From the ¶ Department of Molecular Genetics and Microbiology,
the Department of Neurobiology and Behavior, and the
** Howard Hughes Medical Institute, Institute of Cell and Developmental
Biology, State University of New York at Stony Brook, Stony Brook,
New York 11794-5222 and the §§ Department of
Physiology, University of Colorado, School of Medicine, Health
Sciences Center, Denver, Colorado 80262
Received for publication, April 7, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Fibroblast growth factor receptors (FGFR) are
widely expressed in many tissues and cell types, and the temporal
expression of these receptors and their ligands play important roles in
the control of development. There are four FGFR family members,
FGFR-1-4, and understanding the ability of these receptors to
transduce signals is central to understanding how they function in
controlling differentiation and development. We have utilized signal
transduction by FGF-1 in PC12 cells to compare the ability of FGFR-1
and FGFR-3 to elicit the neuronal phenotype. In PC12 cells FGFR-1 is
much more potent in the induction of neurite outgrowth than FGFR-3. This correlated with the ability of FGFR-1 to induce robust and sustained activation of the Ras-dependent mitogen-activated
protein kinase pathways. In contrast, FGFR-3 could not induce strong
sustained Ras-dependent signals. In this study, we analyzed
the ability of FGFR-3 to induce the expression of sodium channels,
peripherin, and Thy-1 in PC12 cells because all three of these proteins
are known to be induced via Ras-independent pathways. We determined that FGFR-3 was capable of inducing several Ras-independent gene expression pathways important to the neuronal phenotype to a level equivalent of that induced by FGFR-1. Thus, FGFR-3 elicits phenotypic changes primarily though activation of Ras-independent pathways in the
absence of robust Ras-dependent signals.
The fibroblast growth factors
(FGFs)1 play roles in
development, angiogenesis, wound healing, and tumorigenesis (reviewed
in Ref. 1). FGF actions are mediated by activation of FGF receptor (FGFR) tyrosine kinases. FGFRs are a gene family of four members, termed FGFR-1-4. These receptors are widely expressed in many tissues
and different cell types, and the temporal expression of the receptors
and their ligands is regulated during development (reviewed in Ref. 2).
Analysis of naturally occurring mutations in these receptors has
indicated that they control the differentiation of specific cell types
during development. Point mutations within the genes encoding human
FGFR-1, -2, or -3 cause different syndromes that involve bone
development (reviewed in Refs. 3 and 4) and some of these syndromes
(Aperts and thanatophoric dysplasia) may also manifest effects in the
central nervous system. Point mutations in FGFR-3 that cause activation
of its tyrosine kinase activity have been shown to be responsible for
the commonest form of dwarfism in humans (4-7). Recently FGFR-3 has
also been implicated in multiple myeloma, where its abnormal
overexpression because of a chromosomal translocation has been detected
in ~25% of cases (8, 9). However, it is not clear what role this
expression contributes to the phenotype of this disease. Studies
analyzing the consequences of null mutations in FGFRs in mice also
implicated these receptors as playing a role in development. The
knockout of either FGFR-1 or FGFR-2 (10, 11) in mice resulted in
embryonic lethality, whereas that of FGFR-3 was nonlethal. The
FGFR-3-deficient mice developed an overgrowth of the long bones and
abnormal curvature of the spine and tail (12, 13) and were deaf
(12).
The FGFRs are very similar in structure. In particular, their tyrosine
kinase domains are highly conserved, and overlapping subsets of ligands
induce their activation. Regulation appears to take place at two
different levels. Temporal control of the expression of both ligands
and receptors is an important mechanism for regulating signal
transduction during development. In addition, the receptors seem to
have differing signaling capabilities. Studies have indicated that
FGFR-1 is much better at producing mitogenic signals than either FGFR-3
or FGFR-4 when assayed in BaF3 cells (14-16). We have demonstrated
that there is also a difference between FGFR-1 and -3 in their
abilities to induce neurite outgrowth in PC12 cells when activated by
FGF-1 (17, 18). FGFR-3 can barely induce neurite outgrowth, whereas
activation of FGFR-1 induces rapid and robust neurite outgrowth. In the
BaF3 and PC12 cell systems it appears that sustained signals that lead
to the Ras-dependent activation of the extracellular
regulated kinases are necessary for the biological phenotypes observed.
The above results imply that FGFR-3 is not able to induce strong
sustained signals. However, in vivo it obviously plays key roles in development. There are two potential nonexclusive hypotheses that could explain these observations. The threshold for signaling in
these developmental tissues may be low, and sustained signals may not
be mandatory. Alternatively FGFR-3 may induce physiologically relevant
and strong signals using signaling pathways that are distinct from the
Ras-extracellular regulated kinase pathway. In this study we have
chosen to examine this latter possibility. The nature of FGFR-3
signaling and the downstream targets are still not well understood and
are the subject of intense investigation. To establish the signaling
capabilities of FGFR-3 and to determine the nature of FGFR-3 induced
signals, we took advantage of the PC12 cell system in which either NGF
or FGF-1 can induce the elaboration of a neuronal phenotype. Underlying
the growth factor-induced acquisition of neuronal phenotype are a
variety of signaling pathways and gene expression changes that lead to
the specific neuronal traits. Prominent neuronal traits are mediated by
both Ras-dependent and Ras-independent signaling pathways.
For example, gene expression events leading to morphological
differentiation (neurite outgrowth) are predominantly mediated via a
Ras-dependent mitogen-activated protein kinase pathway,
whereas the establishment of a sodium based action potential is
mediated by the expression of voltage-dependent sodium
channel genes in a Ras-independent manner. Normal PC12 cells express
both FGFR-1 and FGFR-3, and both of these receptors can be activated by
FGF-1. This makes it difficult to distinguish pathways activated by
FGFR-1 from those activated by FGFR-3. However, a variant PC12 cell
line, termed fnr-PC12, exists that has lost the expression of
functional FGFR-1 but has retained functional FGFR-3 (17, 18). The use
of these cells allowed us to assay the ability of FGFR-3 to activate
several distinct signal transduction pathways that are important for
the development of the neuronal phenotype. In this paper we report that
although FGFR-3 is not capable of inducing sustained activation of
Ras-dependent pathways, it is as capable of inducing the
activation of Ras-independent pathways to levels equivalent to those
seen with the activation of FGFR-1.
Cell Culture--
PC12, 17N-2 PC12, fnr-PC12 cells, and
fnr-PC12-derived transfectant lines have been described previously (17,
18). Cells were grown on tissue culture dishes in Dulbecco's modified
Eagle's medium supplemented with 10% donor horse serum, 5% fetal
bovine serum, and 1% penicillin/streptomycin in an atmosphere of 10% CO2 at 37 °C. Recombinant human NGF was used at a final
concentration of 50-100 ng/ml. FGF-1 (a kind gift of Dr. M. Jaye) was
added at the final concentration of 50-100 ng/ml together with heparin (50 µg/ml). The Src family member tyrosine kinase inhibitor PP2 was
purchased from Calbiochem.
Western Blot Analysis--
Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40). To dissolve Nonidet P-40 -insoluble peripherin protein, cells were lysed in RIPA buffer (20 mM
Tris, pH 7.6, 135 mM NaCl, 2 mM EDTA, 0.1%
SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM
Na3VO4, and 1 mM
phenylmethylsulfonyl fluoride). The lysates were clarified by
centrifugation. The protein concentration was determined using
Coomassie Plus protein assay reagent (Pierce). Aliquots of supernatant
(100 µg) of each sample were subjected to SDS-polyacrylamide gel
electrophoresis on a 7.5% polyacrylamide gel and transferred onto a
nitrocellulose membrane using a Bio-Rad Trans Blot according to the
manufacturer's instructions. After incubation for 1 h with
blocking solution (5% nonfat dry milk in PBS), the protein blots were
probed with primary antibody for 2 h at room temperature or
overnight at 4 °C. For specific detection of Thy-1 and peripherin
proteins, culture medium of hybridoma clones producing anti-Thy-1
antibody (a kind gift of Dr. J Trimmer) and anti-intermediate filament
protein antibody (19) were used after dilution in blocking solution.
The antibody against phospho-Akt (New England Biolabs) was used
according to the manufacturer's instructions. The blots were probed
with anti-mouse IgG horseradish peroxidase-conjugated antibody
(Amersham Pharmacia Biotech) for 1 h at room temperature. The
blots were treated for 1 min using ECL kit (Amersham Pharmacia Biotech)
and exposed to x-ray film (Kodak).
Northern Blot Analysis--
Isolation of total cellular RNA from
PC12 cells or transfectant lines and Northern blot analysis were
carried out as described previously (20). The concentration of RNA was
determined by measuring the optical density at 260 nm. RNA samples (10 µg/lane) were electrophoresed through agarose gels containing 2.2 M formaldehyde, 40 mM MOPS, pH 7, 10 mM sodium acetate, and 1 mM EDTA and then electrophorectically transferred to nylon membranes (Duralon-UV; Stratagene) at 200 mAmp at 4 °C overnight. The blots were
cross-linked using UV cross-linker (Stratagene). Blots were incubated
in prehybridization buffer (5× SSC, 10× Denhardt's solution, 50 mM sodium phosphate, pH 6.7, 50% formamide, 0.5% SDS, and
0.5 mg of denatured salmon sperm DNA/ml (31) for at least 2 h at
68 °C. The prehybridization solution was completely removed and
replaced with hybridization buffer (5× SSC, 1× Denhardt's solution,
20 mM sodium phosphate, 50% formamide, 0.5% SDS, and 0.1 mg of denatured salmon sperm DNA/ml) supplemented with 5 × 106 cpm/ml [ Immunocytochemistry--
Cells were plated on coverslips coated
overnight with 25 µg/ml poly-L-lysine (Sigma) and 10 µg/ml laminin (Collaborative Biomedical Products), grown for 24 h, and treated with 50 ng/ml of FGF-1 for an additional 3 days.
Cultures were fixed in 3.7% formaldehyde and 0.12 M
sucrose in PBS for 10 min and then rinsed with PBS, permeabilized in
methanol at Sodium Current Recordings--
Whole cell recordings of PC12
sodium current were made by means of an Axopatch 200A amplifier (Axon
Instruments Inc., Burlingame, CA). The recording solution contained 140 mM NaMES, 0.2 mM MgCl2, 0.2 mM CaCl2, 10 mM NaHEPES, pH 7.2. The pipette solution contained 140 mm CsMES, 10 mM CsEGTA,
10 mM CsHEPES, pH 7.2. To record sodium currents the cells
were held at PC12 Survival Assay--
Cells were plated on 24-well plates
precoated with rat tail collagen (Collaborative Research). Prior to
plating the cells were washed five times with serum-free medium and
then resuspended at a concentration of 2 × 105/ml in serum-free medium. 0.5 ml of cells were
plated into each well and were appropriate FGF-1 (50 ng/ml) or NGF (100 ng/ml) was added, cultures were done in triplicate. The cultures were refed with factor containing medium every 2 days, After 8 days of
treatment, the cell both in the medium and attached to the plate were
lysed in 100 µl of 0.1× PBS, 0.5% Triton X-100, 2 mM MgCl2, 0.5% ethylhexadecyldimethylammonium bromide, 0.28%
glacial acetic acid, 2.8 mM NaCl, and bromphenol blue. The
nuclei were counted with a hemocytometer, but only nuclei with a
distinct membrane and nucleoli representative of live cells were counted.
In this study we wanted to compare the abilities of FGFR3 to
activate various signal transduction pathways in PC12 cells. We were
particularly interested in comparing pathways that had been shown
previously to be either activated in a Ras-dependent or
Ras-independent manner. These studies were done using NGF and a PC12
cell line, 17N-2 PC12 cells, that constitutively expresses a
dominant-interfering mutant of Ras (20). NGF was shown to activate the
expression of transin in a Ras-dependent manner, whereas
the type II sodium channel and peripherin were activated in
Ras-independent manner. Therefore, to confirm that FGF-1 activated the
expression of these genes in a similar way, we used the 17N-2 PC12
cells to determine what effect this dominant-interfering mutant of Ras
has on the activation of these genes. Fig.
1A shows a Northern blot
analysis that demonstrates that whereas the sodium channel gene
expression is still induced by addition of FGF-1, the levels of transin
are unchanged. The induction of the sodium channel in these cells by
FGF-1 was reproducible even though it was low (Fig. 1A).
Sodium channel induction in these cells by NGF was also found
previously to be low (20). The induced expression of the peripherin
protein was also not affected by the N17-ras mutant (Fig.
1B). This indicates that just like NGF, FGF-1 induces transin via a Ras-dependent pathway and peripherin and the
sodium channel via Ras-independent pathways. FGF-1 induction of Thy-1 expression was also found to be Ras-independent and to require the
activation of a Src-dependent pathway in a similar manner to NGF activation (data not shown and Fig. 6)
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]UTP-labeled antisense RNA
probes. Hybridization was carried out overnight at 68 °C. RNA probes
were synthesized using the following linearized cDNA clones as
templates: RB211 (21) encoding a conserved sodium channel coding region
and pIB15 (22) encoding cyclophilin subcloned into pSP65; pG7TR1,
encoding transin (23) subcloned into pGEM7Zf (+); pPeripherin, encoding
a partial sequence of rat peripherin (nucleic acid 1159-1503), which
was isolated by polymerase chain reaction from a PC12 dT/random
cDNA library and subcloned into pBluescript II SK(+) plasmid; pST4,
encoding Thy-1 (24) subcloned into pSP65. All riboprobes were generated according to the manufacturer's instruction using SP6 polymerase (Promega) except for peripherin probes, which was synthesized using T7
polymerase. The blots were washed twice in 2× SSC-0.1% SDS at
68 °C and twice in 0.2% SSC, 0.1% SDS for 20 min at 68 °C.
Levels of radiolabeled probe bound to the blot were determined by
PhosphorImager (Molecular Dynamics) analysis, and all values were
normalized to the level corresponding to cyclophilin mRNA.
20 °C for 15 min, and then blocked with 15% goat
serum in PBS for 1 h at room temperature. Cells were incubated
with affinity-purified anti-sodium channel protein antibody (25) that
was diluted in blocking solution overnight at room temperature. After
rinsing twice with blocking solution, cells were incubated with goat
anti-rabbit antibody conjugated with Alexa568 (Molecular
Probes, Inc.) diluted in blocking solution for 1 h at room
temperature. Cultures were rinsed twice with PBS and mounted using
Vectashield mounting medium (Vector Laboratories). Confocal images were
obtained using a Zeis LSM510 laser scanning confocal microscope.
100mV and stepped to positive potentials for 20 ms. PC12
cell sodium currents were digitized at 50 kHz and analyzed off-line
using HEKA Pulse & Pulse Fit software (Instrutech, Great Neck, NY).
Capacitive transients were compensated using a combination of manual
compensation on the amplifier and further processing using either a P/4
or P/10 leak subtraction protocol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FGF-1 induced signal transduction in PC12
cells expressing a dominant-interfering mutant of Ras.
A, 17N-2 PC12 cells were treated with 50 ng/ml FGF-1 for
48 h. Total cellular RNA (10 µg) was electrophoresed through
0.8% agarose gels and transferred onto a nylon membrane. The blot was
hybridized with a probe specific for type II sodium channel (top
panel) or a probe specific for transin (middle panel)
and with a probe specific for the internal control cyclophilin
(bottom panel). B, 17N-2 PC12 cell lysates
treated for 0, 24, or 72 h with FGF-1 were electrophoresed through
a 7.5% SDS-polyacrylamide gel and transferred onto a nitrocellulose
filter membrane. Blots were probed with anti-intermediate filament
protein antibody and exposed to x-ray film as described under
"Experimental Procedures."
Having determined that FGF-1 activation proceeds through both Ras-dependent and Ras-independent pathways we now wanted to determine the abilities of FGFR-1 and FGFR-3 to activate these pathways. To assay for the ability of FGFR-3 to activate different signaling pathways, we compared signals induced by FGF-1 in three different cell lines. Fnr-PC12 cells are the parental cell line that does not express FGFR-1 but does express low levels of FGFR-3. FGFR-3b cells are fnr-PC12 cells that have been transfected with FGFR-3, and FGFR-31b cells are fnr-PC12 cells transfected with a chimeric FGF receptor, composed of the extracellular domain of FGFR-3 fused to the cytoplasmic domain of FGFR-1. The use of this chimeric receptor allowed us to eliminate differences in signaling that could be attributed to FGF-1 binding because both of the transfected receptors have the same FGF-binding domains. We have shown previously that these two transfected cell lines overexpress equivalent amounts of receptors (17). The use of these cells eliminates the possibility that any differences in signaling can be attributed to receptor number. By assaying for FGF-1-induced signals in the fnr-PC12 cells, we will be able to identify signaling pathways that FGFR-3 can activate. Then, by comparing FGF-1 induced signals in the two transfected cell lines, we can compare the efficiency with which FGFR-1 and FGFR-3 can activate these pathways.
We chose to look primarily at the induction of gene expression because
our previous analysis had revealed that the initiation of signaling by
FGFR-1 and -3 was similar (17, 18). However, the downstream
consequences that require robust gene expression were dramatically
different. To initiate these studies, we chose to look at the induction
of the gene transin by FGF-1. Transin is induced via a
Ras-dependent signaling pathway (20). Therefore, its
induction would serve as a control to establish that using induction of
a Ras-dependent gene l would also reveal the differing signaling capabilities of FGFR-1 and -3. Fig.
2 shows analysis of the induction of
transin mRNA using Northern blot analysis. In the fnr-PC12 cells
there was a barely detectable induction of transin mRNA by FGF-1.
In contrast, treatment of the fnr-PC12 cells with NGF did induce
transin mRNA expression (data not shown), indicating that the
signaling pathway to transin mRNA induction is intact in these
cells. In the FGFR-3b cell line that overexpresses FGFR-3, transin
mRNA induction was now detected after 72 h of treatment with
FGF-1. However, analysis of the FGFR-31b cell line revealed that this
receptor could induce much higher levels of transin mRNA after
72 h (Fig. 2). Quantitation of the amount of mRNA present
under the different conditions demonstrated that the level of induction
of transin mRNA at the 72 h time point was ~15-fold higher
in the cells that expressed the FGFR-31b chimeric receptor in
comparison with the FGFR-3b cells. As a loading control the levels of
cyclophilin mRNA was also measured, and this demonstrated that
similar amounts of RNA were loaded in each lane (Fig. 2). Together with
the lack of induction seen in the fnr-PC12 cells (Fig.
3), these results indicate that FGFR-3 is
much less efficient than FGFR-1 at inducing this
Ras-dependent pathway. These observations confirm our
previous results and validate this approach to show potential
differences between signaling by FGFR-1 and -3.
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The ability of FGFR-3 to activate pathways that are known to be Ras-independent was examined next. The activation of the Type II sodium channel gene has been shown to occur through a pathway that is independent of Ras. Fig. 3 shows the induction of mRNA encoding the type II sodium channel as measured by Northern blot analysis. Analysis of the induction of the Type II sodium channel in the fnr-PC12 cells after 60 h of treatment by FGF-1 demonstrates that FGF-1 could induce the increased expression of the type II sodium channel. As a control we also measured transin induction after 60 h of treatment with FGF-1 (Fig. 3). The data indicated that the endogenous FGFR-3 in the fnr-PC12 cells can induce type II sodium channel, albeit weakly. We compared the abilities of the overexpressed receptors to induced type II sodium channel mRNA. As can be seen in Fig. 3, FGFR-3 is as efficient as FGFR-1 in inducing the increased expression of type II sodium channel mRNA, whereas there is a major difference between these receptors in their abilities to induce the Ras-dependent transin mRNA.
We also measured the induction of functional channel expression by
these receptors and the expression of the channels within the cells.
Fig. 4A documents that the
increase in mRNA levels of the type II sodium channel correlates
well with an increase in channel protein levels. Immunofluorescence
localization studies revealed a staining pattern indicative of
localization of the channel proteins to the surface of the
differentiated PC12 cells. To determine that the immunofluorescence
corresponded to functional sodium channels, whole cell patch clamp
recordings were performed. Recordings from FGF-treated FGFR-3b cells
indicated large inward sodium currents when the cells were depolarized
to positive membrane potentials (Fig. 4B). In recordings
from eight FGF-treated FGFR-3b cells, all exhibited inward current, the
overall average corresponding to 357pA. By contrast, in recordings from
11 control cells, only 4 exhibited inward sodium current. All sodium
current in treated and nontreated cells was inhibited by addition of 1 µM tetrodotoxin, an inhibitor of
voltage-dependent sodium channels.
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We next looked at the induction of the protein peripherin. The
expression of this protein is controlled through another
Ras-independent pathway that in this case involves phospholipase C
(PLC
) activation (26).2
Analysis of mRNA levels in the fnr-PC12 cells by Northern blot demonstrated that both FGF-1 and NGF induced peripherin to similar levels (data not shown). Comparison of the induction of peripherin in
the cells overexpressing the two FGFRs showed that in both cases there
was induction of mRNA to similar levels (Fig.
5A). Quantitation of the
levels of induction indicated that FGFR-3b activation gave rise to a
5-fold increase in mRNA levels after 72 h, whereas the FGFR31b
receptor induction was 4-fold. This indicates that FGFR-3 can activate
this pathway as efficiently as FGFR-1. We also looked at the induction
of the expression of peripherin at the protein level by Western
blotting. Fig. 5B shows that FGF-1 can induce peripherin in
the fnr-PC12 cells and that in the cells overexpressing the FGFR-3
receptor the induction of peripherin levels is increased significantly.
This indicates that FGFR-1 and FGFR-3 are equally efficient at the
induction of this PLC
-dependent pathway.
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The Thy-1 protein is a cell surface glycoprotein whose expression is
induced by FGF-1 treatment of PC12 cells via another distinct
Ras-independent pathway that involves a Src-dependent branch point (Ref. 20 and below). Analysis of the induction of mRNA
encoding for Thy-1 in the fnrPC12 cells revealed that at the 72-h time
point FGF-1 could induce a 3-fold increase in Thy-1 mRNA levels
(Fig. 6A), and this was a
similar level to that seen with NGF (data not shown). A time course of
the induction of the expression of Thy-1 mRNA in the FGFR-31b and
FGFR-3b overexpressing fnrPC12 cells revealed that both of these
receptors induced equivalent levels of Thy-1 mRNA (Fig.
6A). By 72 h FGFR31b had induced a 7-fold increase and
FGFR3b had induced a 10-fold increase. Analysis of Thy-1 protein levels
in the fnrPC12 cells and the FGFR-3b overexpressing cells also
demonstrated that FGF-1 could induce Thy-1 protein expression and that
this was greatly increased in the cells expressing more FGFR-3b (Fig.
6B). These data demonstrate that FGFR-3b can induce Thy-1
expression with similar efficiencies to FGFR-31b. To demonstrate that
the induction of Thy-1 by FGFR-3b involves a Src family member we used
the inhibitor, PP2. This class of inhibitor preferentially inhibits Src
family member tyrosine kinases (27) and at the concentration used has
very little effect on FGFR-3b kinase activation (data not shown).
Fnr-Pc12 cells expressing FGFR-3b were activated by FGF-1 either in the
presence or absence of PP2 and the induction of Thy-1 monitored after
48 h by Western blotting. As can be seen in Fig. 6C, in
comparison with no treatment or 10 min treatment Thy-1 protein levels
after 48 h incubation were increased by the addition of FGF-1
(lane 3). However, if the cells were treated with PP2 and
stimulated with FGF-1, there was no induction of Thy-1 (lane
5). Thus, treatment of the cells with PP2 completely blocks the
induction of Thy-1, demonstrating that the induction is via a Src
family member-dependent pathway. These data are in
agreement with earlier studies that indicated that Thy-1 induction by
NGF was via a Src-dependent branch point (7).
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Finally, we tested the ability of FGFR-3 to inhibit cell death
following withdrawal of serum. As shown in Fig.
7A, withdrawal of serum from
fnr-PC12 cells leads to cell death. Stimulation of the cells by NGF
allows cell survival. When the cells were stimulated by FGF-1 there was
some increase in cell survival, but it was not as efficient as NGF.
This indicates that FGFR-3 can activate cell survival pathways but not
as efficiently as the NGF receptor. To compare the abilities of FGFR-1
and FGFR-3 cytoplasmic domains to allow cell survival, we compare the
fnr-PC12 cells overexpressing these two receptors. As shown in Fig.
7A these two receptors are equally able to induce cell
survival when overexpressed. Similar results were also seen when cell
survival was measured using annexinV staining (data not shown).
Although it is not clear which pathways are important for regulating
this effect, the activation of protein kinase B (also known as Akt), via the PI-3 kinase pathway has been shown to inhibit apoptosis in
similar systems. Activation of Akt requires phosphorylation, and there
are antibodies available that are directed against the phosphorylated
and activated Akt. Therefore, we treated fnr-PC12 cells expressing
FGFR-1 and FGFR-3 with FGF-1 and performed a Western blot analysis
using the antibodies that detected activated Akt. As can be seen in
Fig. 7B, FGFR-1 and FGFR-3 induced identical levels of
activation of Akt (compare lanes 3 and 6). This
may explain how these two receptors can both inhibit cell death in PC12
cells.
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DISCUSSION |
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The cytoplasmic signaling domains of FGF receptors are highly conserved; yet data are accumulating that they do not all signal equivalently. A comparison of the ability of FGFR-1 and FGFR-3 to induce proliferation of the lymphoid cell line BaF3 or neurite outgrowth in PC12 cells indicated that only FGFR-1 was able to mediate these things efficiently (14-18). In these cell systems it appears that sustained and robust signals that lead to the Ras-dependent activation of the extracellular regulated kinases are necessary for the biological phenotype analyzed. This implied that FGFR-3 was not able to induce strong sustained signals. However, in vivo it obviously plays key roles in normal development and in several diseases. This indicated that either the threshold for signaling in these developmental tissues is low so that sustained signals are not mandatory or that FGFR-3 can induce physiologically relevant and strong signals using other signaling pathways. To determine whether the latter possibility was correct, we took advantage of the PC12 cell system in which the elaboration of a neuronal phenotype can be induced by FGF-1. This growth factor mediates changes in gene expression that underlie the induction of various neuronal phenotypic changes through both Ras-dependent and Ras-independent pathways. In this report we demonstrate that the FGFR-3 can in fact induce Ras-independent signaling pathways as efficiently as FGFR-1, and this induction leads to important neuronal traits. Thus FGFR-3 is capable of strong physiologically important signaling via Ras-independent pathways.
Previous analysis of activation of Ras-dependent pathways
in PC12 cells indicated that FGFR-3 was unable to induce efficient gene
expression through the Ras-dependent pathways (17, 18). Analysis of the ability of FGFR-3 to induce the
Ras-dependent gene transin in this report confirms that
FGFR-3 is very poor at inducing Ras-dependent genes.
However, as we demonstrate in this report, FGFR-3 was clearly capable
of inducing all the Ras-independent pathways that we analyzed equally
as well as FGFR-1. These included the induction of the expression of
the protein peripherin. The expression of this gene is induced through
a pathway that requires PLC. The activation of PLC
is
accomplished via interaction with a tyrosine autophosphorylation site
within the carboxyl terminus of the FGFRs. This site and its
surrounding amino acid sequence are conserved between FGFR-1 and
FGFR-3; therefore, it is perhaps not so surprising that both the
receptors can induce the expression of genes via
PLC
-dependent pathways. Recent experiments using chimeric receptors that could be activated by platelet-derived growth
factor also demonstrated that PLC
was equally well phosphorylated by
both FGFR-1 and -3 (28). These data agree with ours that the difference
in signaling capabilities between these two receptors cannot simply be
explained by differences in kinase activities.
The two other genes we analyzed for induction by FGFR-3, namely the type II sodium channel and Thy-1, are known to be induced via Ras-independent pathways. We found that FGFR-3 was as good as FGFR-1 in its ability to induce these two genes. The activation of cyclic AMP-dependent pathways has been implicated in sodium channel induction. Similarly, Thy-1 is activated in a Ras-independent manner by NGF, and this involves a novel branch point off a Src-dependent pathway. Our studies using the Src family member kinase inhibitor PP2 indicate that FGF-1 activates Thy-1 expression via a Src-dependent branch point. We also compared the abilities of FGFR-1 and FGFR-3 to inhibit cell death and to activate the protein tyrosine kinase Akt. Again we found that FGFR-3 was equally efficient as FGFR-1 in inhibiting cell death and activation Akt. Therefore, in clear contrast to its inability to activate Ras-dependent pathways, it is clear that FGFR-3 can activate all of the Ras-independent pathways we analyzed equally as well as FGFR-1.
In our analysis we did not identify an FGFR-3-specific signal transduction pathway, in that FGFR-1 was found to also be capable of activating all of the pathways we analyzed. Recently, FGFR-3 has been shown to be able to activate the protein Stat-1, and this is a candidate pathway that may be specific to FGFR-3 among the FGFR family (29, 30). This activation appeared to be cell type-specific because it was only clearly shown in chondrocytes. PC12 cells express high levels of Stat-1; however, we and others were unable to demonstrate activation of Stat-1 by either FGFR-1 or FGFR-3 in the overexpressing cells (30).3 This indicates that the activation of Stat-1 by FGFR-3 may involve cell type-specific adaptor proteins that are absent from PC12 cells. Thus, signaling through FGFR-3, in addition to occurring primarily via Ras-independent pathways, may also involve the use of specific proteins to expand the repertoire of the signaling cascades. The unique ability to signal primarily through Ras-independent pathways may explain why activating mutations in FGFR-3 only have penetrance in certain tissues. This may involve the activation of specific pathways that are not easily duplicated by other receptors and may also involve additional cellular proteins that are only expressed in the tissues affected. The unique signaling ability may also explain why only FGFR-3 of the four family members is overexpressed in multiple myeloma cells.
In summary, we have demonstrated that FGFR-3 can induce the equivalent
activation of various Ras-independent pathways to FGFR-1. In contrast,
as shown previously FGFR-3 could not induce sustained robust activation
of Ras-dependent pathways. This indicates that FGFR-3
induces signal transduction primarily through strong activation of
Ras-independent pathways in the absence of a robust
Ras-dependent signal.
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ACKNOWLEDGEMENTS |
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We thank the various members of our laboratories for helpful comments on the manuscript. We are particularly indebted to Joan Speh for help with the confocal imaging. We thank Michael Jaye for the generous provision of fibroblast growth factor.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Public Service Grants CA28146 and CA42573 (to M. J. H.) and PO1-NS34375 (to S. H., G. M, and S. R. L).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.
§ Supported by Grant RG2959A1-T from the National Multiple Sclerosis Society. Present address: Dept. of Physiology and Biophysics, School of Medicine, University of Seville, 41009 Seville, Spain.
Supported by National Institutes of Health Minority Access
Grant GM08655.
Investigator of the Howard Hughes Medical Institute.
¶¶ To whom correspondence should be addressed. Tel.: 631-632-8792; Fax: 631-632-8891; E-mail: mhayman@ms.cc.sunysb.edu.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M002959200
2 D.-Y. Choi and S. Halegoua, manuscript in preparation.
3 I. Ischenko and M. J. Hayman, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; PLC, phospholipase C.
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