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Laboratoire d’Explorations Fonctionnelles Endocriniennes (N.B., C.G., A.L., R.C., Y.L.), Hôpital Trousseau, 75012 Paris, France, and INSERM Unité U515, Hôpital Saint-Antoine, 75012 Paris, France; INSERM Unité 244 (J.-J.F.), DBMS/BRCE, CEA/Grenoble, 38054 Grenoble cedex 9, France
Address all correspondence and requests for reprints to: Nathalie Boulle, Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hôpital Trousseau, 26 Avenue Arnold NETTER, 75012 Paris, France. E-mail: lab.endoc{at}trs.ap-hop-paris.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various growth factors and cytokines other than IGFs have been shown to
regulate adrenal growth and function in normal adult and fetal
adrenals. These include basic fibroblast growth factor (FGF-2),
transforming growth factor -
and -ß1 (TGF-; TGF-ß1),
epidermal growth factor (EGF), tumor necrosis factor- (TNF-), and
interleukins (7, 8, 9, 10). Of all of these factors, FGF-2 may be a prime
candidate to evaluate in adrenocortical tumor cells. This growth factor
is highly expressed in adrenal tissues and is one of the most potent
mitogens for cultured adult and fetal adrenal cells (10, 11, 12, 13, 14). In human
fetal adrenal glands, Mesiano et al. (15) showed a
cooperative mitogenic effect of IGF-II and FGF-2. Similarly,
interactions between FGF-2 and the IGF system have been described in
various cell models including muscle, chromaffin, hypothalamic, and
neuroblastoma cells (16, 17, 18, 19). Finally, FGF-2 is present in many tumor
cells and cell lines and has been shown to be involved in the
transformation and proliferation of various cell types (20, 21, 22, 23, 24).
Altogether, these observations suggest that FGF-2 may be an important growth factor for adrenocortical tumor cells and may interact with the IGF system in these cells.
In this study, we used the human H259R cell line as a model to examine whether FGF-2 was expressed in adrenocortical tumor cells and whether it affected their proliferation. Because the IGF system has an important role in this tumor model, we also studied the effects of FGF-2 on the expression of IGF-II and IGFBP-2 by H295R cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human FGF-2 was purchased from R&D systems (Minneapolis, MN) and recombinant IGF-II was obtained from Sigma (St. Louis, MO). Anti-IGFBP-2 antiserum, anti-IGF-II monoclonal antibodies and anti-FGF-2 (type II) monoclonal antibodies were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). 125I-IGF-I and 125I-IGF-II were produced in the laboratory by iodination of recombinant IGF-I and IGF-II (kindly supplied by Ciba-Geigy, Basel, Switzerland), using the chloramine-T method (25). The human IGFBP-2 complementary DNA (cDNA) probe was kindly provided by Dr. S. Babajko (INSERM U 515, Paris, France) (26). The human IGF-II cDNA probe was cloned in the laboratory (27). Hybond XL membranes for northern blotting were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK).
Culture medium [1:1 mixture of DMEM and Ham’s F12 medium (F12)], transferrin and sodium selenite were purchased from Sigma. L-glutamine and penicillin were obtained from Life Technologies, Inc. (Paisley, Strathclyde, UK) and ultroser G from Biosepra (Marlborough, MA). Trypsin-EDTA was purchased from Difco (Detroit, MI). Cultures dishes were obtained from Nunc (Roskilde, Denmark). [3H]-thymidine (specific activity 83 Ci/mmol) was supplied by Amersham. Brefeldin A was obtained from Calbiochem (La Jolla, CA).
The BCA protein reagent assay was purchased from Pierce Chemical Co. (Rockford, IL).
Reagents for SDS/PAGE were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA). The ECL Western blotting detection system was purchased from Amersham.
Methods
Cell cultures. NCI H295R cells were grown in T75 flasks in a
1:1 mixture of DMEM/F12 medium supplemented with transferrin (5
µg/ml), sodium selenite (5 ng/ml), L-glutamine (2.5
mM), penicillin (50 IU/ml), and 2% Ultroser G at 37C in a
5% CO2 atmosphere. Confluent cells were
trypsinized using 0.05% trysin-EDTA, washed three times with PBS, and
seeded in the same medium but without ultroser G (serum-free medium) in
78 cm2 dishes (7 x
104 cells/cm2). After a
72-h incubation, the medium was renewed (day 0 of the experiment) and
replaced with serum-free medium without (control) or with various
concentrations of FGF-2 as indicated in the figures. The medium was
then renewed with or without FGF-2 on days 2 and 4 of culture. After 7
days of culture, cells were counted and the conditioned media were
collected for IGF-II and IGFBP assays and stored at -20 C until
analyzed. For kinetic analysis, the same procedure was used except that
cells were counted and the medium collected after various times of
culture as indicated in the figures.
To study the effects of brefeldin A (BfA), cells were grown with or without FGF-2 as described previously except that brefeldin A (1 µg/ml) was added on day 4 of culture. Because BfA had toxic effects if used for prolonged periods (>48 h), the incubation with BfA was stopped after 48 h (day 6 of the experiment), and cell extracts were prepared as indicated below.
[3H]-thymidine incorporation. For [3H]-thymidine incorporation, cells were seeded in serum-free medium in 96-well dishes (105 cells/cm2). Cells were grown with or without various concentrations of FGF-2 as described above and 2.5 µCi/ml [3H]-thymidine were added for the final 72 h of culture. Cells were then rinsed three times with PBS and lysed for 3 h at 37 C using 100 µl 0.6 M NaOH. Fifty microliters of the cell suspension was counted in a scintillation counter to determine the amount of radioactivity incorporated into DNA.
Cell extracts. Cells grown in 78 cm2 dishes (25 to 40 x 106 cells) were rinsed four times with PBS and scraped into ice-cold RIPA buffer (50 mM Tris-HCl pH 8, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS; 500 µl per dish) containing protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM PMSF). After a 15-min incubation at 4 C, cell lysis was completed by three freeze-thaw cycles, after which cell lysates were centrifuged at 100,000 x g for 30 min at 4 C. The protein content of the supernatants was determined by the BCA protein assay. Cell extracts were then stored at -80 C until use. Protein determination in various experiments indicated that 200 µg of protein extract from H295R cells treated or not treated with FGF-2 correspond to about 2.4 x 106 cells.
For FGF-2 analysis, nuclear extracts and cytosol were prepared as previously described (28).
Immunoblotting. The conditioned media were desalted on Sephadex G-25 columns, lyophilized, and submitted to SDS-PAGE under nonreducing conditions (11% acrylamide for IGFBP-2 and 15% acrylamide for IGF-II). The amount of conditioned medium loaded on the gel was adjusted to obtain the same cell number for each condition (4 x 105 cells equivalent for IGFBP-2 analysis and 2 x 106 cells equivalent for IGF-II analysis). For cell extract analysis, the same amount of protein was loaded for the different conditions (200 µg protein for most experiments). After electrophoresis, the proteins were electrotransferred onto nitrocellulose, which was then incubated overnight at 4 C with anti-IGFBP-2 antiserum (1/2000) or anti-IGF-II monoclonal antibodies (1/500). After incubation with a second anti-IgG antibody coupled to horseradish peroxidase, the complexes were visualized by chemiluminescence (ECL system).
FGF-2 immunoblotting was carried out in the same conditions (15% SDS-PAGE) except that electrophoresis was performed under reducing conditions. The monoclonal FGF-2 antibody was used at a dilution of 1/200.
Western ligand blotting. Western ligand blotting was performed as previously described (29). Briefly, nitrocellulose membranes were prepared as for IGFBP-2 immunoblotting and incubated with a mixture of 125I-IGF-I and 125I-IGF-II (5 x 105 cpm each) at 4C for 48 h. The membranes were then rinsed and autoradiographed at -80 C for 3 to 5 days.
RNA extraction and northern blotting. Total RNA was extracted from H295R cells by the guanidium thiocyanate extraction method (3). Total RNA (15 µg) was size-fractioned on a 1.2% agarose formaldehyde gel, transferred to Hybond XL membranes, and hybridized as previously described with 32P-labeled cDNA probes for h-IGF-II and h-IGFBP-2 (30). The results obtained for IGF-II and IGFBP-2 messenger RNAs (mRNAs) were normalized with the corresponding 28S RNA detected by ethidium bromide staining of the gel.
Densitometry. Western ligand blots, immunoblots, and Northern blots were analyzed by scanning with a GS700 imaging densitometer and the molecular analyst data system (Bio-Rad Laboratories, Inc., Richmond, CA).
Statistical analysis. Data in the text are expressed as means ± SD of at least three independent experiments. One way ANOVA was used for statistical evaluation of the data. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This antibody detected three major
bands of about 18, 22, and 24 kDa in whole cell extract and cytosol,
the 18 kDa FGF-2 isoform being predominant in the cytosol. In nuclei,
only the 22 and 24 kDa bands were detected and the 24-kDa band appeared
as a doublet. Thus, in H295R tumor cells, various isoforms of FGF-2 are
expressed in different subcellular compartments.
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). In H295R cells treated
with 10 ng/ml FGF-2 for 7 days, cell number increased by 38.2 ±
5% (mean ± SD from six different experiments).
Similar results were obtained with
[3H]-thymidine incorporation. The effect of
FGF-2 was time dependent, the maximum effect occurring after 7 days of
culture (Fig. 2A). The mitogenic effect of FGF-2 was also dose
dependent, reaching a plateau around 1 ng/ml (Fig. 2B).
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Northern blot analysis of total RNA from H295R cells showed that FGF-2
had no significant effect on the levels of the different IGF-II mRNA
species (6, 4.8, and 2.2 kb) (Fig. 3A).
In contrast, major changes in the IGF-II protein were observed in
response to FGF-2, as shown by immunoblotting (Fig. 3, B and C).
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. Three immunoreactive IGF-II peptides were detected in extracts from
control H295R cells: a major band of 18 kDa apparent molecular mass and
two fainter bands in the 24- to 26-kDa range. A similar profile was
observed for H295R cells treated with FGF-2, but there was a marked
increase in the total amount of intracellular IGF-II protein (193
± 17% of control cells), mostly due to an increase in levels of the
18 kDa pro-IGF-II peptide (Fig. 3B).
Figure 3C shows the profile of secreted IGF-II protein. Various
immunoreactive IGF-II peptides were detected in conditioned media from
control H295R cells, the most abundant being mature IGF-II, about 7.5
kDa in size. Higher molecular mass forms of IGF-II were also detected,
with one group of peptides migrating around 24–26 kDa and another
group ranging in size from 14–18 kDa. In the presence of FGF-2, the
total amount of secreted IGF-II greatly diminished (45 ± 6% of
control cells). Mature IGF-II (7.5 kDa) and the 24–26 kDa pro-IGF-II
peptides became barely detectable. The decrease in secreted 14- to
18-kDa IGF-II was less pronounced, with a shift toward the higher
molecular mass forms (18 kDa) (Fig. 3C).
Thus in H295R cells, FGF-2 greatly decreased the levels of secreted IGF-II protein, particularly those of mature IGF-II, whereas it increased the levels of intracellular pro-IGF-II. This observation suggested that FGF-2 inhibited the correct engagement of pro-IGF-II into its secretory pathway.
The effects of FGF-2 on secreted and intracellular IGF-II protein were
both time and dose dependent (Fig. 4, A
and B). These modifications occurred between days 2 and 4 of culture,
being completed after 4 days of culture in the presence of FGF-2. On
day 4, high levels of pro-IGF-II of molecular mass 14 to 18 kDa were
secreted into the conditioned medium (Fig. 4A and data not shown). In a
dose-response experiment, FGF-2 effects were detected at a
concentration of 1 ng/ml FGF-2, and were maximal at a concentration of
10 ng/ml (Fig. 4B).
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). This suggests
that the low levels of the 7.5-kDa IGF-II peptide in presence of FGF-2
were not due to enhanced proteolysis of the growth factor by proteases
present in the conditioned medium.
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The intracellular levels of IGF-II detected by immunoblotting reflects
both production and secretion of IGF-II. Thus, under these conditions,
it is not possible to determine whether the increased amount of
intracellular IGF-II in FGF-2-treated cells results from increased
synthesis of the growth factor or from the inhibition of IGF-II
secretion resulting in accumulation of the protein in the cell. To
discriminate between these two hypotheses, we used brefeldin A (BfA) to
specifically inhibit protein secretion, so that intracellular IGF-II
levels in H295R cells treated or untreated with FGF-2, would only
reflect IGF-II synthesis (Fig. 5B).
In the absence of BfA, FGF-2-treated cells contained larger amounts of
intracellular IGF-II protein (+95%) than control cells, contrasting
with a decrease in secreted IGF-II (Fig. 5B). In cells treated with
BfA, no IGF-II was detected in the conditioned media and intracellular
IGF-II appeared as a doublet of apparent molecular mass 22–24 kDa,
suggesting inhibition of pro-IGF-II processing as previously described
(Fig. 5B) (31). In the presence of BfA, there was no significant
difference in the amount of intracellular IGF-II between control cells
and cells treated with FGF-2, indicating that FGF-2 did not increase
the synthesis of IGF-II protein in H295R cells. Thus, the increased
amount of intracellular IGF-II detected in the presence of FGF-2
probably reflects accumulation of the growth factor due to reduced
secretion rather than increased synthesis.
FGF-2 reduces IGFBP-2 expression
The effects of FGF-2 on the secretion of IGFBPs by H295R cells
were also examined (Fig. 6). As
previously described, western ligand blot analyses showed that control
H295R cells secreted almost exclusively a 34-kDa IGFBP identified as
IGFBP-2 by immunoblotting (3). No additional IGFBP was detected in
conditioned media from cells treated for 7 days with 10 ng/ml FGF-2.
However, there was a marked decrease in the amount of secreted IGFBP-2
(21 ± 7% of control cells) (Fig. 6A).
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), but rather to a decrease in the
levels of IGFBP-2 mRNA (33 ± 10% of control cells) (Fig. 6B).
These results suggest that in H295R tumor cells, FGF-2 reduces IGFBP-2
expression at the transcriptional level.
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) and was maximal for a concentration of 10 ng/ml FGF-2 (Fig. 7B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three isoforms of FGF-2 were detected in H295R cells by immunoblotting: the 18-kDa cytoplasmic isoform and two higher molecular mass forms of 22 and 24 kDa located in the nucleus. A similar profile has been described in bovine adrenocortical cells and in various cell models, including tumor cells (10, 24, 32). Various effects have been assigned to these isoforms of FGF-2: the 18 kDa form is secreted and promotes cell proliferation and migration after binding to FGF receptors, whereas the 22–24 kDa forms, targeted to the nucleus, are thought to act within cells, favoring cell transformation and growth in serum-free medium (24, 33). Although weaker, the mitogenic effect of FGF-2 on H295R cells was consistent with results obtained for normal adult and fetal adrenal cortical cells, with a similar dose-response curve for FGF-2 (10, 14). This proliferative effect occurred late (7 days of culture), but this may be due to the long doubling time of the H295R cell line (3). The proliferative response of H295R tumor cells to FGF-2 also suggested that these cells expressed functional FGF receptors.
We have previously shown that IGF-II is highly expressed in H295R cells and is an autocrine growth factor for these cells (3). It was therefore important to evaluate the effects of FGF-2 on IGF-II mRNA and protein levels. FGF-2 had no significant effects on IGF-II mRNA levels. The effects on IGF-II protein were more complex, with a marked increase in the levels of intracellular IGF-II precursors, contrasting with a marked decrease in the amount of secreted IGF-II. This suggested that in H295R cells, FGF-2 induced modifications of the processing of IGF-II protein.
Recent studies have characterized the various steps involved in the posttranslational processing of the 156-amino acid pro-IGF-II precursor (31, 34).
These include the glycosylation of four major Ser and Thr residues
(Ser71, Thr72,
Thr75, and Thr139) and
three sequential cleavages at Arg104,
Lys88, and Arg68 (Fig. 8). In the human embryonic kidney 293
cell line, the first cleavage, at Arg104, occurs
within the cell and involves a protease from the subtilisin-related
proprotein convertase (SPC) family (31). The next two cleavages leading
to mature 7.5 kDa IGF-II probably occur during or shortly after the
secretion of pro-IGF-II (1–104) (35). The apparent molecular masses of
glycosylated forms of pro-IGF-II-(1–156) and pro-IGF-II-(1–104) are
23–27 kDa and 17–18 kDa, respectively (31, 34). In H295R cells,
immunoblot analysis of intracellular IGF-II indicated the presence of
three IGF-II peptides: the 24- and 26-kDa bands probably correspond to
glycosylated forms of pro-IGF-II-(1–156) and the 18 kDa IGF-II peptide
to glycosylated pro-IGF-II-(1–104) (Fig. 8) (31, 34). Similar
molecular forms were observed in the presence of FGF-2, with a
predominant 18 kDa pro-IGF-II peptide suggesting that, in H295R tumor
cells, FGF-2 did not alter the endoproteolysis of IGF-II precursor at
Arg104 by the SPC-like protease.
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) has been observed before in
embryonic 293 cells (31) and probably reflects impaired activation of
the SPC-like protease due to disassembly of the Golgi complex.
Our results also suggest that FGF-2 altered the final processing of
pro-IGF-II. In conditioned medium from control H295R cells, the major
form detected was the 7.5 kDa mature IGF-II, indicating that the
proteases involved in the cleavage of pro-IGF-II-(1–04) at residues
Lys88 and Arg68 are
functional in these tumor cells. IGF-II peptides of higher molecular
mass were also detected including one group of peptides of 24–26 kDa,
corresponding to glycosylated forms of pro-IGF-II-(1–156) and another
group of peptides 14–18 kDa in size which are consistent with
glycosylated isoforms of IGF-II-(1–87) and IGF-II-(1–104). FGF-2
modified the profile of the secreted forms of IGF-II, with a marked
decrease in the amount of mature IGF-II which was not due to
proteolytic degradation of the growth factor. FGF-2 also induced the
predominant secretion of pro-IGF-II of about 18 kDa, likely
glycosylated IGF-II-(1–104). The accumulation of high molecular mass
forms of IGF-II in FGF-2-conditioned medium suggested abnormalities in
the final steps of pro-IGF-II processing. Such abnormalities were also
suggested by experiments with brefeldin A, indicating that the
increased amount of intracellular IGF-II protein in presence of FGF-2
was due to reduced secretion of pro-IGF-II rather than increased
synthesis of the growth factor. Thus, in our tumor model, FGF-2 appears
to inhibit both the secretion of pro-IGF-II-(1–104) and the final
steps of pro-IGF-II processing (endoproteolysis at residues
Lys88 and Arg68), which
probably occur at the time of secretion (Fig. 8).
FGF-2 has been shown to have various effects on the expression of IGF-II, depending on the cell types. However, such modulation of pro-IGF-II processing is not common. In human fetal adrenals, FGF-2 had no effect on the level of IGF-II mRNA, but the effects on IGF-II protein were not studied (36). In BC3H-1 muscle cells, FGF-2 decreased the levels of both IGF-II mRNA and secreted protein, suggesting transcriptional regulation of IGF-II expression (16). FGF-2 also decreased the level of IGF-II mRNA in cultured bone cells, but with no similar decrease in secreted protein level (37). In this model, FGF-2 may have regulated IGF-II via posttranscriptional mechanisms, but no information was given about the forms of IGF-II secreted into the medium.
On the other hand, there are numerous examples of incomplete processing of pro-IGF-II precursor by tumor cells and it is possible that, similar to our observation, some unknown growth factors regulate the last steps of IGF-II processing in these tumor models (38, 39, 40, 41).
FGF-2 also decreased the levels of secreted IGFBP-2 but, in contrast to IGF-II, this effect likely occurred at the transcriptional level, with a concomitant decrease in IGFBP-2 mRNA and protein levels. No IGFBP-2 proteolysis was detected in the presence of FGF-2, as shown by Western immunoblotting. This result contrasts with observations for a neuroblastoma cell line (19) and suggests that the effects of FGF-2 on IGFBP expression are probably cell-type specific.
The relationship between the mitogenic effect of FGF-2 and the modifications of the IGF system in H295R cells is not clear. One possibility would be that FGF-2 proliferative effect is related to the decrease in IGFBP-2, which could increase IGF-II bioavailability. The precise role of IGFBP-2 in the proliferation of adrenocortical tumor cells is not known, but IGFBP-2 has been shown to inhibit IGF-dependent cell proliferation in various models (42, 43, 44, 45). In this way, the reduced production of IGFBP-2 in response to FGF-2 might favor both the autocrine effects of IGF-II secreted by the tumor and endocrine and paracrine effects of IGFs arising from other sources. However, the marked decrease in secreted IGF-II by H295R cells in response to FGF-2 does not support the hypothesis of increased IGF-II bioavailability. The modest proliferative effect of FGF-2 on adrenocortical tumor cells is thus probably independent of the IGF system and specifically related to FGF-2. In this study, we focused on the effects of the 18 kDa isoform of FGF-2, but specific roles for the intracellular 22–24 kDa FGF-2 isoforms have been shown in various cell lines and should also be considered in our tumor model (24).
FGF-2 effects were studied in an in vitro model of adrenocortical carcinoma but the physiological relevance of these effects in vivo is unknown. The observation of high amounts of IGF-II and IGFBP-2 protein in adrenocortical carcinoma contrasts with the effects of FGF-2 on H295R cells described here (1, 2, 5). However, malignant adrenocortical tumors have been shown to express high amounts of pro-IGF-II peptides of 18–24 kDa apparent molecular mass, similar to the profile observed in conditioned medium from FGF-2-treated H295R cells (3, 5). This observation and preliminary data indicating that adrenocortical tumors express FGF-2 protein suggest that FGF-2 may potentially have a role in vivo.
In conclusion, we have shown that FGF-2 is expressed by adrenocortical tumor cells and is mitogenic for these cells. Moreover, FGF-2 regulates the expression of IGF-II and IGFBP-2 and modulates the processing of pro-IGF-II. This effect of FGF-2 on pro-IGF-II processing may represent a new mechanism regulating IGF-II expression and activity.
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Footnotes |
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Received January 21, 2000.
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References |
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and
interferon-
inhibit insulin-like growth factor II gene expression in
human fetal adrenal cell cultures. Mol Cell Endocrinol 91:59–65[CrossRef][Medline]
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or with • 10 ng/ml FGF-2 as indicated in
Materials and Methods and cells were counted after
various culture periods. Data are expressed as means ±
SD of triplicates. Although a weak proliferative effect was
detected after 4 days of culture in presence of FGF-2 (+ 17%), this
effect was maximum after 7 days of culture (+ 36%). Similar results
were obtained in three different experiments. *, P
< 0.05; **, P < 0.01. B, H295R cells seeded in
96-well plates were cultured for 7 days with various concentrations of
FGF-2 and [3H]-thymidine incorporation was
assayed as described in Materials and Methods. Results are
expressed as a percentage of control values (mean ±
SD of six wells). **, P <
0.01
represents
glycosylation sites. The molecular masses of the various forms of
IGF-II are indicated on the right. The putative inhibitory
effects of FGF-2 on the final steps of pro-IGF-II processing (secretion
of pro-IGF-II (1–104) and endoproteolysis at K88 and R68) are
indicated by arrows, with the symbol 
.