(Received for publication, May 22, 1995; and in revised form, October 30, 1995)
From the
The expression of fibroblast growth factor (FGF) 1, a potent neurotrophic factor, increases during differentiation and remains high in adult neuronal tissues. To examine the importance of this expression on the neuronal phenotype, we have used PC12 cells, a model to study FGF-induced neuronal differentiation. After demonstrating that FGF1 and FGF2 are synthesized by PC12 cells, we investigated if FGF1 expression could be a key element in differentiation. Using the cell signaling pathway to determine the effects of FGF1 alone, FGF1 plus heparin, or a mutated FGF1, we showed an activation to the same extent of mitogen-activated protein (MAP) kinase kinase and MAP kinase (extracellular regulated kinase 1). However, only FGF1 plus heparin could promote PC12 cell differentiation. Thus, the MAP kinase pathway is insufficient to promote differentiation. Analysis of the PC12 cells after the addition of FGF1 plus heparin or FGF2 demonstrated a significant increase in the level of FGF1 expression with the same time course as the appearance of the neuritic extensions. Transfection experiments were performed to enhance constitutivly or after dexamethasone induction the level of FGF1 expression. The degree of differentiation achieved by the cells correlated directly with the amount of FGF1 expressed. The MAP kinase pathway did not appear to be involved. Interestingly, a 5-fold increase in FGF1 in constitutive transfected cells extended dramatically their survival in serum-free medium, suggesting that the rise of FGF1 synthesis during neuronal differentiation is probably linked to their ability to survive in the adult. All of these data demonstrate that, in contrast to the MAP kinase cascade, FGF1 expression is sufficient to induce in PC12 cells both differentiation and survival. It also shows that auto- and trans-activation of FGF1 expression is involved in the differentiation process stimulated by exogenous FGFs through a new pathway which remains to be characterized.
FGF1 ()and 2 are widely distributed in the peripheral
and central nervous systems in the adult. In rat brain, FGF2 is present
in most neurons within the cerebral cortex(1) ,
hippocampus(2) , and cerebellum(3) . High levels of
FGF1 expression have been observed in motor neurons, primary sensory
neurons, and retinal ganglion neurons(4, 5) . In chick
brain, the expression of FGF1 is developmentally regulated(6) .
In bovine and rat embryonic retina, all neuronal layers express FGF1
with an appearance corresponding to their sequential
differentiation(7, 8) . In rat, the level of FGF1
expression remains uniformly low throughout the embryonic period until
postnatal day 7. Thereafter, it increases rapidly, reaching a maximum
in the adult retina. In the intermediate central nervous system,
subclasses of FGF receptors appear to be down-regulated during
development(9) , and during retinal embryonic development, the
expression of FGFR1 and FGFR2 follows the retinal
layering(10) . These patterns of FGF expression suggest that
these growth factors are involved in the integrity, development, and
differentiation of the central nervous system. In fact, in vitro studies have shown that FGF1 promotes the survival of
photoreceptors (11) and the neuritic outgrowth of dissociated
retinal ganglion cells(12) . FGF1 also inhibits pigmentation of
immature pigmented epithelium cells of embryonic chick retina and
stimulates ganglion cell differentiation (13) . FGF2 promotes
the survival of neurons of the peripheral (14) and central
nervous systems (15, 16) and delays photoreceptor
degeneration in a retinal degeneration model(17, 18) .
These FGF activities in in vitro systems together with the temporal and spatial expression patterns of FGF in embryonic and adult neuronal tissues suggested that the FGF expressed by neuronal cells could be involved in the mediation of their neurotrophic activity. To investigate whether the expression of FGF1 by neuronal cells was implicated in the differentiation and neuronal survival, we have used PC12 cells as an in vitro model. This cell line was derived from a rat adrenal tumor (24) and responded to NGF and FGF by the extension of neurites and the acquisition of sympathetic neuronal phenotype. The transition of chromaffin phenotype to neuronal phenotype by NGF and FGF is accompanied by events mediated by the activation of high affinity tyrosine kinase receptors and the activation of the MAP kinase cascade. This is a main signaling pathway for cell proliferation, differentiation, and transformation and appears to mediate differentiation of PC12 cells induced by NGF and FGF2 (25, 26, 27, 28) .
In this study,
we show that PC12 cells expressed both FGF1 and FGF2. These cells were
treated with different stimuli: FGF1-heparin, FGF2, and NGF (promotors
of differentiation) or Lys-132-mutated FGF1 (FGF1) and
FGF1 alone (which do not promote differentiation). The expression of
FGF1 at the transcript and protein level in parallel with the
activation of the MAP kinase cascade was investigated in stimulated
cells as a function of neuronal differentiation. The effect of FGF1
expression on neuronal differentiation was also examined in transfected
cells in which FGF1 expression was under the control of a constitutive
or dexamethasone-inducible promotor.
We show that the expression of
FGF1 was activated only by exogenous FGF stimuli (FGF2 and
FGF1-heparin) which are neurotrophic for PC12 cells and was unchanged
when cells remained undifferentiated upon treatment with FGF1 alone or
mutated FGF1. In contrast, the MAP kinase cascade was
similarly activated in stimulated cells whether or not they
differentiated in sympathetic neurons. Accordingly, in transfected
cells the expression of FGF1 strictly correlated with the
differentiated phenotype and increased the survival of PC12 cells. We
thus propose that the activation of the MAP kinase cascade is
insufficient to induce the differentiation and survival of PC12 cells,
and that the expression of FGF1 stimulated by exogenous FGF stimuli is
a key element in the neurotrophic activities of the FGF.
Figure 1:
Expression of FGF1 and FGF2 transcripts
during the morphological differentiation process of PC12 cells treated
with FGF1 and heparin. A, PC12 cells express FGF1 and FGF2
transcripts. 1 µg of total RNA from PC12 cells and 170 pg of
nitrate reductase (NR) transcripts were reverse transcribed
and 1/10 of reverse transcripts amplified by PCR, using
oligonucleotides specific for FGF1 (rFGF1S, rFGF1AS), FGF2, and NR.
After 28 cycles of amplification, the PCR products were
electrophoresed, analyzed by Southern blotting, and hybridized with
FGF1, FGF2, and NR-specific probes. B, C, and D, PC12 cells were treated with FGF1 (100 ng/ml) and heparin
(10 µg/ml) during 1 h, 2 h, 1 day, 3 days, and 5 days. Total RNA
from treated cells was extracted and analyzed for FGF1 (B) or
FGF2 (D) expression by the RT-PCR assay as in A. PC12
cells cultured in the absence of FGF1 and heparin during 1, 3, and 5
days were used as control cells and analyzed for FGF1 (C)
expression. After 29 cycles of amplification, the intensity of the
amplified FGF1 and FGF2 products hybridized with specific probes was
quantified by densitometric analysis, and expressed as a percentage of
the intensity of the amplified FGF products at day 5. This experiment
has been repeated independently three times with similar results. E, PC12 cells were treated with 100 ng/ml FGF1 and 10
µg/ml heparin. Neuritic extension formation was examined after 1,
2, 3, and 5 days of treatment (magnification
85).
Figure 2:
Effects
of FGF2, FGF1, and FGF1 on neuritic extension in PC12
cells. PC12 cells were plated onto poly-L-lysine-coated dishes
in DMEM supplemented with 10% fetal calf serum and 5% horse serum.
Twenty-four hours after plating, the cells were treated with 100 ng/ml
FGF1 (2), 100 ng/ml FGF1 plus 10 µg/ml heparin (3), 10 ng/ml FGF2 (4), 50 ng/ml NGF (5), 5
µg/ml FGF1
plus 10 µg/ml heparin (6),
5 µg/ml FGF1
(7), or 10 µg/ml heparin (8). Untreated cells were used as a control (1). The
morphology of the cells was examined after 4 days of treatment
(magnification
75).
Figure 3:
Analysis by RT-PCR of FGF1 transcripts in
differentiated and undifferentiated PC12 cells. Total RNA was extracted
from PC12 cells after 3 days of treatment with 100 ng/ml FGF1, in the
absence (lane 2) or presence (lane 3) of heparin (10
µg/ml), with 10 ng/ml of FGF2 (lane 4), with 100 ng/ml of
NGF (lane 5) or with 100 ng/ml FGF1 plus
heparin (10 µg/ml) (lane 6). Untreated cells cultured for
3 days were used as control (lane 1). One µg of each RNA
preparation was assayed along with nitrate reductase transcripts to
RT-PCR as in Fig. 1A. After 28 cycles, the
amplification products were electrophoresed, analyzed by Southern
blotting, and hybridized with FGF1 and nitrate reductase-specific
probes. This experiment has been independently performed three times
with similar results.
Figure 4:
PC12 cells transfected with constitutive
FGF1 expression vectors. PC12 cells were cotransfected with 10 µg
of pSVL-FGF1-134 vector DNA and 1 µg of PSVL2-neo using Lipofectin
reagent as described under ``Experimental Procedures'' and
stable transfected lines were isolated after 15 days of selection in
geneticin (0.5 mg/ml) containing medium. The established clones were
cultured in serum containing DMEM, and their morphology was examined
after 3 days of culture. An undifferentiated transfected clone (B7) (1 and 3) and a differentiated one (B12) (2 and 4) are shown at magnification 70 (1 and 2) and
180 (3 and 4).
Figure 5:
Analysis of FGF1 expression in FGF1
transfected PC12 cells. A, analysis by RT-PCR of human FGF1
transcripts. 1 µg of total RNA from PC12 cells (1),
differentiated B12, B18 (3 and 4) and
undifferentiated B7 (5) transfected cells were assayed along
with nitrate reductase transcripts by RT-PCR using hFGF1S1S and hFGF1AS
primers, as described in Fig. 1A. RNA isolated from
human mammary epithelial cell line MDA-MB-231 was used as positive
control (2). The amplification products derived from
transfected cells (3-5) after 30 cycles, from PC12 cells (1) and MDA-MB-231 (2) after 40 cycles were analyzed
by Southern blotting and hybridized with FGF1 and nitrate
reductase-specific probes. Three independent RT-PCR were performed with
similar results. B, analysis by RT-PCR of rat FGF1
transcripts. RNA from PC12 cells (1), MDA-MB-231 (2),
differentiated B12, B18 (3, 4), and undifferentiated
B7 (5)-transfected cells were assayed by RT-PCR using rFGF1S1S
and rFGF1AS primers. Amplified products were analyzed as in A except
that hybridization was performed with an internal rat specific FGF1
primer. C, analysis by EIA of FGF1 protein levels. 1 mg of
protein lysate from native PC12 cells, from PC12 cells stimulated for 3
days with FGF1 (100 ng/ml) in presence or absence of heparin (10
µg/ml), from differentiated clones (B12, B18), and
nondifferentiated clone (B7)-transfected PC12 cells was concentrated on
heparin-Sepharose column affinity, and FGF1 present in the lysates was
quantified by an EIA as described under ``Experimental
Procedures.'' The results are expressed as nanograms of FGF1 per
mg of total protein and are the mean value of three independent assays. D, undifferentiated transfected cells retained the capacity to
extend neurites. Undifferentiated FGF1-transfected PC12 cells (clone
B7) were cultured for 3 days in DMEM supplemented with 10% fetal calf
serum and 5% horse serum without exogenous stimulus (1) or in
presence of 100 ng/ml FGF1 plus 10 µg/ml heparin (2)
(magnification 115).
To confirm the role of an increase of FGF1 expression during the neurotrophic process, PC12 cells were transfected with the dexamethasone-inducible expression vector pLK-FGF1-134. Different stable clones were isolated. In response to dexamethasone, somes clones (as the D49 clone) extended neurites (Fig. 6B), whereas other clones maintained an undifferentiated phenotype (as the D1 clone). FGF1 synthesis in these clones was analyzed by Western blot (Fig. 6, A and C). As expected, only the D49 clone, induced by dexamethasone to differentiate, depicted an increased level of FGF1 to the same extent as constitutive transfected cells, B12 (Fig. 6A). This increase appeared 1 day after treatment and before the appearance of the extension of neurites which followed the same time course as in FGF1-treated cells (Fig. 6C). Dexamethasone, by itself, had no effect on the process of differentiation promoted by exogenous FGF1 (data not shown) and had no influence on FGF1 expression in control PC12 cells (Fig. 6A).
Figure 6:
Analysis of PC12 cells transfected with
inducible FGF1 expression vectors. A, 150 µg of protein
lysates isolated from PC12 cells (1 and 2), from
inducible FGF1-transfected cells nondifferentiated D1 clone (3 and 4) and differentiated D49 clone (5 and 6), and from constitutive FGF1-transfected cells
nondifferentiated B7 clone (7) and differentiated B12 clone (8) were concentrated on heparin-Sepharose and the level of
FGF1 produced by these cells was examined by Western blot as described
under ``Experimental Procedures.'' PC12 cells and inducible
transfected cells (D1 and D49) were maintained in glucocorticoid
depleted medium (1, 3, and 5) or treated
with dexamethasone (5 10
M) for 3
days (2, 4, and 6). Constitutive transfected
cells (B7 and B12) were cultured in DMEM for the same time. Under these
conditions, only the inducible transfected clone D49 in presence of
dexamethasone (6) and the constitutive B12 transfected clone (8) extended neurites. B, the morphology of the D49
cells nontreated (1) or treated with dexamethasone (2) for 3 days was presented. C, in the
differentiated inducible transfected cells D49, the level of FGF1 was
examined after 1, 3, and 5 days of dexamethasone treatment
(respectively lanes 2, 3, and 4). Nontreated
cells were presented in lane 1. Fifty µg of protein
lysates were concentrated on heparin-Sepharose and FGF1 detected by
Western blot as in A.
Figure 7: Neurotrophic markers in constitutive transfected PC12 cells. A, PC12 cells treated for 3 days by exogenous FGF1 (100 ng/ml) in the presence or absence of heparin and differentiated transfected cells (B18) were lysed by 0.5% Triton X-100 in 50 mM phosphate buffer, and the ChAT activity was measured (see ``Experimental Procedures''). The results are expressed as a multiplication factor compared to the ChAT activity detected in nontreated PC12 cells. The results are the mean values of two experiments run in triplicate. B, PC12 cells and differentiated transfected cells (B12 and B18) were lysed as above, and the AchE activity was measured (see ``Experimental Procedures''). As for A, the activities in transfected clones were reported to control PC12 cells activity and expressed as a multiplication factor. The result are the mean values of two experiments run in triplicate. C, total RNA was extracted from PC12 cells and from undifferentiated (B7) and differentiated (B12 and B18) transfected clones expressing FGF1 constitutively. 10 µg of each RNA sample were blotted and hybridized with Thy-1-specific cDNA probe.
Figure 8:
Cell
survival of constitutive FGF1 transfected cells. Cultures of PC12
cells, differentiated (B12) and undifferentiated (B7)
transfected clones were washed with PBS, dissociated with 10 mM EDTA, and plated in DMEM without serum. After 1, 2, 3, 4, and 7
days of culture, cells were stained with trypan blue. Viable and dead
cells were counted, and the results are expressed as a percentage of
cell viability (viable cells/viable and dead cells). The total number
of cells (dead and alive cells) was unchanged even after 7 days of
culture. The results are the mean values of triplicates, and this
experiment was performed three times with similar results. Key:
&cjs2098;, PC12; &cjs2112;, B12; ,
B7.
Exogenous FGF1 induced
the activation of ERK1 to the same extent as did NGF and FGF2 whether
or not cells were induced to differentiate, that is in presence or
absence of heparin (Fig. 9A). Similarly, cells treated
with the mutated FGF and heparin, which remained
undifferentiated, induced the same extent of ERK1 activity as
differentiated cells treated with FGF2 or FGF1 and heparin.
Figure 9:
Activation of ERK1 by FGF1,
FGF, FGF2 and NGF stimulation. A, PC12 cells
were treated for 5 min with heparin (10 µg/ml), FGF1 (100 ng/ml),
FGF1 (100 ng/ml) plus heparin (10 µg/ml), FGF1
(FGF1 Lys132-) (100 ng/ml) plus heparin (10 µg/ml),
FGF2 (10 ng/ml), and NGF (50 ng/ml). ERK1 activity (phosphorylation of
exogenously added myelin basic protein) in the cell lysates was
measured (see ``Experimental Procedures''). NGF stimulation,
used as control, represents 100% ERK1 activity. The activities of ERK1
after FGF stimulation were calculated in comparison to NGF stimulation.
The results are the mean values of three independent experiments, each
run in triplicate. B, PC12 were treated with FGF1, FGF1 plus
heparin, FGF1
, and NGF at the same concentrations as in A for different periods of times and ERK1 activity was
measured. NGF stimulation for 5 min represented the 100% ERK1 activity.
The results are the mean values of triplicates, and this experiment was
performed three times with similar results. C, control PC12
cells (1 and 4) and PC12 cells treated for 15 min
with FGF1 (2), FGF1 plus heparin (3), and NGF (5) were fixed with 4% paraformaldehyde, incubated in the
presence of 0.3% Triton X-100 with antibodies to anti-ERK1 (1, 2, 3, and 5) and nonimmune serum (4) for 1 h. The antigen-antibody complexes were detected with
FITC-rabbit antibodies and observed by fluorescent
microscopy.
The
kinetic pattern of ERK1 activation (Fig. 9B) was
studied after stimulation with NGF, FGF1, and FGF1 in
the presence or absence of heparin. FGF1 and the mutated FGF1
stimulated ERK1 activity with a different pattern from the NGF
one. After 5 min of stimulation the peak of activity was identical in
NGF-, FGF1-, and FGF1
-treated cells, but after 15 min,
in FGF1-treated cells, ERK1 activity had returned to basal level, while
in NGF-treated cells, it remained elevated. Heparin, which was
essential for differentiation, did not change the profile of ERK1
activation by FGF1 (Fig. 9B).
It has been shown previously that NGF, in contrast to epidermal growth factor which is a mitogenic factor for the PC12 cells, induced nuclear translocation of ERK1(27) . Therefore, we examined the subcellular localization of ERK1 in PC12 cells stimulated by FGF1 in the presence or the absence of heparin (Fig. 9C). The immunohistochemical analysis showed that, in both conditions, FGF1 was unable to induce nuclear translocation, while after 3 days of treatment with FGF1-heparin cells extended neurites but remained undifferentiated in presence of FGF1 alone.
While ERK1 has been shown to be involved in the neurotrophic activity of NGF, considering the diversity of MAP kinases, it was plausible that another MAP kinase could be implicated in the differentiating action of FGF1. Therefore we analyzed the activity of the upstream regulator, MAPKK. As for ERK1 activity, we showed that in undifferentiated cells treated with FGF1 alone, the MAPKK is activated to the same extent as in differentiated cells treated with FGF1 and heparin or with NGF (Fig. 10A).
Figure 10: Activities and expression of ERK1 and MAPKK in constitutive FGF1 transfected PC12 cells. A, ERK1 and MAPKK activities in undifferentiated (B7) and differentiated (B12 and B18) FGF1-transfected cells were measured (see ``Experimental Procedures''). The activities detected in nontreated or treated PC12 cells by NGF (50 ng/ml) or FGF1 (100 ng/ml) for 3-5 min were used as negative and positive controls. The activities of PC12 cells stimulated by NGF represent 100% activity. The results are the mean values of triplicates, and this experiment was performed three times with similar results. B, ERK1 and MAPKK expressions in FGF1-transfected PC12 cells. Lysates from PC12 cells (1) or from undifferentiated B7 (2) or differentiated B18 (3) FGF1-transfectants were analyzed for ERK1 and MAPKK expression by Western blotting with specific antibodies (see ``Experimental Procedures''). C, immunolocalization of ERK1 in FGF1 transfected PC12 cells. Undifferentiated B7 (1 and 2) and differentiated B18 (3 and 4) FGF1-transfected cells were cultured on poly-L-lysine-coated slides, fixed with 4% paraformaldehyde, and incubated in the presence of 0.3% Triton X-100 with antibodies to anti-ERK1 (1 and 3) and nonimmune serum (2 and 4) for 1 h. The antigen-antibody complexes were detected with FITC-rabbit antibodies and observed by fluorescent microscopy. This experiment and the immunolocalization presented in Fig. 9C were performed together, thus the positive control (ERK1 translocation induced by NGF treatment) in Fig. 9C is the same for both.
In summary, it appears that the activation of the MAP kinases by FGF1 is independent of the differentiation state of the cells, and that, in contrast to NGF, FGF1 does not sustain ERK1 activity described as a key element of PC12 cell differentiation.
In the absence of exogenous stimuli, the transfected undifferentiated (B7) or differentiated (B12 and B18) cells exhibited a low basal ERK1 and MAPKK activities (Fig. 10A). This absence of activity was not due to an inhibition of ERK1 or MAPKK expression, since Western blot analysis showed that both were expressed at the same level in the transfected cells and in PC12 cells (Fig. 10B). Modification of the localization of ERK1 did not occur, as immunohistochemical analysis showed that ERK1 was localized mostly in the cytoplasm in both the transfected clones and PC12 cells (Fig. 10C). ERK1 could also be activated by treating the transfected clones by exogenous NGF for 5 min. In differentiated PC12 cells (treated with NGF for 3 days and then NGF and serum-depleted medium for one night) and in differentiated transfected cells (B18), 5 min of NGF stimulation activated ERK1 to the same extent (Fig. 11). The presence of potentially active FGF receptor in differentiated transfected clones was examined using FGF-saporin, a cytotoxic complex which enters the cells via the high affinity FGF receptors (45) (Fig. 12). Clones B7 and B12 and the PC12 cells were treated with 1 nM FGF-saporin, and after 3 days of treatment 47% of PC12 cells, 60% of B7 cells, and 43% of B12 cells were killed, confirming the presence of functional FGF receptors in the transfected clones (Fig. 12). These results demonstrate that the absence of differentiation observed under the various conditions described above could not be attributed to a default in FGF-R activation.
Figure 11: Activation by NGF of ERK1 in constitutive FGF1 transfected cells, in NGF-differentiated PC12 cells and in undifferentiated control PC12 cells. PC12 cells were treated with NGF for 3 days to obtain differentiated PC12 cells. These cells (NGF-treated differentiated PC12 cells) and the undifferentiated (B7) or differentiated (B18) FGF1-transfected cells were depleted overnight in a low serum medium, 0.25% FCS, and 0.2% bovine serum albumin, and then stimulated with 100 ng/ml NGF for 5 mn. ERK1 activity was measured by phosphorylation of myelin basic protein. The results are the mean values of triplicates, and this experiment was performed twice with similar results.
Figure 12:
Activity of FGF receptors in constitutive
FGF1 transfectants. Cytotoxic effect of FGF-saporin. PC12 cells and
differentiated (B12) and undifferentiated (B7)
FGF1-transfected cells were treated with 1 nM FGF2-saporin,
FGF2, and saporin. After 3 days of treatment, cells were trypsinized
and counted. The result are expressed as a percentage; the untreated
cell number represents 100%. The results are the mean values of
triplicates, and this experiment was performed three times with similar
results. Key: , control; &cjs2098;, FGF;
&cjs2100;, FGF-saporin; &cjs2090;, saporin
Figure 13:
Analysis by RT-PCR of NGF expression in
PC12 cells. A, total RNA was extracted from PC12 cells after 3
days of treatment with 100 ng/ml FGF1 in the presence of 10 µg/ml
heparin (lane 2), 10 ng/ml FGF2 (lane 3), 100 ng/ml
FGF1 plus 10 µg/ml heparin (lane 4) or 100
ng/ml NGF (lane 5). Untreated cells cultured for 3 days were
used as a control (lane 1). One µg of each RNA preparation
was assayed along with nitrate reductase transcripts by RT-PCR with
NGF-specific primers. After 35 cycles, the amplified products were
electrophoresed, analyzed by Southern blotting, and hybridized with NGF
and nitrate reductase-specific probes. B, total RNA was
extracted from PC12 cells (lane 1), differentiated
constitutive FGF1-transfected cells B12 (lane 2),
differentiated D49 (lanes 3 and 4) or
undifferentiated D1 (lanes 5 and 6) inducible
FGF1-transfected cells not treated (lanes 3 and 5) or
treated with 5
10
M dexamethasone (lanes 4 and 6) and from rat hippocampus (lane
7). NGF expression was analyzed by RT-PCR as in A, except
that 40 cycles were performed.
PC12 cells have been widely used as a model system for examining the molecular mechanisms by which FGF2 and NGF induce neuronal differentiation. In the present study, instead of comparing the effects of different neurotrophic (NGF/FGF) versus mitogenic (epidermal growth factor) factors, we have taken advantage of the heparin requirement in the neurotrophic process (43, 46) mediated by FGF1, to distinguish the specific responses implicated in the neurotrophic activity of this growth factor.
We show that FGF1 alone or the mutated FGF1,
known to have decreased affinity for heparin(29, 40) ,
does not induce neuronal differentiation, in contrast to FGF1 plus
heparin. This indicates that FGF1 and FGF1-heparin may have distinct
cellular targets. This is supported by data showing that FGF2
internalized by heparan sulfate proteoglycan or by the complex heparan
sulfate proteoglycan/FGF-receptor is not targeted to the same
intracellular compartments(47) . In PC12 cells, the different
intracellular fates of FGF1 and FGF1-heparin may determine its
neurotrophic action.
A variety of cellular mechanisms are presumably
involved in the process of differentiation induced by neurotrophic
factors. The most important signaling pathway thought to be implicated
in PC12 cell differentiation, promoted by NGF and FGF2, is the Ras/MAP
kinase signaling
system(26, 28, 48, 49) . It has been
argued that it is the duration of MAP kinase activation (25) and the nuclear translocation of ERK1 (26) induced
by NGF and FGF2, but not by epidermal growth factor, which plays a key
role in the generation of the neurotrophic action. In the present
study, we show that FGF1, FGF1, and FGF1-heparin, which
have distinct effects on PC12 cell differentiation, have the same
profile of activation of ERK1 and do not alter the ERK1 subcellular
localization. This suggests that the activation of the MAP kinases
alone is not sufficient to induce differentiation and that FGF1
neurotrophic activity could be either mediated by a different cascade
or could diverge from the MAP kinase signal beyond ERK1. In both cases
an additional element must be involved.
Such an element could be the increased expression of FGF1 which, in contrast to the activation of the MAP kinase cascade, correlates strictly with the differentiation of PC12 cells, either treated with exogenous FGF or transfected with FGF1 expression vectors. In cells treated with exogenous FGF2 or FGF1 and heparin, kinetic analysis of FGF transcripts shows an increased amount of FGF mRNAs which had already occurred at day 1, before any morphological modifications of PC12 cells. This increase then followed the same time course as the neuritic extension process. In contrast, the level of FGF1 remained unchanged during the differentiation of NGF-treated cells. A similar situation was observed in expression of NGF mRNA during the differentiation of FGF-treated cells, suggesting different signaling pathways for FGF and NGF neurotrophic activities.
To confirm that the FGF increase, produced by PC12 cells stimulated by exogenous FGF, was sufficient to promote neuronal differentiation, constitutive and inducible FGF1-transfected cells were established. In PC12 cells treated with exogenous NGF or FGF, differentiation occurs after several days when cell proliferation ceases. Thus, stable transfection with a putative neurotrophic factor under the control of a constitutive expression promoter was performed assuming that the proliferation phase which precedes the differentiation process is enough to isolate colonies. As expected, several stable transfected clones which presented a morphological differentiated phenotype and an increase in ChAT and AChE activities and of Thy-1 mRNA were isolated. The proliferation rate of these differentiated clones and of PC12 cells were identical, but as expected, differentiated cells ceased to proliferate earlier than the undifferentiated cells (data not shown). In the absence of any exogenous neurotrophic stimuli, FGF1 constitutive transfected cells or transfected cells under the control of the murine mammary tumor virus-inducible promotor, expressed a differentiated phenotype with neuritic extensions only when the concentration of FGF1 reached a certain level of expression. Some constitutive transfected cells expressing an intermediate level of FGF1 displayed an undifferentiated or an incompletely differentiated phenotype (data not shown). In inducible transfected clones the extension of neurites followed the same time course of that in exogenous treated cells and in both cases the increase in FGF1 preceded the morphological changes. All of these data imply that FGF1 expression either stimulated by exogenous FGF or under the control of strong promotors in transfected cells is responsible for the neurotrophic activity.
In fact, increased expression of FGF1, in constitutive transfected PC12 cells, not only induces differentiation but also promotes the long term survival of differentiated cells in a serum-free medium, while control PC12 cells die rapidly and exhibit the characteristic pattern of DNA fragmentation associated with apoptosis(50) . As exogenous FGF are known to increase survival of neuronal cells such as photoreceptors (11, 17, 18) , cholinergic neurons, and retinal ganglion cells(51) , in view of our data, the survival activity of exogenous FGF should depend on an increase in the expression of FGF1 or FGF2 in these neuronal cells.
Previous data have already demonstrated that the expression of FGF induced by the exogenous forms (autoactivation and transactivation of FGF) could be involved in the mediation of FGF biological activities. These forms of expression would stimulate cell proliferation in astrocytes and hippocampal neuronal cells and myogenic differentiation (21, 22) . In lens epithelial cells, the increased expression of FGF1 prevents apoptosis, while the addition of FGF1 antisense oligonucleotides provokes cell death(33) .
Our data also underline that the neuronal differentiation and survival activities of FGF1 could thus be controlled by a precise quantitative regulation of its level of expression. The importance of quantitative aspects of FGF has also been described for exogenous FGF. Guillemot and Cepko (13) demonstrated that the choice of fate of the bipotential neuroepithelium depends on the concentration of exogenous FGF1. A 2-10 times difference in FGF2 concentration also controls the choice of ventral type mesoderm or notochord differentiation(52) , and the proliferative, migratory, or differentiation responses of rat lens epithelial cells in vitro is dependent on the dose of FGF2 to which they are exposed(53) .
The observation that an increase in FGF1 promotes neuronal differentiation and increases survival of PC12 cells suggests that in vivo the expression of FGF1 which reaches a maximum in adult neuronal tissues could be a key control step in the induction or inhibition of differentiation as well as the survival of nervous tissues. This may explain the need for a precise regulation of FGF1 gene expression which involves multiple promoters (54) as well as some regulatory elements located in the long and complex 5`- and 3`-untranslated regions of FGF1 transcripts(55, 56, 57) . Studies demonstrating that endogenous FGF are localized in the nuclei of several types of cultured cells and tissues (20, 35, 58, 59) and that exogenous FGF can translocate to the nuclei at certain phases of the cell cycle (19, 60) suggest that FGF could act directly in the cell (23) possibly as nuclear transcription factors. This hypothesis is supported by cell-free experiments in which FGF2 was shown to modulate the transcription of Pgk-1 and Pgk-2 genes(61) . In PC12 cells, FGF could be involved in the activation of genes necessary for neuronal differentiation and survival. In this context, the results of ongoing experiments to establish the subcellular localization of FGF1 in FGF1-treated cells and transfected PC12 cells as a function of differentiation would be of interest.
Neuronal cell death occurs naturally during development and is also a consequence of insult, aging, and degenerative disorders. Increasing tissue FGF levels might thus be beneficial in certain chronic and progressive neurodegenerative disorders.
Addendum-While this paper was submitted, an analysis of the PC12 differentiation pathway demonstrated, in accordance with our results, that MAP kinase activation is insufficient for growth factor receptor-mediated PC12 differentiation(62) .