulation, agrees with that hypothesis. In addition to the control exerted by FGF-2 isoforms at FGFR-1 level, it will be of biological relevance to analyze also their regulations on the different second messenger levels involved in the transduction pathway evoked by exogenous FGF-2.