From the Department of Medicine, Vanderbilt
University School of Medicine, the § Department of Molecular
Physiology and Biophysics, Vanderbilt University, and the ¶ Flow
Cytometry Pathology Laboratory, Vanderbilt Veterans Affairs
Medical Center, Nashville, Tennessee 37232
Received for publication, September 20, 2002, and in revised form, February 19, 2003
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ABSTRACT |
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Activated fibroblast growth factor
receptor 1 (FGFR1) propagates FGF signals through multiple
intracellular pathways via intermediates FRS2, PLC Fibroblast growth factors
(FGFs)1 comprise a family of
23 polypeptides that induce mitogenic, angiogenic, and chemotactic
responses in cells of mesodermal and neuroectodermal origin (1-3). The FGF signaling pathway also plays a significant role in normal development, and increased FGF production is associated with chronic immunologic injury as well as tumor development and metastasis (4-6).
Such a diverse array of biological effects occurs through ligand
interaction with high affinity cell surface receptors (FGFR1-4) that
are structurally similar and exhibit a high degree of sequence homology
at the amino acid level (3, 7). The full-length FGFR exhibits three
extracellular immunoglobulin-like domains, a single transmembrane
domain, and a split intracellular tyrosine kinase domain (7). Ligand
binding causes receptor dimerization allowing trans
autophosphorylation of intracellular tyrosine residues and
activation of intrinsic kinase activity (8).
Activation of FGFR1 results in tyrosine phosphorylation of multiple
signaling and adaptor proteins, including FRS2 (FGF receptor substrate
2/SNT-1), Shc, Grb2, Ras/Raf, Crk, phosphatidylinositol 3-kinase,
SHP-2, and Src (reviewed in Ref. 9). A constitutive association of the
multidocking protein FRS2 at the juxtamembrane segment of FGFR1 occurs
independently of receptor activation (10). Interaction of FRS2 with Ras
is associated with cell cycle progression (11). Numerous other studies
provide evidence that FGF activation of the Ras/MAPK pathway
contributes mainly to cellular proliferation and differentiation (10,
12, 13).
Multiple studies have demonstrated activation of the non-receptor Src
family tyrosine kinase members in response to activated FGFR (14-16).
An association of c-Src and FGFR1 has been reported by
immunoprecipitation of recombinant receptor (14), however, this finding
has not been reported elsewhere. Transient exposure of 3T3 cells to
FGF-1 induces continuous activation of the Src kinase pathway, which in
turn stimulates increases in the level of c-myc (12). In
contrast, Landgren et al. (17) have shown that mutation of
FGFR1 tyrosine residue 766 to phenylalanine causes an increase in
activation of Src family members, suggesting a regulatory role of
protein kinase C in Src activation. The involvement of c-Src in
migration of rat bladder carcinoma cells and murine fibroblasts in
response to FGF-1 has also been reported (12, 18, 19). Similarly, the
direct association of phosphatidylinositol 3-kinase and c-Src in an
SH3-dependent manner suggests that these intracellular
signaling proteins may act in concert downstream of FGFR1 to
co-ordinate cellular motility (20).
The nine known Src family kinase members exhibit functional redundancy
as evident from mice that are homozygous deficient for single
versus multiple Src kinases. The predominant abnormality of
Src To assess the role of c-Src in cells with physiologic levels of FGFR1,
we examined the effect of varying levels of c-Src expression on
FGF-1-induced proliferation of murine embryonic fibroblasts deficient
for Yes and Fyn. These fibroblasts express either no c-Src, endogenous
levels, or 10-fold excess c-Src (24). Cells with endogenous levels of
c-Src exhibited enhanced proliferation to FGF when compared with
c-Src-deficient cells. Unexpectedly, overexpression of c-Src also
inhibited FGF-1-proliferative responses. These findings were confirmed
using deficient cells that were transfected and sorted by FACS for
varying levels of c-Src. Similarly, endogenous levels of c-Src resulted
in maximal response to EGF while overexpression was inhibitory. In
contrast, cell growth in response to PDGF was minimally affected by
c-Src overexpression. The data suggest that these two classes of growth
factors differ in their regulation of MEF-proliferative responses by
c-Src.
Cell Lines and Culture--
Mouse embryonic fibroblasts (MEF)
deficient in c-Src, Yes, and Fyn (SYF) were obtained from the ATCC and
kindly provided by Dr. Graham Carpenter, Vanderbilt University.
Src++ (MEF deficient for Yes and Fyn with endogenous c-Src
expression) and cSrc (SYF cells deficient for Yes and Fyn but
overexpressing c-Src) cells were obtained from the ATCC. Src4 cells
(SYF cells transfected to express endogenous levels of c-Src protein
comparable to Src++ cells) were kindly provided by Dr.
Leslie Cary, Fred Hutchinson Cancer Research Center. These MEF have
been described previously and were also determined to be deficient in
Lck and Lyn (24).2 The cells
were maintained at 37 °C in DMEM containing 4.5 g/liter glucose and
1.5 g/liter sodium bicarbonate supplemented with 1 mM
sodium pyruvate, 10 mM HEPES, 10% FBS, and 500 µg/ml
G418. For experimentation, SYF and cSrc cells were plated at 0.5 × 106, whereas Src++ and Src4 cells were
plated at 0.2 × 106 cells per 100-mm2
dish (unless otherwise specified) in 10% FBS/DMEM supplemented with 20 µg/ml gentamicin. Subconfluent cultures (70%) were serum-starved overnight in 0.1% FBS/DMEM. Cells were stimulated by addition of 0.2%
BSA/DMEM supplemented with 10 units/ml heparin ± 10 ng/ml recombinant human FGF-1 (R&D Systems) for 10 min (unless otherwise specified) at 37 °C. For detection of phosphotyrosine proteins, cells were washed with ice-cold PBS containing 100 µM
sodium orthovanadate and scraped into boiling 2× SDS sample buffer for
sonication. Samples were stored at RNA Extraction and Reverse Transcriptase-PCR--
Total RNA was
isolated from fibroblasts using TRI Reagent® (Molecular
Research Center, Inc.) according to the manufacturer's specifications.
First-strand cDNA was synthesized from 2 µg of total RNA using
oligo-dT and SUPERSCRIPT RNase H-reverse transcriptase (Invitrogen).
Each PCR reaction contained cDNA equivalent to 80 ng of template
DNA. FGFR1 transcripts were detected using oligonucleotide PCR primers
designed to amplify the transmembrane and kinase domains of hFGFR1 as
previously described (4). Sense (5'-GAC AA(A/G) GA(G/A) ATG GAG GTG
CT-3') and antisense (5'-GTT (G/A)TA GCA GTA (T/C)TC CAG CC-3')
primers each exhibited 90% homology to the murine sequences
(human/mouse). FGFR2 transcripts were detected using oligonucleotide
primers designed against the transmembrane and kinase domains of
hFGFR2. Sense (5'-GTC CAT CAA (T/C)CA CAC (G/C)TA CCA CCT G-3') and
antisense (5'-AAT CAT CTT CAT CAT CTC CAT CTC T-3') primers were used
as previously described (4) with slight modification of cycling
parameters as follows: 94 °C for 1 min, 56 °C for 30 s, and
72 °C for 30 s (35 cycles). All PCR reactions included
H2O only (no DNA) as a negative control. The housekeeping
transcript glyceraldehyde-3-phosphate dehydrogenase was used as a
positive control of RNA expression and was amplified over 30 cycles
using sense (5'-ATC GAG CTC ATC CCA TCA CCA TCT TCC AGG-3') and
antisense (5'-ACA TCT AGA GCC ATC ACG CCA CAG TTT CCC-3') primers.
Amplified products were analyzed by separation on 1% agarose gels
containing 0.4 µg/ml ethidium bromide.
Flow Cytometry with Fc-FGF--
FGFR1 expression at the cell
surface was detected by FACS analysis using a modified method of
enzymatic amplification staining (Flow-Amp Systems, Ltd.). Briefly,
1 × 106 cells were washed with Hanks' buffered
saline solution and resuspended in PBS containing 0.2% BSA and 2.5 µg/ml heparin. Fc-FGF (26) was added (100 ng/ml), and the cells were
rotated at 4 °C for 1 h. Cells were collected by brief
microcentrifugation and rapidly washed with PBS containing 250 µg/ml
heparin to remove ligand from low affinity cell surface heparan sulfate
proteoglycan binding sites. Samples were subsequently incubated with
HRP-anti-human IgG (diluted 1:200 in 2% BSA/PBS) for 20 min at
4 °C. The cells were collected by microcentrifugation, washed with
PBS, and stained with Flow-Amp EAS amplifier and
Streptavidin-fluorescein isothiocyanate according to the
manufacturer's protocol. Samples were fixed in 1% paraformaldehyde
and analyzed by flow cytometry.
Antibodies and Western Immunoblotting--
Whole cell lysates
(20 µg/lane) from MEF cultures incubated in the presence or absence
of FGF-1 (10 ng/ml) were separated by 10% SDS-PAGE and transferred to
Immobilon nitrocellulose (Millipore) for protein immunodetection. The
anti-Src clone GD11 monoclonal antibody (Upstate Biotechnology) was
used according to the manufacturer's recommendations. Briefly,
membranes were blocked for 20 min at RT in 3% milk/PBS and antibody
was diluted 1:1000 in block solution for membrane incubation overnight
at 4 °C. The PY20 monoclonal anti-phosphotyrosine antibody
(Transduction Laboratories) was used at 1:1000 in 1% BSA/TBS-T (10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween 20)
for 1 h at RT. Flg (C-15) rabbit polyclonal anti-FGFR1 (Santa Cruz
Biotechnology) was diluted 1:500 in 5% nonfat dry milk/TBS-T for
1 h of membrane incubation at RT. Blots were subsequently incubated with either horseradish peroxidase-conjugated (HRP)-goat anti-mouse IgG (Chemicon International) or HRP-goat anti-rabbit IgG
(Southern Biotechnology Associates, Inc.) diluted 1:5000 in the
appropriate block solution for 45 min at RT. Protein bands of interest
were detected by enhanced chemiluminescence. Membranes were stripped
for 30 min at 70 °C using stripping solution (62.5 mM
Tris (pH 6.8), 2% SDS, and 100 mM Thymidine Incorporation Assay--
SYF cells were plated at
2.5 × 103 cells/well in 96-well flat-bottom plates.
Src++ and cSrc cells were plated at 1.25 × 103 cells per well. Cultures were incubated in 10%
FBS/DMEM for 24 h, washed with sterile PBS, and serum-starved in
0.1% FBS/DMEM overnight. Cells were subsequently incubated for 22 h in 0.2% BSA/DMEM with 10 units/ml heparin supplemented with either
FGF-1, EGF (R&D Systems), PDGF-B (R&D Systems), or LPA (Sigma) as
indicated. [3H]Thymidine was added at 1 µCi/well for
the final 17 h of culture, and incorporation was determined by
liquid scintillation counting. Experiments were performed four times.
For each condition, the mean incorporation of six wells was determined
and normalized as a -fold increase over the basal average ± S.E.
For experiments comparing various growth factors, cell lines were
plated at equal density (1.25 × 103 cells per well)
for thymidine incorporation. Data are expressed as mean incorporation
(total counts per minute) ± S.E. of six individual wells.
Cellular Proliferation--
MEF cells were plated in 24-well
dishes at 2.5 × 104 cells/well in 10% FBS/DMEM.
After 24 h the media was aspirated and the cells were washed with
SF-DMEM. Cultures were incubated in 0.2% BSA/DMEM supplemented with 10 units/ml heparin ± FGF-1, EGF, PDGF-B (all at 10 ng/ml), or 10%
FBS for 5 days. Unless otherwise specified, cells were harvested with
trypsin/EDTA, collected by microcentrifugation, and resuspended in 10%
FBS/DMEM for visual counting every day. Culture media was replaced at
day 3 of stimulation. Data are expressed as mean ± S.E. of three
independent experiments.
Transient Transfection of SYF Cells--
The pLSXH.mSrc
retroviral vector containing mouse non-neuronal Src (mSrc) was made by
Dr. Lionel Arnaud and kindly provided by Dr. Leslie Cary, both of the
Fred Hutchinson Cancer Research Center (Seattle, WA). The mSrc sequence
was excised from pLSXH by BamHI digestion and ligated
to the BamHI sites of pIRES2-EGFP expression vector
(Clontech, kindly provided by Dr. David Piston, Vanderbilt University) (mSrc.EGFP). SYF cells were grown to 70% confluency in 75-cm2 plastic tissue culture flasks and
transiently transfected with empty vector or mSrc.EGFP DNA using
SuperFect transfection reagent (Qiagen) according to the
manufacturer's specifications. Briefly, DNA incubated with 1:5
SuperFect for 10 min at RT was found to provide optimal EGFP expression
and cellular survival. The cultures were transfected for 3 h at
37 °C and incubated with fresh media overnight prior to
experimentation. FACS analysis determined transfection efficiency to be
~19% in the vector control population and ~35% in the mSrc.EGFP
population (not shown).
Immunofluorescent Detection of c-Src--
SYF cells were plated
on ethanol-sterilized glass coverslips in 6-well culture plates at
7.5 × 104 cells/well for 24 h (~70%
confluency). Cells were transiently transfected as described
previously. At twenty-four hours post-transfection the cells were
washed with RT sterile PBS, fixed in 4% paraformaldehyde (10 min, RT),
permeabilized with 0.2% Triton X-100/PBS (5 min, RT), and blocked for
1 h at RT with 10% normal goat serum/block buffer (1% BSA in
PBS). The coverslips were then incubated overnight at 4 °C with 10 µg/ml anti-Src antibody and for 45 min at RT with 1:500 Alexa 568 goat anti-mouse (Molecular Probes). Antibodies were diluted in blocking
buffer. Samples were imaged using the 40×, 1.3-numerical
aperture F-Fluar objective lens of a Zeiss LSM 510 confocal microscope.
Multiline scans (488- and 543-nm laser lines) were used to eliminate
detection bleed-through for the green (bandpass 505-530 filter) and
red (longpass 560 filter) fluorophores. Transfected cells were
identified by expression of EGFP and c-Src expression was confirmed by
visualization of c-Src-associated Alexa fluorescence. The intensity of
EGFP- and c-Src-associated fluorescence was quantified using Scion
Image Software, version 3.6 (Scion Corp.). Internal, non-transfected control cells were also examined as were coverslips incubated in the
absence of primary antibody, and these showed negligible fluorescence.
FACS Sort of c-Src-expressing SYF Cells--
Transiently
transfected SYF cells were washed with serum-free media, harvested with
T/E, and collected by centrifugation prior to resuspension at
106/ml in 1% BSA/PBS with Ca2+ and
Mg2+. EGFP-positive populations were determined by FACS
analysis comparing transfected cells to the SYF parental line. Samples
were gated for sorting of low and high level EGFP-expressing cells,
with gates being maintained between vector and mSrc.EGFP populations. Sorted cells were plated in 10% FBS/DMEM for 48 h to assess
viability. To determine the ability of each population to respond to
FGF-1, cells were harvested with T/E and replated at 2 × 103 cells/well (96-well plates), and
[3H]thymidine incorporation was assessed as previously described.
Functional FGFR1 Is Expressed at the Cell Surface of MEF--
FGFR
expression has not been characterized in SYF, Src4, Src++,
or cSrc mouse embryonic fibroblasts. Therefore, we examined the MEF for
FGFR1 and FGFR2 mRNA expression by reverse transcriptase-PCR. Jurkat C2-14, a T cell tumor line expressing stably transfected FGFR1,
was used as a positive control for FGFR1 expression. BALB/c mouse
aortic endothelial cells (MAECs), which express only FGFR2, were used
as the positive control for FGFR2 expression (25). FGFR1 mRNA was
expressed by all MEF (Fig. 1,
top) but FGFR2 mRNA was not (Fig. 1,
middle).
To confirm expression of FGFR1 protein capable of ligand binding, FACS
analysis was performed using an Fc-FGF fusion protein (26). After
incubation with Fc-FGF, the fibroblasts were washed with heparin to
remove ligand from low affinity heparan sulfate proteoglycan binding
sites on the cell surface. Significant binding of Fc-FGF to high
affinity cell surface receptor binding sites was observed for all
fibroblasts (Fig. 2A). The
mean fluorescence intensity, a reflection of the number of receptors
per cell, increased modestly with enhanced c-Src expression.
Derivation of the multiply deficient SYF cell lines required
immortalization of the primary cells with SV40 large T antigen (24).
c-Src was reintroduced into SYF to produce cSrc, a line with 10-fold
greater expression than wild-type cells but which has the same clonal
origin as SYF (24). The Src++ line was derived from a
different embryo and therefore may have clonal differences from SYF and
cSrc in addition to differences arising from levels of c-Src protein
expression. To overcome this problem, we also examined the Src4 MEF
line, which was derived from SYF cells and expresses endogenous levels
of c-Src protein. Expression of 60-kDa c-Src was confirmed by
immunoblotting with anti-c-Src antibody (Fig. 2B). As
expected, c-Src was not detected in SYF cells, was present at
comparable levels in Src4 and Src++ cells, and was
overexpressed in cSrc cells. Expression of the non-glycosylated and
glycosylated forms of full-length FGFR1 was confirmed in all cell lines
as proteins of ~120-125 and 145 kDa (Fig. 2C). A slight
retardation of mobility was observed for the 145-kDa fully glycosylated
form with FGF stimulation, reflecting tyrosine phosphorylation
predominantly of the cell surface receptor. A protein of ~90-kDa
molecular mass was visualized in all FGF-1-stimulated MEF by
phosphotyrosine immunoblotting (Fig. 2D, arrow).
Molecular size suggests this is FRS2, and its phosphorylation confirms
activation of FGFR1. As expected, overexpression of c-Src also caused a
marked increase in total tyrosine phosphorylation.
FGF-1-induced Mitogenic Activity Is Regulated by the Level of c-Src
Expression--
Experiments using neutralizing antibodies or
dominant-negative forms of c-Src have shown that, in fibroblasts,
mitogenic responses to EGF, PDGF, and CSF-1 depend upon Src kinase
activity (27, 28). In contrast, Zhan et al. (14) have
reported that FGF-induced proliferation of NIH 3T3 cells was not
dependent on c-Src, and Klinghoffer et al. (24) suggest that
c-Src, Yes, and Fyn are largely dispensable for PDGF-induced
proliferation and signaling, because SYF cells exhibit mitogenic
responses to this growth factor. To examine MEF mitogenic response to
FGF-1, serum-starved cells were stimulated with FGF-1, and DNA
synthesis was measured by [3H]thymidine incorporation. A
dose response was observed, and FGF-1 at 1 ng/ml stimulated maximal
incorporation in all cell lines (Fig. 3).
Src++ cells, however, demonstrated significantly enhanced
incorporation compared with either the deficient SYF cells or the
overexpressing cSrc cells. Src4 cells, which have the same clonal
derivation as SYF and cSrc but express endogenous levels of c-Src, also
exhibited greater thymidine incorporation than SYF or cSrc cells at all concentrations of FGF-1 examined (data not shown). These results suggested that endogenous levels of c-Src expression are optimal for
transition of quiescent fibroblasts from G0 to
G1 phase of the mitotic cycle in response to FGF-1.
MEF growth was examined to determine the effects of c-Src expression
and FGF-1 stimulation on later phases of the mitotic cycle (Fig.
4). SYF, Src++, and cSrc
cells were plated at equal density in serum and exposed to FGF-1 for 5 days. In the absence of serum or FGF (Fig. 4A, black
bars), the number of viable cells ultimately declined. Not surprisingly, the most rapid loss of viability occurred in
c-Src-deficient SYF cells. However, in response to FGF,
Src++ cells unexpectedly showed significantly greater
proliferation than cells overexpressing c-Src (Fig. 4A,
middle versus lower panels). To
confirm that these results were not a consequence of the
Src++ cell line being derived from a different embryo (24),
the study was repeated and included Src4 cells that were derived from
the same embryo as SYF and cSrc cells (Fig. 4B). Src4 and
Src++ cells expressing endogenous levels of c-Src again
demonstrated significantly greater cell growth than either SYF or
overexpressing cSrc cells. In contrast, growth in FBS correlated
directly with the quantity of c-Src, and no inhibition was seen with
overexpression (Fig. 4C).
These data demonstrate that, although c-Src is not essential for
FGF-1-stimulated MEF growth, endogenous levels of expression are
optimal for maximal proliferation. To quantify this observation and
eliminate clonal variability, the deficient SYF cells were transiently
transfected with a control pIRES2-EGFP expression vector or vector
containing the non-neuronal mSrc sequence. Cells were sorted based on
c-Src expression, and [3H]thymidine incorporation of the
subpopulations was measured. Transfected cells were identified by EGFP
immunofluorescence (Fig. 5, B
and E). Cells expressing c-Src were identified by
immunostaining (Fig. 5, C and F). The intensity
of EGFP expression was an excellent indicator of c-Src expression in
mSrc.EGFP cultures (Fig. 5G, r2 = 0.953). Cells with low EGFP expression ("Low") and high
EGFP expression ("High") were isolated by sorting (Fig.
6A, populations collected as
indicated by bars). Sorted subpopulations were resuspended in 10% FBS/DMEM for 48 h to determine viability. Vector control cells exhibited reduced viability and fewer cells were recovered than those that were plated, whereas mSrc.EGFP cells exhibited a 4.6- (Low) and 2.6-fold (High) increase in total cell
number. Consequently, insufficient pIRES vector control cells were
recovered to include all three FGF-1 concentrations in the
[3H]thymidine incorporation assay.
Low level expression of c-Src significantly enhanced FGF-1-induced DNA
synthesis at all concentrations of FGF-1 examined (Fig. 6B).
A high level of expression of c-Src suppressed
[3H]thymidine incorporation compared with low level
c-Src-expressing cells, and these values were no different than vector
control cells. No significant dose response to FGF-1 was observed in
control cells. The results show directly that either overexpression or deficiency of c-Src results in impaired initiation of the mitotic cycle
in response to FGF-1.
Effects of Quantitative Differences in c-Src on Other Growth
Factors--
The results with FGF-1 suggested that there is an
"optimal" level of c-Src for growth factor-induced mitogenesis. To
determine whether this phenomenon is unique to FGF or common to other
growth factors, we examined the effects of varying c-Src expression on response to EGF, PDGF-B, and LPA (Fig.
7A). Compared with both deficient cells and cells overexpressing c-Src, Src4 cells that express
endogenous levels of c-Src showed maximal DNA synthesis to all stimuli,
but some striking differences were apparent. Restoration of endogenous
levels of c-Src resulted in significantly enhanced response to FGF and
EGF compared with deficient cells. For both FGF-1 and EGF, the response
of overexpressing cSrc cells was not different from deficient cells. In
contrast, responses to PDGF-B and LPA were only minimally enhanced by
restoration of endogenous levels of c-Src. Comparison to basal DNA
synthesis of these cell lines in the absence of growth factors showed
that varying levels of c-Src expression had little effect on response
to PDGF-B and LPA (Fig. 7A, values above bars).
The latter data for cSrc and SYF cells are consistent with results
reported previously by Klinghoffer et al. (24) for PDGF.
Distinct effects of c-Src overexpression between PDGF versus
FGF-1 and EGF were also apparent when we assessed proliferation (Fig.
7B). Maximal proliferation to all individual growth factors was seen in Src4 cells that express endogenous levels of c-Src. However, overexpressing cSrc cells showed markedly reduced
proliferation to FGF-1 and EGF but only minimal reduction to PDGF-B.
This was not due to a generalized defect in overexpressing cells as
evident from the fact that the greatest increase in cell number
occurred in overexpressing cSrc cells grown in FBS.
Experiments using neutralizing antibodies or dominant-negative
forms of Src have shown that Src family kinases are required for EGF-,
PDGF-, and CSF-1-stimulated entry to the S-phase of the mitotic cycle
in fibroblasts (27-29). Similarly, it has been reported that c-Src,
Fyn, and Yes are activated at mitosis and play a role in cellular
division at the G2-M transition phase (29, 30). Here we
show that SYF, Src4, Src++, and cSrc MEF express functional
FGFR1, but not FGFR2, similar to human fetal fibroblasts (31). All MEF
responded to FGF-1 as measured by thymidine incorporation and increased
cell number, indicating that FGF-1 can induce proliferation even in the
absence of Src family kinases. Surprisingly, however, cells expressing endogenous levels of c-Src showed greater responsiveness to FGF than
both Src-deficient and Src-overexpressing cells. This was the case for
both clonally derived cell lines and Src-transfected SYF cells,
demonstrating that the effect is due to Src and not simply a result of
clonal origin.
Our results differ from Liu et al. (19) who reported that
Src deficiency did not affect FGF-1-induced proliferation in cells from
Src In contrast to the experiments with neutralizing antibodies and
dominant-negative Src, Klinghoffer et al. (24) reported that
c-Src is dispensable for PDGF- and LPA-induced proliferation. They
found no significant difference in thymidine incorporation between
deficient SYF cells and overexpressing cSrc cells. The 5-fold increase
in total cell number for cSrc versus 3-fold increase for SYF
cells in response to PDGF was not specifically addressed, and cells
expressing endogenous levels of c-Src were not examined. Our results
for PDGF-induced DNA synthesis (Fig. 7A) and proliferation (Fig. 7B) of cSrc versus SYF cells are consistent
with those of Klinghoffer. Using cells with endogenous levels of c-Src,
we now show that the quantity of c-Src also has little effect on
PDGF-induced proliferation. An important caveat is that all of these
MEF lines are derived after transformation with SV40 large T antigen.
Broome and Courtneidge (33) have reported that MEF responses to PDGF occur due to the independent effect of SV40 large T antigen on c-myc induction rather than c-Src expression. Their
study showed data for NIH 3T3 fibroblasts and wild-type MEF expressing
an interfering form of Src, and data for Src-deficient or Src
overexpressing cells was not provided.
The unanticipated observation in our experiments was the finding that
quantitative differences in c-Src expression have major effects on
FGF-1 induced entry into cell cycle and cell growth. MEF with
endogenous levels of c-Src showed maximal DNA synthesis and
proliferation, whereas cells with 10-fold overexpression of c-Src
behaved identically to those that lack c-Src when stimulated by FGF-1.
This is in contrast to cells in the absence of growth factor
stimulation, where overexpression of c-Src clearly rescues cells from
death (Figs. 4A and 7B). This indicates that
c-Src has negative as well as positive regulatory effects on FGFR1
signaling and suggests that there is an "optimal" level of c-Src
for maximal FGF-induced proliferation. The pronounced negative effect
of c-Src overexpression was also observed for EGF but not for PDGF-B,
LPA, or serum. This may indicate that there are similar mechanisms for
Src regulation of FGF and EGF signaling pathways that differ from PDGF.
The mechanisms underlying the negative effects of c-Src overexpression
are not addressed by our data. At least for FGFR1, it is unlikely to
involve quantitative effects on cell surface receptor density or
maximal ERK1/2 activation, because these were similar in cells with
endogenous levels of c-Src and overexpression (Fig. 2A and
data not shown).
, and Ras.
Conflicting reports exist concerning the interaction between FGFR1 and
Src family kinases. To address the role of c-Src in FGFR1 signaling, we
compared proliferative responses of murine embryonic fibroblasts (MEF)
deficient in c-Src, Yes, and Fyn to MEF expressing either endogenous
levels or overexpressing c-Src. MEF with endogenous c-Src had
significantly greater FGF-induced DNA synthesis and proliferation than
cells lacking or overexpressing c-Src. This was related directly to
c-Src expression by analysis of c-Src-deficient cells transfected with
and sorted for varying levels of a c-Src expression vector. This
suggests an "optimal" quantity of c-Src expression for FGF-induced
proliferation. To determine if this was a general phenomenon for growth
factor signaling pathways utilizing c-Src, responses to epidermal
growth factor (EGF), platelet-derived growth factor (PDGF), and
lysophosphatidic acid (LPA) were examined. As for FGF, responses
to EGF were clearly inhibited when c-Src was absent or overexpressed.
In contrast, varying levels of c-Src had little effect on responses to
PDGF or LPA. The data show that mitogenic pathways activated by FGF-1 and EGF are regulated by c-Src protein levels and appear to differ significantly from those activated by PDGF and LPA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice is osteopetrosis (21) and mice deficient in
other individual Src family kinases exhibit mild phenotypes (reviewed
in Ref. 22), whereas multiply deficient animals demonstrate severe
phenotypes or lethality (23, 24). With such overlap in function, it is difficult to delineate the exact role of Src family kinases in FGFR
signaling. Therefore, the absence of one family member may not
eliminate FGFR-induced biological responses if other members are
available to compensate.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. BALB/c mouse
aortic endothelial cells (MAECs) have been described previously (25)
and were provided by Dr. Keith Bishop.
-mercaptoethanol) for
reprobing with relevant antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Murine embryonic fibroblasts express FGFR1
mRNA. Total RNA was extracted from fibroblast cultures and
reverse-transcribed to cDNA. Primers were designed to amplify
regions encoding the transmembrane and kinase domains of either FGFR1
(top) or FGFR2 (middle). Products were separated
by 1% agarose containing 0.4 µg/ml ethidium bromide.
Glyceraldehyde-3-phosphate dehydrogenase cDNA was amplified as a
loading control (bottom). M, molecular weight
markers; Y, SYF; 4, Src4; +,
Src++; c, cSrc; J, C2-14 Jurkat R1
(FGFR1 control); E, MAEC (FGFR2 control); ,
H2O.
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[in a new window]
Fig. 2.
FGFR1 expressed at the cell surface of MEF
are capable of activation by ligand binding. A,
detection of cell surface FGFR1 expression by flow cytometry.
Shaded tracings are cells incubated with Fc-FGF, anti-human
IgG-HRP and streptavidin-fluorescein isothiocyanate. Open
tracings are cells stained in the same way without Fc-FGF.
B, Western immunoblotting of SYF, Src4, Src++,
and cSrc whole cell lysates (20 µg/lane) in the absence ( ) or
presence (+) of 10 ng/ml FGF-1 (10 min) using anti-cSrc antibody.
C, the same membrane immunoblotted for FGFR1. D,
total cellular tyrosine phosphorylation detected by immunoblotting with
anti-phosphotyrosine antibody. The arrow denotes enhanced
phosphorylation of 90-kDa FRS2 in the presence of FGF-1. The molecular
weight marker is indicated at the left. Blots are
representative of three independent experiments.
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Fig. 3.
Fibroblasts expressing endogenous levels of
c-Src are most sensitive to FGF-1 induction of the mitotic cycle.
Thymidine incorporation was determined following 22 h of
stimulation with FGF-1 (0.1-10 ng/ml) and is expressed as average
-fold increase over basal incorporation ± S.E. Values represent
data from four separate experiments and are derived from sextuplet
wells in each experiment.
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Fig. 4.
Fibroblasts expressing endogenous levels of
c-Src are significantly more sensitive to FGF-1 induced cellular
proliferation. A, cells were plated at equal density
(2.5 × 104) in 10% FBS/DMEM for 24 h. Cells
were subsequently stimulated for 5 days with 0.2% BSA ± 10 ng/ml
FGF-1, and media were replaced at day 3. Total adherent cells were
harvested and counted every day. B, similar experiment as
described in A, including the Src4 cell line. Cells were
counted at day 5. C, similar experiment as described in
B with cells grown in 10% FBS. Data represent the mean ± S.E. for duplicate wells from three separate experiments. Dark
bars in A and B represent basal cultures
(0.2% BSA); hatched bars represent FGF-1-stimulated
cultures.
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Fig. 5.
Correlation between EGFP fluorescence and
c-Src expression. SYF cells transfected with control pIRES
vector (A-C) or mSrc.EGFP (D-F)
were immunostained for c-Src. Differential interference images
(A and D); EGFP expression (B and
E); c-Src expression (C and F).
G, a strong linear relationship was determined between the
intensity of EGFP and c-Src-associated Alexa fluorescence
(r2 = 0.953). Scale bar represents 10 µm.
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Fig. 6.
SYF fibroblasts expressing low level c-Src
kinase are more sensitive to FGF-1 induction of the mitotic cycle than
high level c-Src-expressing cells. A, detection of EGFP
expression by flow cytometry. Black tracing represents pIRES
vector control cells; gray tracing represents mSrc.EGFP
cells; dotted tracing represents the SYF parental
population. Bars indicate gated populations isolated by
sorting. B, low level c-Src expression enhanced
[3H]thymidine incorporation in SYF cells in the presence
of FGF-1 (0.1-10 ng/ml) compared with either high level c-Src
expression or control pIRES expression. Data represent the average
-fold increase in thymidine incorporation over basal values ± S.E. of sextuplet wells.
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Fig. 7.
EGF-induced proliferation is also maximal in
fibroblasts expressing endogenous levels of c-Src. A,
thymidine incorporation was determined following 22 h of
stimulation with FGF-1, EGF, PDGF-B, or LPA as indicated and is
expressed as average total counts per minute ± S.E. Values
above bars represent the same data expressed as -fold increase
over basal ± S.E. and represent six individual wells. White
bars represent SYF cultures; light gray hatched bars
represent Src4 cells; dark gray hatched bars represent cSrc
cells. B, cells were plated at equal density (2.5 × 104) in 10% FBS/DMEM for 24 h. Cultures were
subsequently stimulated with 0.2% BSA/DMEM ± FGF-1, EGF, PDGF-B
(all at 10 ng/ml), or 10% FBS. Cells were counted on day 4, and data
represent the average total cell number ± S.E. of three
individual wells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice compared with Src+/+ mice. The
difference likely reflects the compensatory effects of Fyn and Yes that
were expressed in Src
/
cells used previously
versus the absence of other Src family kinases in the cells
used here. In addition, inclusion of high concentrations of insulin in
their medium may have activated insulin and IGF receptors thereby
stimulating the Ras/Raf/MEK pathway and masking the effects for FGF
(reviewed in Ref. 32).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Graham Carpenter, Vanderbilt University, for providing SYF cells and Dr. Keith Bishop, University of Michigan, for providing MAECs. We also thank Dr. Leslie Cary, Fred Hutchinson Cancer Research Center, for the gift of Src4 cells and the pLSXH.mSrc retroviral vector made by Dr. Lionel Arnaud, also from the Fred Hutchinson Cancer Research Center. Dr. David Piston, Vanderbilt University, generously provided the pIRES2-EGFP expression vector.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL53771 (to G. G. M.) and Training Grant T32-HL69765-01 (to D. M. K.) and aided by the Vanderbilt Cell Imaging Resource supported by Grants P30CA68485 and DK20593.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.
To whom correspondence should be addressed:
Vanderbilt University Medical School, A3310 Medical Center North,
Nashville, TN 37232-2605. Tel.: 615-322-2305; Fax: 615-343-6160;
E-mail: Geraldine.Miller@vanderbilt.edu.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M209698200
2 D. Kilkenny, unpublished result.
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ABBREVIATIONS |
---|
The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; MAPK, mitogen-activated protein kinase; FACS, fluorescence-activated cell sorting; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; RT, room temperature; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; MEF, murine embryonic fibroblast; SYF, c-Src, Yes, and Fyn; MAECs, mouse aortic endothelial cells; LPA, lysophosphatidic acid.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Powers, C. J.,
McLeskey, S. W.,
and Wellstein, A.
(2000)
Endocr. Relat. Cancer
7,
165-197 |
2. | Yamashita, T., Yoshioka, M., and Itoh, N. (2000) Biochem. Biophys. Res. Commun. 277, 494-498[CrossRef][Medline] [Order article via Infotrieve] |
3. | Basilico, C., and Moscatelli, D. (1992) Adv. Cancer Res. 59, 115-165[Medline] [Order article via Infotrieve] |
4. | Zhao, X. M., Frist, W. H., Yeoh, T. K., and Miller, G. G. (1994) J. Clin. Invest. 94, 992-1003[Medline] [Order article via Infotrieve] |
5. |
Miller, G. G.,
Davis, S. F.,
Atkinson, J. B.,
Chomsky, D. B.,
Pedroso, P.,
Reddy, V. S.,
Drinkwater, D. C.,
Zhao, X. M.,
and Pierson, R. N.
(1999)
Circulation
100,
2396-2399 |
6. | Dickson, C., Spencer-Dene, B., Dillon, C., and Fantl, V. (2000) Breast Cancer Res. 2, 191-196[CrossRef][Medline] [Order article via Infotrieve] |
7. | Johnson, D. E., and Williams, L. T. (1993) Adv. Cancer Res. 60, 1-41[Medline] [Order article via Infotrieve] |
8. | Jaye, M., Schlessinger, J., and Dionne, C. A. (1992) Biochim. Biophys. Acta 1135, 185-199[Medline] [Order article via Infotrieve] |
9. | Klint, P., and Claesson-Welsh, L. (1999) Front. Biosci. 4, d165-d177[Medline] [Order article via Infotrieve] |
10. |
Ong, S. H.,
Hadari, Y. R.,
Gotoh, N.,
Guy, G. R.,
Schlessinger, J.,
and Lax, I.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
6074-6079 |
11. | Kouhara, H., Hadari, Y. R., Spivak-Kroizman, T., Schilling, J., Bar-Sagi, D., and Schlessinger, J. (1997) Cell 89, 693-702[Medline] [Order article via Infotrieve] |
12. |
LaVallee, T. M.,
Prudovsky, I. A.,
McMahon, G. A.,
Hu, X.,
and Maciag, T.
(1998)
J. Cell Biol.
141,
1647-1658 |
13. | Klint, P., and Claesson-Welsh, L. (1999) Oncogene 18, 179-185 |
14. |
Zhan, X.,
Plourde, C.,
Hu, X.,
Friesel, R.,
and Maciag, T.
(1994)
J. Biol. Chem.
269,
20221-20224 |
15. | Landgren, E., Klint, P., Yokote, K., and Claesson-Welsh, L. (1998) Oncogene 17, 283-291[CrossRef][Medline] [Order article via Infotrieve] |
16. | Yayon, A., Ma, Y. S., Safran, M., Klagsbrun, M., and Halaban, R. (1997) Oncogene 14, 2999-3009[CrossRef][Medline] [Order article via Infotrieve] |
17. | Landgren, E., Blume-Jensen, P., Courtneidge, S. A., and Claesson-Welsh, L. (1995) Oncogene 10, 2027-2035[Medline] [Order article via Infotrieve] |
18. | Rodier, J. M., Valles, A. M., Denoyelle, M., Thiery, J. P., and Boyer, B. (1995) J. Cell Biol. 131, 20221-20224 |
19. | Liu, J., Huang, C., and Zhan, X. (1999) Oncogene 18, 6700-6706[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Fincham, V. J.,
Brunton, V. B.,
and Frame, M. C.
(2000)
Mol. Cell. Biol.
20,
6518-6536 |
21. | Soriano, P., Montgomery, C., Geske, R., and Bradley, A. (1991) Cell 64, 693-702[Medline] [Order article via Infotrieve] |
22. | Lowell, C. A., and Soriano, P. (1996) Genes Dev. 10, 1845-1857[CrossRef][Medline] [Order article via Infotrieve] |
23. | Stein, P. L., Vogel, H., and Soriano, P. (1994) Genes Dev. 8, 1999-2007[Abstract] |
24. |
Klinghoffer, R. A.,
Sachsenmaier, C.,
Cooper, J. A.,
and Soriano, P.
(1999)
EMBO J.
18,
2459-2471 |
25. |
Bastaki, M.,
Nelli, E. E.,
Dell'Era, P.,
Rusnati, M.,
Molinari-Tosatti, M. P.,
Parolini, S.,
Auerbach, R.,
Ruco, L. P.,
Possati, L.,
and Presta, M.
(1997)
Arterioscler. Thromb. Vasc. Biol.
17,
454-464 |
26. |
Dikov, M. M.,
Reich, M. B.,
Dworkin, L.,
Thomas, J. W.,
and Miller, G. G.
(1998)
J. Biol. Chem.
273,
15811-15817 |
27. |
Twamley-Stein, G. M.,
Pepperkok, R.,
Ansorge, W.,
and Courtneidge, S. A.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7696-7700 |
28. | Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtneidge, S. A. (1995) Mol. Cell. Biol. 15, 1102-1109[Abstract] |
29. | Roche, S., Fumagalli, S., and Courtneidge, S. A. (1995) Science 269, 1567-1569[Medline] [Order article via Infotrieve] |
30. | Chackalaparampil, I., and Shalloway, D. (1998) Cell 52, 801-810 |
31. | Tartaglia, M., Fragale, A., and Battaglia, P. A. (2001) DNA Cell Biol. 20, 367-379[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Bevan, P.
(2001)
J. Cell Sci.
114,
1429-1430 |
33. | Broome, M. A., and Courtneidge, S. A. (2000) Oncogene 19, 2867-2869[CrossRef][Medline] [Order article via Infotrieve] |