(Received for publication, June 9, 1995; and in revised form, August 3, 1995)
From the
fgf3 has been implicated in the embryonic and fetal
development of the mouse and as an oncogene in murine breast cancer. We
describe a procedure to purify the product of the mouse fgf3 gene and show it to be a potent mitogen for some epithelial cell
lines. Using a receptor binding competition assay, Fgf3 was shown to
bind with high affinity to the IIIb isoforms of Fgf receptor (FgfR) 1
and FgfR2 (ID =
0.8 nM) and with a
lower affinity to the IIIc variant of FgfR2 (ID
=
9 nM). No competition for the binding of
I-Fgf1 was observed for FgfR1 (IIIc), FgfR3 (IIIb and
IIIc), or FgfR4. Mitogenicity assays using BaF3 cells containing
individual Fgf receptors showed a pattern of response in agreement with
the receptor binding results. A comparison of two mammary epithelial
cell lines showed a marked difference of potency and dependence upon
heparin in their response to mouse Fgf3, suggesting a complex
interaction between the ligand and its low and high affinity receptors.
The fibroblast growth factors constitute a family of nine
proteins that share 35-55% amino acid identity over a core region
(Refs. 1 and 2; reviewed in (3) and (4) ). The
prototypic members, Fgf1 and Fgf2, have been ascribed a number of
properties including the induction of cell proliferation,
differentiation, migration, and cell survival, consistent with roles as
autocrine and paracrine signaling molecules. For Fgf2 and Fgf3 there is
also good evidence for the translocation of the protein directly to the
cell nucleus(5, 6, 7, 8) ; however,
the biological significance of this event is not known. The common
route for Fgf signaling is through an interaction of an extracellular
Fgf with cell surface receptors (reviewed in (9) and (10) ). Two classes of Fgf receptor have been identified: a low
affinity receptor typified by heparan sulfate proteoglycans that bind
Fgfs to high capacity but seem not to signal (11) and a high
affinity receptor with intrinsic tyrosine kinase activity (reviewed in (9) and (10) ). Four tyrosine kinase receptor genes (fgfr1 to fgfr4) have been identified in mammals that
encode an extracellular ligand binding domain composed of two
(-form) or three (
-form) immunoglobulin-like motifs, a
transmembrane segment, and a cytoplasmic portion that encompasses a
tyrosine kinase domain. FgfR1, (
)FgfR2, and FgfR3 but not
FgfR4 have a choice of exon encoding the second half of the third Ig
loop, termed IIIb and IIIc, respectively. This alternative splicing
changes the ligand binding specificity of the
receptors(12, 13, 14, 15) . Both
receptor classes are needed for signal transduction. The low affinity
receptors are required for high affinity ligand binding and appear to
facilitate the dimerization of the tyrosine kinase receptor-Fgf
complexes(16, 17, 18) . Dimer formation
results in tyrosine autophosphorylation of the receptor providing
suitable sites for second messenger interactions and consequent signal
transduction (reviewed in (19) ).
fgf3 was identified as a proto-oncogene activated by proviral insertion in mouse mammary tumors(20, 21) . Expression of fgf3 was not detected in the normal mammary glands, suggesting that its inappropriate expression contributed to tumorigenesis. This notion gained considerable support from transgenic mouse studies(22, 23, 24) . Thus, constitutive ectopic expression of fgf3 led to abnormal mammary gland development manifest as multifocal pregnancy-sensitive epithelial hyperplasia, with the stochastic appearance of frank neoplasia. These observations prompted us to identify the receptors responsible for Fgf3 signaling and to determine which isoforms are present on mammary epithelial cells.
In this study, we describe a purification procedure for Fgf3, determine its receptor preferences, and show that these correlate with its ability to induce DNA synthesis in BaF3 cells expressing a single introduced receptor isoform. Mammary epithelial cells are shown to express receptors for and respond mitogenically to Fgf3. Using two mammary cell lines, we observed different response profiles for Fgf3 and other Fgfs that are dependent upon the concentration of heparin.
Figure 1:
Purity
of mouse Fgf3. a, mouse Fgf3 (FGF3 = 1
nM), recombinant human Fgf7 (rhFGF7 =
4.5
nM), and mouse Fgf7 (mFGF7; partially purified from
DMI-1 cells and tested at a 1/50 dilution) were added in the
absence(-) or presence (+) of 5 µg/ml of the
Fgf7-neutralyzing antibody 1G4 to quiescent BALB/MK cells in a
mitogenicity assay. The response is plotted as the fold stimulation
over antibody-treated or -untreated control cells (the antibody had no
significant effect on the unstimulated cells). The mean value of
duplicate determinations is shown. Silver stain (b) and
Western blot (c) analyses of Fgf3 eluted from a
heparin-Sepharose column loaded with DMI-1 cell-conditioned medium (see
``Materials and Methods''). Fraction numbers are indicated on top, and the sizes of molecular weight markers (Life
Technologies, Inc.) on the left.
COS-1 cells were transfected with the appropriate Fgf receptor and
seeded at 5 10
cells/well into 48-well tissue
culture dishes pre-treated with poly-L-Lysine (Sigma) as
described by the manufacturer. After 48 h, the cell monolayers were
washed twice with ice-cold binding medium (DMEM containing 50 mM Hepes, pH 7.4, 1 mg/ml bovine serum albumin, and 1 µg/ml
heparin) and incubated for 3 h at 4 °C with the indicated amounts
of
I-Fgf1 or
I-Fgf3 in binding medium.
Competition binding was performed in the presence of up to 200-fold
excess of unlabeled ligand. The cell monolayers were then rinsed twice
with cold binding medium, and solubilized by incubating in 0.1% SDS and
0.3 M NaOH for 30 min at 37 °C, and the associated gamma
radiation was counted. To determine specific binding, the radioactivity
bound to cells transfected with empty vector was subtracted from that
of cells receiving Fgf receptors. The amount of
I-Fgf
bound was plotted against the concentration of competitor, and the dose
that inhibits the binding by 50% (ID
) was then calculated.
MIRB-FgfR plasmids
were transfected into BaF3 cells and selected in the presence of 600
µg/ml G418 (Life Technologies, Inc.). Individual clonal cell lines
were isolated by limiting dilution and screened for responsiveness to
Fgf1. These cell lines were used in quantitative proliferation assays,
measuring [H]thymidine incorporation into DNA as
described previously(13, 33, 35) .
Recombinant Fgf1 (provided by K. Thomas, Merck) was used as a positive
control in each experiment because Fgf1 is the only Fgf ligand that can
activate all splice variants of all Fgf receptors. (
)
BALB/MK and NIH3T3 cells were transferred to 48-well
tissue culture plates (2 10
cells/well) in 0.5 ml
of growth medium and left for 9 or 7 days, respectively, to become
confluent and quiescent(29) . The culture medium was then
replaced with serum-free medium containing the test samples and
processed as described previously(36) . C57MG and HC11 cells
were made quiescent by replacing the medium after 24 h of growth with
DMEM containing 0.1% new born calf serum. After a further 72 h, the
cells were treated with the test samples in fresh medium containing
0.1% serum for 22 h. [
H]Thymidine incorporation
assays were performed as described previously(36) .
Figure 2:
Competition between Fgf3 and I-Fgf1 for binding to different Fgf receptors. COS-1
cells expressing the IIIb (open circles) or IIIc (closed
circles) isoforms of the different Fgf receptors as indicated were
incubated with
I-Fgf1 in the presence of increasing
concentrations of Fgf3. Cells were then washed and lysed, and specific
binding was determined as described under ``Materials and
Methods.'' The calculated ID
values are listed in Table 1.
Figure 3:
Competition between Fgf3 and I-Fgf1 or
I-Fgf3 for receptor binding.
COS-1 cells expressing the indicated isoforms of FgfR1 and FgfR2 were
incubated with
I-Fgf1 (open circles) or
I-Fgf3 (closed circles) in the presence of
increasing concentrations of Fgf3. Cells were processed as described in Fig. 2. The calculated ID
values are listed in Table 1.
Figure 4:
Fgf3 induction of DNA synthesis in Fgf
receptor expressing BaF3 cells. BaF3 cells expressing the indicated
isoforms of FgfR1 and FgfR2 were used to compare the mitogenic activity
of Fgf1 (open squares), Fgf3 (filled circles), and
Fgf7 (open circles) in the presence of 2 µg/ml heparin.
The mitogenic response, determined by
[H]thymidine incorporation, is expressed as the
mean value ± S.D. of duplicate assays. These data are
representative of two independent
experiments.
Figure 5:
Effect of heparin on the induction of DNA
synthesis by Fgfs. In the top panels (C57MG and HC11 as
indicated), quiescent cell cultures were treated with different
concentrations of Fgf3 in the absence (open circles) or
presence (filled circles) of 2 µg/ml heparin and
[H]thymidine incorporation measured as described.
In the lower left panel, quiescent C57MG cells were treated or
not (filled triangles) with 0.1 nM Fgf1 (open
squares) or 1 nM Fgf3 (filled circles) in the
absence or presence of increasing heparin concentrations as indicated.
In the lower right panel, quiescent HC11 cells were treated or
not (filled triangles) with 0.1 nM Fgf1 (open
squares), Fgf3 (filled circles), or Fgf7 (open
circles) in the absence or presence of increasing heparin
concentrations as indicated. The mean value of duplicate determinations
is shown.
The differential
sensitivity of the two mammary cell lines toward Fgf3 could reflect a
difference in the expression of its high affinity receptors. To gain
some insight into the Fgf receptor repertoire of these and other
Fgf3-responsive cell lines, total cell RNA was prepared and used for
RT-PCR analyses with receptor-specific oligonucleotide primers. The
entire extracellular domain of each Fgf receptor was amplified, and
different splice variants ( and
forms) were detected by
ethidium bromide staining of the PCR products fractionated on agarose
gels (Fig. 6, a and b). IIIb and IIIc variants
of FgfR2 were distinguished by digesting the PCR products with
restriction endonucleases, which cleave exclusively one or the other
variant (Fig. 6b and (30) ). For the
corresponding isoforms of FgfR1, a different approach was needed that
relied on one primer hybridizing specifically to the IIIb or IIIc
spliced isoform (Fig. 6a). Three size variants of FgfR1
and FgfR2 were detected in all the cell lines tested. Previous sequence
analysis of FgfR2 PCR products has shown these forms represent the
,
, and
with acid box receptor variants(29) .
The results show that HC11 and BALB/MK express the IIIb and IIIc
isoforms of FgfR1 and FgfR2 (IIIb), whereas C57MG and NIH3T3 express
only the IIIc variant of these receptors. Thus, the greater sensitivity
of HC11 toward Fgf3 in the proliferation assay can be explained by the
presence of two receptor isoforms with an approximately 10-fold greater
affinity for Fgf3 (see Table 1).
Figure 6:
Fgf receptor expression by Fgf3-responsive
cell lines. cDNAs from HC11, C57MG, BALB/MK, and NIH3T3 cells were used
for PCR amplification of the extracellular domain using primers
specific for FgfR1, FgfR1 (IIIb), and FgfR1 (IIIc) (a) or
FgfR2 (b) as indicated. The ethidium bromide-stained agarose
gels show three prominent PCR products consistent with the sizes for
the ,
, and
plus acid box variants. FgfR2 PCR products
were digested or not (U) with enzymes that uniquely digest the
IIIb or IIIc exon; AvaI (A) cuts in IIIb and EcoRV (RV) in IIIc, respectively(30) . The
labels NIH3T3 and BALB/MK in panel a and top of panel b have been inadvertently switched in this figure.
The mitogenic potential of
Fgf3 was then compared with that of Fgf1 and Fgf7 on the mammary cell
lines (Fig. 7). Fgf1 gave a similar response on both cell lines
(ED =
0.1 nM), whereas Fgf7 induced
DNA synthesis only in HC11 cells (ED
=
0.2
nM), as expected from the previous receptor expression study.
Fgf1 showed a higher efficiency than Fgf3 in triggering a mitogenic
response in C57MG cells (ED
=
0.1 nMversus 1 nM), probably as a result of having a higher
affinity than Fgf3 for FgfR2 (IIIc) (ID
=
2
nMversus 9 nM; Table 1and data not
shown), of having a broader range of Fgf receptor interactions, and of
being potentiated by heparin, which was included in this assay. On the
contrary, the dose-response curves obtained with HC11 cells indicated
that Fgf3 was more efficient than the other Fgfs in stimulating DNA
synthesis (ED
=
0.01 nM). As HC11
cells express FgfR2 (IIIb), which has a similar affinity for the three
Fgfs tested ( Table 1and (13) ), the variation in potency
can be explained in this case by the use of heparin at a concentration
of 2 µg/ml, which was found to be optimal for Fgf3 but not for Fgf1
and Fgf7 (see Fig. 5). However, the maximum responses obtained
with these Fgfs were in the same range.
Figure 7:
Comparative
mitogenic activity of Fgf1, Fgf3, and Fgf7. Quiescent C57MG or HC11
cells were treated with increasing concentrations of Fgf3 (filled
circles), Fgf1 (open squares), or Fgf7 (open
circles) in the presense of 2 µg/ml heparin, and the
stimulation of DNA synthesis was measured by the incorporation of
[H]thymidine as described under ``Materials
and Methods.'' The mean value of duplicate determinations is
shown. Results are representative of at least two
experiments.
The results described herein show that biologically active
mouse Fgf3 can be purified from culture supernatants of NIH3T3 cells
engineered to secrete high levels of this factor. Immunological and
bioactivity criteria were used to show that differential salt elution
from a heparin-Sepharose column was effective in separating a mixture
of Fgf7 and Fgf3 that occurs in the conditioned medium of these cells.
In previous studies, recombinant Fgf3 made in bacterial or baculovirus
expression systems was found to be mostly insoluble. However, the small
amount of soluble recombinant Fgf3 showed no mitogenic activity on
cells that we show here to be responsive to Fgf3. ()Using a
cell-free translation system we were able to make biologically active
Fgf1, Fgf4, and Fgf7, but Fgf3 gave equivocal
results(39, 40, 41) . This could have
resulted from the use of inappropriate assay cells, or alternatively
the Fgf3 was inactive; the studies reported here suggest the latter
explanation.
The competition for receptor binding experiments ( Fig. 2and Fig. 3) clearly show that mouse Fgf3 has the
highest affinity for the IIIb isoforms of FgfR1 and FgfR2 and a 10-fold
lower affinity for FgfR2 (IIIc). There was no detectable competition
for I-Fgf1 binding to FgfR1 (IIIc), FgfR3 isoforms, or
FgfR4. It is also worth noting that we detected no significant
differences between the binding of Fgf3 to the
and
isoforms
of FgfR2 (data not shown). The lower apparent affinity of mouse Fgf3
for FgfR2 (IIIc) was observed using either
I-Fgf1 or
I-Fgf3 as tracer, indicating that mouse Fgf3 has indeed a
lower intrinsic affinity for this receptor isoform. The relative
binding affinities correlated well with the mitogenic activity of Fgf3
on BaF3 cells expressing single isoforms of the various Fgf receptors (Fig. 4) and are consistent with the mitogenic potential of Fgf3
for different cell lines that express combinations of these Fgf
receptors ( Fig. 5and Fig. 7). In contrast to mouse Fgf3,
the Xenopus homologue was previously shown to have a similar
high affinity for the IIIb and IIIc isoforms of FgfR2 and, as expected,
is a more potent mitogen for cells expressing FgfR2 (IIIc) ( (29) and (36) and data not shown).
Analysis of the mitogenic potential of Fgf3 on two mammary epithelial cell lines showed that C57MG cultures required at least 10 times more ligand compared with those of HC11 to give a half-maximum response (Fig. 5). This difference in sensitivity can be largely explained by the expression on HC11 cells of the IIIb variants of FgfR1 and FgfR2, which show a higher affinity toward Fgf3 than that of the IIIc variants found in C57MG cells (Fig. 6). An unexpected observation was the opposite effect of heparin on the mitogenic response elicited by Fgf3 on the two cell lines: heparin progressively increased the potency of Fgf3 on HC11 cells but decreased it on C57MG cells (Fig. 5). This differential modulation by heparin does not simply correlate with the presence or absence of the IIIb or IIIc variants of the FgfR1 and FgfR2 receptors. For instance, NIH3T3 cells are similar to C57MG in that they express the Fgf receptor IIIc isoforms; however, they show an enhanced response to Fgf3 in the presence of heparin (data not shown). Furthermore, we found that heparin also differentially modulated the effect of other Fgfs on the same cell line. For example, heparin decreased Fgf7 mitogenic activity on HC11 cells, whereas it potentiated Fgf1 and Fgf3, albeit at different concentrations. Recently, a similar observation was reported by others using BALB/MK cells(42) . Thus, different Fgfs may require distinct combinations and/or concentrations of low and high affinity receptors to achieve an optimal signaling effect. Response potentiation by heparin has been proposed to occur through the stabilization of the Fgf molecule and of Fgf-Fgf receptor complexes(18, 43, 44, 45, 46) . Oligomerization of Fgfs by heparin may further increase the affinity of Fgf for the Fgf receptor and results in dimerization and activation of the Fgf receptor(16, 17, 35) . However, the mechanism of inhibition by heparin as shown here and by others (42) suggests a more complex role for the low affinity receptors in modulating Fgf responses.
The receptor specificity of
Fgf3 encompasses that found for Fgf7 (13, 14, 15) . It is therefore interesting
that in some cases where Fgf7 has been implicated as a
mesenchyme-secreted growth/differentiation factor acting on the
adjacent epithelium, such as the prostate and seminal vesicle, fgf3 ectopically expressed as a transgene has been shown to act as an
oncogene(22, 24, 47, 48, 49) .
A similar situation may also exist for the mammary gland where the high
affinity receptors for Fgf3 are present and provide an explanation for
its role in virally induced breast cancer. Thus, virally mediated
activation of Fgf3 directly in the epithelial cells could result in
autocrine growth stimulation (reviewed in (21) ). Additionally,
recent evidence suggests that growth factors can protect cells from
undergoing apoptotic death, a process that occurs throughout the
mammary gland after lactation. Therefore, Fgf3 may act in a dual
capacity to facilitate the retention and accumulation of cells
susceptible to further somatic mutation. The presence of Fgfs and Fgf
receptors in the mammary gland (50) ()also implies
that the normal development and differentiation of the mammary gland
during pregnancy and lactation may involve steps mediated by members of
the Fgf family. We are presently investigating this possibility to gain
further insight into the role of Fgfs in normal breast development.