c-Src regulation of fibroblast growth factor-induced proliferation in murine embryonic fibroblasts.

Activated fibroblast growth factor receptor 1 (FGFR1) propagates FGF signals through multiple intracellular pathways via intermediates FRS2, PLCgamma, 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.

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)(2)(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 nonreceptor 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 Ϫ/Ϫ 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 FGFRinduced biological responses if other members are available to compensate.
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-1proliferative 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.

EXPERIMENTAL PROCEDURES
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 ϩϩ (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 ϩϩ 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 g/ml G418. For experimentation, SYF and cSrc cells were plated at 0.5 ϫ 10 6 , whereas Src ϩϩ and Src4 cells were plated at 0.2 ϫ 10 6 cells per 100-mm 2 dish (unless otherwise specified) in 10% FBS/DMEM supplemented with 20 g/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 Ϯ 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 M sodium orthovanadate and scraped into boiling 2ϫ SDS sample buffer for sonication. Samples were stored at Ϫ20°C. BALB/c mouse aortic endothelial cells (MAECs) have been described previously (25) and were provided by Dr. Keith Bishop.
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 g 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 (5Ј-GAC AA(A/G) GA(G/A) ATG GAG GTG CT-3Ј) and antisense (5Ј-GTT (G/A)TA GCA GTA (T/C)TC CAG CC-3Ј) 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 (5Ј-GTC CAT CAA (T/C)CA CAC (G/C)TA CCA CCT G-3Ј) and antisense (5Ј-AAT CAT CTT CAT CAT CTC CAT CTC T-3Ј) 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 H 2 O only (no DNA) as a negative control. The housekeeping transcript glyceraldehyde-3phosphate dehydrogenase was used as a positive control of RNA expression and was amplified over 30 cycles using sense (5Ј-ATC GAG CTC ATC CCA TCA CCA TCT TCC AGG-3Ј) and antisense (5Ј-ACA TCT AGA GCC ATC ACG CCA CAG TTT CCC-3Ј) primers. Amplified products were analyzed by separation on 1% agarose gels containing 0.4 g/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 ϫ 10 6 cells were washed with Hanks' buffered saline solution and resuspended in PBS containing 0.2% BSA and 2.5 g/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 g/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 g/ 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 HRPgoat 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 ␤-mercaptoethanol) for reprobing with relevant antibodies.
Thymidine Incorporation Assay-SYF cells were plated at 2. 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-cm 2 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 ϳ19% in the vector control population and ϳ35% in the mSrc.EGFP population (not shown).
Immunofluorescent Detection of c-Src-SYF cells were plated on eth-2 D. Kilkenny, unpublished result.

c-Src Regulation of FGFR1 Signaling
anol-sterilized glass coverslips in 6-well culture plates at 7.5 ϫ 10 4 cells/well for 24 h (ϳ70% 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 g/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 40ϫ, 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-Srcassociated 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 10 6 /ml in 1% BSA/ PBS with Ca 2ϩ and Mg 2ϩ . 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 ϫ 10 3 cells/well (96-well plates), and [ 3 H]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 ϩϩ , 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 ϩϩ 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 ϩϩ 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 ϳ120 -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 ϳ90-kDa c-Src Regulation of FGFR1 Signaling 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 [ 3 H]thymidine incorporation. A dose response was observed, and FGF-1 at 1 ng/ml stimulated maximal incorporation in all cell lines (Fig. 3). Src ϩϩ 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 G 0 to G 1 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 ϩϩ , 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 ϩϩ 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 ϩϩ cell line being derived from a different embryo (24), the study was repeated

c-Src Regulation of FGFR1 Signaling
and included Src4 cells that were derived from the same embryo as SYF and cSrc cells (Fig. 4B). Src4 and Src ϩϩ 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 [ 3 H]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, r 2 ϭ 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 [ 3 H]thymidine incorporation assay.
Low level expression of c-Src significantly enhanced FGF-1induced DNA synthesis at all concentrations of FGF-1 examined (Fig. 6B). A high level of expression of c-Src suppressed [ 3 H]thymidine incorporation compared with low level c-Srcexpressing 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. DISCUSSION 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)(28)(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 G 2 -M transition phase (29,30). Here we show that SYF, Src4, Src ϩϩ , 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 Ϫ/Ϫ mice compared with Src ϩ/ϩ mice. The difference likely reflects the compensatory effects of Fyn and Yes that were expressed in Src Ϫ/Ϫ 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).
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, be- c-Src Regulation of FGFR1 Signaling cause these were similar in cells with endogenous levels of c-Src and overexpression ( Fig. 2A and data not shown).