From the INSERM U 531, Institut Louis Bugnard, CHU
Rangueil Bat L 3, 31403 Toulouse Cedex 4, France and ** Division of
Endocrinology, Diabetes and Metabolism, University of California,
Irvine, California 92697
Received for publication, February 11, 2000, and in revised form, August 19, 2000
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ABSTRACT |
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The high molecular weight
(HMW) fibroblast growth factor (FGF)-2 isoform of 210 amino
acids initiated at a CUG start codon possesses a nuclear localization
sequence and is not secreted. In contrast, the low molecular weight
(LMW) isoform of 155 amino acids initiated at the AUG start codon can
be secreted and activates the cell surface FGF receptors. The two
isoforms possess different biological properties; however, little is
known about the intracrine regulatory mechanisms involved in the
biological effects of the HMW FGF-2 isoform. Using pancreatic cells
stably transfected with cDNAs leading to the expression of either
the HMW FGF-2 (A3 cells) or the LMW form (A5 cells), we provide
evidence that the two FGF-2 isoforms differentially modulate PKC
levels. The LMW FGF-2 up-regulated the PKC Basic FGF1 (FGF-2) is a
protein belonging to the family of heparin-binding growth factors and
is produced by many cell types either normal or malignant (for review
see Ref. 1). In tumors, it is one of the key factors regulating growth,
blood supply, and invasiveness. These pleiotropic effects have been
related to the binding of FGF-2 to high affinity FGF receptors or their splice variants and to low affinity binding sites (2). However, FGF-2
possesses some peculiar features supporting the involvement of other
regulatory mechanisms in the final biological effects. Indeed, FGF-2 is
synthesized from a single mRNA as five different isoforms with
molecular masses of 18, 22, 22.5, 24, and 34 kDa through alternative
translation at AUG and CUG start codons (3-5) and through internal
ribosomal entries (6). The initiation of translation at the AUG codon
gives rise to a peptide of 155 amino acids, and the initiation at the
four CUG codons is responsible for the synthesis of the other isoforms
that possess N-terminal extensions containing nuclear
localization sequences (5, 7). Confocal and immunohistochemical
analysis show that these nuclear localization sequence-containing
isoforms are predominantly localized in the nucleus and more precisely
in the nucleoplasmic compartment; no labeling is found at the cell
surface and in the extracellular spaces (1, 8-10). Usually the import
process across the nuclear pore complex involves the activity of
transport factors not yet characterized for FGF-2. All FGF-2 isoforms
lack leader consensus secretory sequences. However, the LMW peptide
(155 aa) can be secreted through still unclear mechanisms requiring
energy and cooperation with heat shock proteins and
Na+,K+-ATPase (11, 12). After an endocytotic
process small amounts of this LMW peptide can be specifically targeted
to the nucleolar compartment (13). The nuclear translocation of FGF-2
seems to be correlated with the nuclear import of the type 1 FGF
receptor (FGFR1) (for review see Ref. 14). The selective expression of either the LMW or the HMW form of 210 amino acids in different cell
types induces different and even opposite effects. For instance, expression of the LMW form enhances cell migration (10), integrin, and
tissue plasminogen activator expressions (15), whereas the HMW
FGF-2 does not affect these properties. By contrast, the HMW form
specifically promotes, at the difference of the LMW form, growth at low
serum concentration (0-1%) (10, 16, 17), reduces laminin B1 (8),
tissue plasminogen activator and plasminogen activator
inhibitor-1 expression (18), adenylate cyclase activity (19),
and inhibits interleukin-6 promoter activity (20). Furthermore, neutralizing anti-FGF-2 antibodies recognizing the common sequence of
all FGF-2 forms (10, 21), suramin (18), a well established inhibitor of
FGF-2 binding to FGF receptors (22), and expression of high level of
dominant negative FGFR (10) inhibit the biological effects of the
extracellular FGF-2, but they do not modify those evoked by the HMW
FGF-2. Thus, the LMW isoform regulates cell functions predominantly
through auto-paracrine mechanisms, whereas the larger ones exert their
biological effects via an "intracrine" pathway, independent of the
activation of cell surface FGF receptors (for reviews see Refs. 1, 14,
and 23). The signaling pathways involved in intracrine regulations are
still poorly known. It has been postulated that the colocalization of
HMW FGF-2 and FGFR1 in the nuclear matrix might reflect the existence
of FGF receptor activation at the nuclear level (14). We previously
reported a reduced adenylate cyclase activity in cells expressing the
HMW peptide, which was found to play a positive role on cell growth (19). However, this modification cannot fully explain the increased proliferation in low serum.
Because some PKC isotypes are involved either positively or negatively
in the proliferation rate of many cell types (24), we sought to examine
the effects of the HMW FGF-2 peptide of 210 aa on PKC levels and
functions. For that purpose, we analyzed either stably transfected
cells or cells transiently transfected with a tetracycline-regulated
gene expression system (25), and the cell model used was the well
differentiated pancreatic cell line AR4-2J. These cells were
transfected with point mutated FGF-2 cDNAs leading to the
expression either of the LMW of 155 aa or of the HMW isoform of 210 aa
(16). The results reported here demonstrate that the HMW peptide
modified the expression of PKC Cell Culture--
AR4-2J cells and the different transfected
cells were maintained in DMEM containing 4.5 g/liter glucose (Life
Technologies, Inc.) and supplemented with 10% fetal calf serum (Life
Technologies, Inc.). Trypsin (0.05%) and EDTA (0.02%) (Biowhittaker,
France) were used to dissociate the cells for successive passages, and medium was replaced every 2 days. Cultures were regularly checked for
the absence of contamination by mycoplasma. Cells were routinely plated
at the density of 15 000 cells/cm2 in DMEM containing 10%
fetal calf serum. Media were changed the day before PKC or ERK analysis
to standardize the experimental conditions. For FGF-2 stimulation,
after an overnight attachment medium was replaced by serum-free DMEM
and buffered at pH 7.3 with 20 mM Hepes containing 2 nM FGF-2.
To analyze the effect of the PKC inhibitors, cells were cultured in
DMEM and buffered at pH 7.2 with 20 mM Hepes containing 0 or 2.5% fetal calf serum. Gö 6976 (Calbiochem, La Jolla, CA) was
used at the concentration of 50 nM to inhibit the
conventional PKCs. GF109203X (Calbiochem), which belongs to the group
of bisindolylmaleimide derivatives, was used at the concentration of 1 µM to inhibit PKC Plasmids and Transfections--
The retroviral infection of
AR4-2J cells was described previously (16). Geneticin-resistant cells
were shown to express low levels of FGF-2. Transfected cells
synthesizing the HMW FGF-2 isoform of 210 aa were called A3, and cells
expressing the LMW isoform of 155 aa were called A5. Control CAT cells
were obtained by transfecting AR4-2J cells with the vector containing
the chloramphenicol acetyltransferase reporter gene. Any of
these transfected cell lines has been cloned.
The pTet-Off vector expresses the tetracycline repressor tTA
(CLONTECH, Palo Alto, CA) (25). The HMW FGF-2
cDNA (a generous gift of Dr H. Prats, INSERM U 397, Toulouse,
France) was inserted in the multiple cloning site of the pTRE vector
(CLONTECH). In this gene expression system the HMW
FGF-2 expression is induced by decreasing the concentrations of the
tetracycline analogue doxycycline, in the culture medium. Control CAT
cells were transiently cotransfected with the two vectors in the
presence of 2000 ng/ml doxycycline. After 8 h of transfection, the
medium was removed, and cells were grown in the presence of decreasing
concentrations of doxycycline (from 2000 to 0 ng/ml). Native PKC Preparation of Rat PKC Probes--
Total RNA was prepared by the
guanidinium isothiocyanate method (28). Total RNA, previously heated 10 min at 94 °C, was reverse transcribed at 39 °C in the presence of
1 mM each dNTP, 1 unit/µl RNasin (Promega,
Charbonnières, France), 0.25 µg/µl oligo(dT) and 2 units/µl
Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Inc.). The reverse transcriptase buffer contained 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 2 mM MgCl2 and 1 mg/ml nuclease-free bovine serum
albumin. After 10 min at 23 °C and a 1-h incubation at 39 °C, the
reaction was stopped at 94 °C, 5 min. Amplification was then carried
out in a final volume of 100 µl, with 0.5 µg of cDNA, 2.5 units/µl Taq polymerase (PerkinElmer Life Sciences) in the
same buffer, and 500 pmol of each oligonucleotide primer. All reactions
were performed for 35 cycles with cycling times of 1 min at 95 °C, 1 min at 65 °C, and 1.30 min at 72 °C. Primers were synthesized by
Eurogentec (Liège, Belgium). Primers were chosen on the basis of
the cDNA sequences of the rat PKC isotypes (GenBankTM
data base), and the absence of cross-reactivity among the different PKCs was checked.
The following primers were used: for PKC Northern Blots--
Total RNA was prepared by the guanidinium
isothiocyanate method, and Northern blot analysis of PKCs was performed
as described previously (29). Nylon sheets were prehybridized for
6 h at 42 °C in 50% formamide, 1% SDS, 5× Denhardt's, 6×
SSC, 5 mM EDTA, and 100 µg/ml salmon sperm DNA.
Hybridizations were carried out at 42 °C in the same medium with PKC
probes (lengths from 409 to 573 bp) 32P-labeled by nick
translation. Blots were washed twice for 30 min at 55 °C in 0.1×
SSC and 0.5% SDS, then dried, and exposed to Kodak X-Omat AR films at
Western Blots and ERK Activity--
Cells were grown 24 h
in a serum-free medium buffered at pH 7.3 with 20 mm Hepes. After
washings with phosphate-buffered saline, cells were harvested in lysis
buffer (1% Nonidet-P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA) containing both protease
(Pefabloc; Roche Molecular Biochemicals) and phosphatase inhibitors (25 mM NaF and 10 mM sodium orthovanadate). Lysates
were clarified by centrifugation for 10 min at 10 000 × g. Protein was determined on the supernatant using the BCA
assay (Pierce). FGF-2, PKCs or ERK-1 and -2 were analyzed by SDS-PAGE
on a 12% polyacrylamide gel then electroblotted onto a polyvinylidene
difluoride membrane (Polyscreen; PerkinElmer Life Sciences). The
different proteins were then revealed by using specific antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA), directed against FGF-2, PKC
isotypes, or ERK-1/2. The anti-ERK-1/2 antibody (sc-1647) reacts to a
lesser extent with ERK-1 (p44) than with ERK-2 (p42). An anti- Cell Treatment with Suramin and Neutralizing Anti-FGF-2
Antibodies--
Cells plated at 100,000 cells/35-mm dish were grown in
Hepes-buffered (20 mM) serum-deprived medium at pH 7.3. Cells were incubated with suramin (400 µM) or
neutralizing anti-FGF-2 antibodies (5 µg/ml) (Upstate Biotechnology,
Lake Placid, NY) in the absence or presence of 0.1 nM
rFGF-2 for 24 h. Cells were harvested, and ERK1/2 phosphorylation
was analyzed as described above.
PKC Translocation--
Cells seeded at the concentration of
100,000 cells/35-mm dish were transfected with the tet-off
expression system. After transfection, cells were grown in
Hepes-buffered (20 mM) serum-deprived medium at pH 7.3, with or without doxycycline. 24 h after the beginning of
doxycycline treatment, cells were washed twice with cold
phosphate-buffered saline and then harvested at 4 °C in 0.5 ml of
the 25 mM hypotonic Tris-HCl buffer, pH 7.5, containing
inhibitors of proteases (Pefabloc) and phosphatases (25 mM
NaF and 10 mM sodium orthovanadate). Then cells were
sonicated three times for 5 s at 4 °C and spun at 100 × g for 5 min to discard the nonlyzed cells. The supernatant
corresponding to the total PKC was then centrifuged at 100,000 × g for 30 min. The 100,000 × g resulting
pellet was resuspended in the same buffer containing 1% Triton X-100.
This 100,000 × g pellet corresponds to the particulate
fraction (31). The two fractions were subjected to SDS-PAGE
electrophoresis, transferred to polyvinylidene difluoride membranes,
and revealed with the anti-PKC Statistics--
Statistical significance was determined
according to the unpaired Student's t test.
FGF-2 Expression Modifies PKC Levels--
Confocal analysis of
AR42J cells stably transfected with the mutated cDNA encoding the
FGF-2 of 210 aa (A3 cells) showed its localization in the nucleus but
not at the cell surface or in the extracellular compartment (8). We
first analyzed total cell extracts by Western blots to find out whether
FGF-2 expression modified PKC levels. PKC
To further define the role of the HMW FGF-2 in the modulation of the
PKC levels, cells were transiently transfected with the tet-off
transcription regulation system to regulate the expression of the HMW
FGF-2 (25). In this system the transactivator tTA regulates a synthetic
promoter composed of the tetracycline operator. The plasmid expressing
the tTA transactivator was cotransfected with the plasmid containing
the HMW FGF-2 cDNA inserted downstream the tetracycline operator.
The presence of tetracycline or its analogue doxycycline prevents
binding of the transactivator to the promoter and reduces the
expression of the HMW FGF-2. Transfected cells displayed a
doxycycline-dependent down-regulation of the HMW FGF-2
expression (Fig. 2). Only this FGF-2
isoform was found produced whatever the doxycycline concentration (Fig.
2), indicating that the HMW FGF-2 did not modulate in the transfected
cells the expression of endogenous FGF-2. The maximal level of HMW
FGF-2 expressed in transiently transfected cells was comparable with that of A3 cells, as observed by SDS-PAGE (data not shown). PKC FGF-2 Expression Modifies PKC mRNA Levels--
To analyze
whether these modifications were related to mRNA changes, the
levels of the PKC transcripts were determined by using rat-specific PKC
probes synthesized by reverse transcriptase-polymerase chain reaction.
Northern blot analysis revealed that PKC The HMW FGF-2 Modifies ERK-1 and -2 Phosphorylations and
Activities--
ERK-1/2 are involved in cell proliferation. We were
then interested on the possible modifications of ERK-1 and 2 levels and activities in cells expressing the HMW FGF-2. ERK-1 and -2 protein levels were found to be comparable in A3 and CAT cells (Fig.
4). By contrast, the anti-active ERK
antibody revealed an hyperphosphorylation state of both proteins in A3
cells (Fig. 4). Similar results were obtained in control CAT cells
transiently transfected with the cDNA encoding the HMW FGF-2 under
the control of the tetracycline-dependent transactivator.
Indeed, ERK-1/2 levels were not modified (Fig. 5), whatever the concentration of the HMW
FGF-2. In addition, a close relationship was observed between HMW FGF-2
levels and ERK-1/2 hyperphosphorylation state (Fig. 5). For control
purposes CAT cells were transfected with the basal
tetracycline-regulated expression vectors and treated with doxycycline.
The phosphorylation state of ERK was not modified by doxycycline, as
expected according to the results obtained in stably transfected cells
that were not treated with doxycycline (Fig. 4). All of the data
indicate that ERK phosphorylation was indeed the result of the
expression of the HMW peptide. The hyperphosphorylation reflected an
increased kinase activity as observed on the MBP substrate (Fig.
5).
Activation of FGFRs Is Not Responsible for the Modifications
Observed in Cells Producing the HMW FGF-2--
The question arises of
whether the HMW FGF-2 exerts its effects after release, for instance by
cell lysis. The results obtained by confocal analysis (8) that are
against a significant release of the HMW FGF-2 as well as the opposite
effects of HMW and LMW FGF-2 on PKC
Taking into account both the opposite effects of high and low molecular
weights FGF-2 on PKC
To analyze the involvement of intracellular FGFRs in the effects of the
HMW FGF-2, A3 cells were transfected with increasing concentrations of
a cDNA encoding the dominant negative FGFR1 truncated in the
cytoplasmic domain (FGFR1dn) (35). The dominant negative FGFR1dn can
dimerize with the FGFR1 and conceivably with the other FGFRs and
inhibit their signaling pathways (10, 35). FGFR1dn
concentration-dependently inhibited ERK-1/2 phosphorylation induced by rFGF-2, in CAT (Fig.
8A) and A3 cells (Fig.
8B). Additionally, the expression of increasing amounts of
the truncated receptor did not modify the basal phosphorylation state
of ERK-1/2 in the presence of anti-FGF-2 antibodies, either in control
CAT cells that do not produce FGF-2 (Fig. 8A) or in A3 cells
(Fig. 8B) producing the HMW FGF-2. These results did not
support the implication of intracellular FGFRs in the intracrine
regulation. They also confirm the previous results excluding the
activation of the cell surface FGFRs in the action of the HMW
FGF-2.
ERK-1 and -2 Phosphorylation Is PKC
According to the fact that phosphorylation and translocation to the
particulate cell fraction is recognized as an index of the PKC The results presented here demonstrate that the expression of the
HMW FGF-2 can induce modifications of some intracellular signaling
pathways through the regulation of calcium-independent PKC It is well established in different cell types that the HMW FGF-2 is
not exported and exerts some specific biological effects different from
those induced by the secreted form (1, 14, 23). However, small amounts
of the growth factor might be present in the culture medium following
cell lysis. It has been shown that when added extracellularly, the
210-aa HMW peptide induces biological effects resembling those evoked
by the LMW one, but these effects differ from those induced by the
constitutive expression of the HMW peptide (43). Thus, the regulatory
mechanisms underlying the PKC expression in cells producing the HMW
FGF-2 should be independent of the activation of cell surface FGFRs.
Indeed, by using suramin and neutralizing anti-FGF-2 antibodies to
inhibit the activation of FGFRs by exogenous FGF-2, the biological
effects induced by the large form of FGF-2 were not modified,
confirming the involvement of intracrine regulation in these effects.
It has been suggested that the intracrine regulation can involve the
direct intracellular activation of nuclearized FGFR-1 (14). However,
expression of inactive FGFR1 reduces ERK-1/2 phosphorylation under
rFGF-2 stimulation but does not modify ERK-1/2 phosphorylation induced
by the HMW FGF-2. These data do not support the hypothesis of an
intracellular activation of FGFR1 receptors by the endogenous HMW
FGF-2.
PKCs Three lines of evidence point to the involvement of PKC PKC The observation that the HMW FGF-2 exerts opposite effects on PKC The present data and our previous ones on the same cells provide a
better understanding of the signaling pathways modulated by the HMW
FGF-2. First, the HMW FGF-2 induces the overexpression of FGFRs
in vitro (33) as also observed in vivo in
different tumor types (51), thereby leading to an increased response to the extracellular FGF-2. Second, the HMW FGF-2 reduces the adenylate cyclase activity and thus reverses the growth inhibition induced by
cAMP (19). Third, as shown in the present study, the HMW FGF-2
modulates the levels of PKC levels by 1.6-fold; by
contrast the HMW isoform down-regulated the level of this PKC isotype
by about 3-fold and increased the amount of PKC
by 1.7-fold. PKC
mRNAs were also modified, suggesting that PKC expression was
regulated at a pretranslational level. Additionally, expression of
different levels of the HMW FGF-2 with an inducible expression system
confirmed the role of this isoform on PKC
and
expressions.
Increased activation of ERK-1 and -2 was also observed in cells
expressing the HMW FGF-2. By using different PKC inhibitors and a
dominant negative PKC
, it was found that ERK activation was PKC
-dependent. These data indicate that expression of HMW
FGF-2 can modify PKC levels by acting at the intracellular level and
that the overexpression of PKC
induces ERK-1/2 activation. The
expression of a dominant negative FGFR1 did not reduce ERK-1/2
activation by the HMW FGF-2, suggesting that ERK activation does not
require FGFR activity. The signaling cascade downstream of ERK might be
involved in the known mitogenic effect exerted by this FGF-2 isoform.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
and ERK-1 and -2 activities
via a receptor-independent pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
, and rottlerin
(Calbiochem), which specifically inhibits PKC
, was used at the
concentration of 6 nM (26, 27). These drugs were
solubilized in Me2SO and used in cultures at the
final concentration of 0.1% Me2SO. Control cells were
grown in the presence of the solvent.
cloned in pCO2 vector and the mutated T505A PKC
cloned in
pcDNA3 were kindly provided by Dr P.J. Parker (Imperial Cancer
Research Fund, London). The dominant negative FGFR1 construct
(FGFR1dn) corresponds to FGFR1 truncated at residue 405. The resulting
protein contains the transmembrane region but is devoid of most of the
intracellular domain and lacks kinase activity. The FGFR1dn construct
was used to transfect CAT and A3 cells. All transfections were
performed by using FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions.
, 5'-GATGAAATGCGACACCTGCG-3' from 387 to 406 bp and
5'-AGGGCTGATGACTTTGTTTCC-3' from 940 to 960 bp; for PKC
,
5'-CCCTGAGTGGAAGTCAACATTCG-3' from 156 to 178 and
5'-GAAGATGGTGTCCCGGCTATTGG-3' from 638 to 660; for PKC
,
5'-CAAAATCTGCGAGGCCGTGAGC-3' from 30 to 51 and
5'-GCATCCGCTCCCTAAACACTCG-3' from 433 to 454 bp; and for PKC
,
5'-AGGTGACCCTTGTACTGTGTCC-3' from 192 to 213 pb and
5'-CATCGTTCTTGTCATCTACTGG-3' from 580 to 601 pb. Sizes of
amplified products corresponded to those expected according to the
position of the primers in the PKC cDNA sequence. The specificity
of the polymerase chain reaction products was further checked by using
restriction enzymes selected according to the sequence of each cDNA
by using computer analysis (PC/GENE, IntelliGenetics, Mountain View,
CA). For each PKC, two sets of restriction endonucleases were used: one
able to cut only at one restriction point, and the second one unable to
cut the amplified product.
70 °C or quantified with a PhosphorImager (Molecular Dynamics).
The rat glyceraldehyde-3-phosphate dehydrogenase probe (kindly provided
by Dr. J. M. Blanchard, Institut de Génétique Moléculaire, Montpellier) was used as internal standard.
-actin
polyclonal antibody (Sigma) was used to evaluate equivalent loadings.
Antigen-antibody complexes were detected using an ECL assay kit
(Amersham Pharmacia Biotech) and quantified with a laser densitometer
(Molecular Dynamics). ERK-1 and -2 phosphorylation was analyzed with an
anti-phosphorylated ERK-1/2 antibody (Santa Cruz Biotechnology). ERK
activity was measured on the myelin basic protein (MBP) (Sigma) as
substrate (30). After cell lysis, equal amounts of protein were
incubated overnight at 4 °C with the anti-ERK-1/2 antibody and
protein A-agarose beads (Sigma). The immunoprecipitated complexes were
incubated with MBP in the presence of [
-33P]ATP
(1 mCi/assay). The labeled MBP was quantified with a PhosphorImager.
antibody as described above. To
analyze the serine-phosphorylated PKC
, equal amounts of protein
were immunoprecipitated with the anti-
PKC antibody, separated on
SDS-PAGE, and revealed with an anti-serine antibody (Zymed
Laboratories Inc. Laboratories, San Francisco, CA)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
were found
expressed in parental AR4-2J and FGF-2 producing cells, and they
exhibited apparent molecular masses of 80, 76, and 96 kDa,
respectively. Fig. 1 summarizes the
results obtained in A3 cells expressing the FGF-2 of 210 aa, compared
with those of A5 cells expressing the FGF-2 of 155 aa and with those of
control CAT cells. In A3 cells PKC
was significantly up-regulated
by a factor of 1.7, whereas PKC
was strongly decreased by about a
factor of 3. In contrast, in A5 cells only the level of PKC
was
significantly different from that in control CAT cells, with a 1.6-fold
increase.
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Fig. 1.
Effect of FGF-2 expression on PKC protein
levels. PKC levels were measured by Western blots on cell extracts
prepared from control CAT cells, A3 cells expressing the HMW FGF-2, and
A5 cells expressing the LMW FGF-2. Cell lysates corresponding to 30 µg of protein for PKC and
and 60 µg of protein for PKC
were loaded on SDS-PAGE and immunoblotted with antibodies specific for
the different PKC isotypes. Western blots from a single experiment are
shown. Quantifications were done with a laser densitometer. Values are
the means ± S.D. of five independent experiments in triplicate.
PKC levels in control CAT cells were given an arbitrary value of 1. *,
p < 0.05; **, p < 0.002, compared
with control CAT cells.
and
levels were modulated by the concentration of the HMW FGF-2 (Fig.
2), and as observed in A3 cells, these PKCs were differentially regulated. Taken together, these data show that the HMW isoform regulates PKC
and
levels and also indicate that the secretory and nonsecretory FGF-2 behave differently with respect to PKC levels.
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Fig. 2.
Dose-dependent inhibition of HMW
FGF-2 expression by doxycycline and modulation of PKC levels.
Control CAT cells were transfected with the tet-off gene expression
system. The plasmid containing the TRE encoded the HMW FGF-2. The
transfected cells were incubated for 24 h with increasing
concentrations of doxycycline and then lysed. Cell lysates
corresponding to 40 µg of protein were loaded on SDS-PAGE and
immunoblotted with antibodies specific for the different PKC isotypes
or with anti-human FGF-2 recognizing all FGF-2 isoforms. Western blots
from a single experiment are shown. PKC and HMW FGF-2 relative levels
were determined with a laser densitometer. Values are the means ± S.D. of three independent experiments. Protein levels in the absence of
doxycycline were given a value of 1. *, p < 0.05; **,
p < 0.01, compared with values obtained in the absence
of doxycycline.
and
were expressed as
transcripts of about 3.4 kilobases, and PKC
was expressed as a
transcript of about 7 kilobases (32). A PKC
mRNA species of
about 8 kilobases (32) was also detected as a faint band. As shown in
Fig. 3, the level of the
isotype mRNA was decreased by a factor of 2.5 in A3 cells; by contrast, that of PKC
was increased by a factor of 2. In contrast, in A5
cells the PKC
mRNA was found increased by a factor of 1.6 (Fig.
3). Thus, the expression of FGF-2 modifies both PKC protein and
mRNA levels.
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Fig. 3.
mRNA levels of the PKC isotypes in
control CAT and FGF-2 expressing cells (A3 and A5). Total RNA (30 µg) was hybridized with specific rat PKC probes prepared by reverse
transcriptase-polymerase chain reaction and 32P-labeled as
described under "Experimental Procedures." A rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was
used as internal standard. Northern blots from a single experiment are
shown. The quantitative analysis of mRNA levels was performed with
a PhosphorImager. Transcripts of control CAT cells were given an
arbitrary value of 1. Values are the means ± S.D. of four
independent experiments. *, p < 0.05; **,
p < 0.01, compared with control CAT cells.
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Fig. 4.
ERK-1 and -2 levels and phosphorylations in
cells expressing the HMW FGF-2. Cells extracts from CAT, A3, and
A5 cells, respectively, were prepared in parallel, and equal amounts of
protein (20 µg) were analyzed by SDS-PAGE. Membranes were
immunoblotted with an antibody directed against ERK-1 and -2 (upper panel). Blots were stripped and reprobed with
anti-phosphorylated ERKs (lower panel). Data from a single
experiment are shown. The relative intensities of bands were determined
by laser densitometry. Values are the means ± S.D. of three
independent experiments. *, p < 0.02; **,
p < 0.001, compared with CAT cells.
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Fig. 5.
ERK-1 and -2 levels and phosphorylations in
CAT cells expressing the HMW FGF-2 under the control of
doxycycline. Control CAT cells were transfected with the tet-off
system leading to the expression of the HMW FGF-2 under the control of
tTA. Cells were then incubated for 24 h with increasing
concentrations of doxycycline before cell lysis. Equal amounts of
protein (20 µg) were analyzed by SDS-PAGE, and the membranes probed
with an antibody recognizing ERK-1 and -2 (top panel). Blots
were reprobed with an anti-phosphorylated ERK-1 and -2 antibody
(middle panel). ERK activity was determined on MBP substrate
(bottom panel). Data from a single experiment are shown in
the panels. Similar results were obtained in three independent
experiments. The relative intensities of bands were determined with a
laser densitometer. Levels in the absence of doxycycline were given a
value of 1. *, p < 0.05; **, p < 0.01, compared with values obtained in the absence of
doxycycline.
and
expressions do not
support the involvement of FGFR activation in the effects of the HMW
form as also reported for other cell lines (1). Anyway, activation of
FGFRs by the HMW form present outside the cells can be determined by
analyzing ERK-1/2 phosphorylation because the secreted FGF-2 strongly
activates the MAPK pathway. We should notice that expression of LMW and HMW FGF-2 does not modify the subtypes of the FGFR or the variant of
FGFR1 (33). CAT cells were transfected with the tet-off expression system and then grown in serum-free medium and treated with 400 µM suramin, which is a well established inhibitor of
FGF-2 binding and activation of the cell surface FGF receptors (34).
This treatment was found to strongly inhibit ERK-1/2 phosphorylation induced by the rFGF-2 added to the culture medium (Fig.
6A). By contrast, suramin did
not modify the kinase phosphorylation in cells expressing the HMW FGF-2
(Fig. 6B). Similar results were obtained by adding to the
culture medium neutralizing antibodies recognizing all FGF-2 forms
(Fig. 7). The antibody treatment strongly decreased ERK phosphorylation induced by rFGF-2 but did not affect the
basal ERK phosphorylation observed either in control cells (Fig. 7;
2000 ng/ml doxycycline) or in cells expressing the HMW FGF-2 (Fig. 7; 0 ng/ml doxycycline).
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Fig. 6.
Effect of suramin on ERK-1 and -2 phosphorylation by the HMW FGF-2. Control CAT cells were
transfected with the tet-off system leading to the expression of the
HMW FGF-2 under the control of doxycycline. Cells were then incubated
for 24 h with 0 or 2 µg/ml doxycycline, with or without suramin
(400 µM), and with or without rFGF-2 (0.1 nM). Equal amounts of protein (20 µg) were analyzed by
SDS-PAGE, and the membranes were probed with an anti-phosphorylated
ERK-1 and -2 antibody. Membranes were stripped and reprobed with an
antibody recognizing ERK-1 and -2. The relative intensities of bands
were determined by laser densitometry. A, suramin inhibits
phosphorylation of ERK-1 and -2 induced by exogenous rFGF-2 in cells
grown with 2 µg of doxycycline to inhibit the expression of the HMW
FGF-2. B, effect of suramin on ERK-1/2 phosphorylation in
cells expressing (0 ng of doxycycline) or not (2000 ng/ml doxycycline)
the HMW FGF-2. Values are the means ± S.D. of two independent
experiments performed in duplicate.
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Fig. 7.
Effect of neutralizing anti-FGF-2 antibodies
on ERK-1 and -2 phosphorylation by the HMW FGF-2. Control CAT
cells were transfected with the tet-off system leading to the
expression of the HMW FGF-2 in the absence of doxycycline. Cells were
then incubated for 24 h with 0 or 2000 ng/ml doxycycline, with or
without neutralizing anti-FGF-2 antibodies (5 µg/ml). To check the
efficiency of the anti-FGF-2 antibody, cells were stimulated with
rFGF-2 (0.1 nM). Equal amounts of protein (30 µg) were
analyzed by SDS-PAGE, and the membranes were probed with an anti-active
ERK-1 and -2 antibody. Membranes were stripped and reprobed with an
antibody recognizing ERK-1 and -2. The relative intensities of bands
were determined by laser densitometry. Values are the means ± S.D. of two independent experiments performed in duplicate.
and
expressions and the above results on
ERK phosphorylation, all of the data indicate that the activation of
cell surface FGFRs is not involved in the effects of the large form of
FGF-2, as already reported in other cell types (1).
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Fig. 8.
Effect of FGFR1dn on ERK-1/2 phosphorylation
by the HMW FGF-2. Control CAT cells were cotransfected with the
tet-off system leading to the expression of the HMW FGF-2 in the
absence of doxycycline and with increasing concentrations of the
FGFR1dn vector (0-3 µg/ml). Cells were then incubated for 24 h
with 2000 (A) or 0 ng/ml (B) doxycycline. Cells
were stimulated with rFGF-2 (0.1 nM) to establish the
efficiency of the transfected FGFR1dn. For experiments performed in the
absence of rFGF-2, anti-FGF-2 antibodies (5 µg/ml) were added to
culture medium. Equal amounts of protein (20 µg) were analyzed by
SDS-PAGE, and the membranes were probed with an anti-phosphorylated
ERK-1 and -2 antibody. Membranes were stripped and reprobed with an
antibody recognizing ERK-1 and -2, and the relative intensities of
bands were determined by laser densitometry (n = 3).
-dependent--
It is known that the secretory FGF-2
activates different PKC isotypes through the occupancy of FGFRs (36,
37), and PKCs have been involved in the activation of the MEK pathway
(38, 39). Then the question arises of whether ERK phosphorylation by
the HMW FGF-2 is PKC-mediated. To answer that question, three PKC
inhibitors were used: GF109203X, which inhibits most of the PKC
isozymes, among them PKC
,
, and
(26-27); Gö 6976, which only inhibits the conventional PKCs
,
, and
; and
rottlerin, a specific inhibitor of PKC
when used at the
concentration of about 6 µM (40). Because the
conventional PKC isotypes
1 and
were not found by reverse
transcriptase-polymerase chain reaction in any of the cell lines used
in the present study, Gö 6976 must only decrease PKC
activity. At the concentrations used in the present study, any of these
compounds modified cell shapes or attachment, indicating the absence of
toxic effects as reported for other cell types (26, 41). Gö 6976 added for 24 h to the cell medium slightly decreased ERK-1 and -2 phosphorylation whatever the doxycycline concentration (Fig.
9). By contrast, GF109203X strongly
reduced ERK-1 and -2 phosphorylation in cells producing the HMW FGF-2
(Fig. 9), suggesting that ERK activity was under the control of an
unconventional PKC. Rottlerin decreased ERK-1 and -2 phosphorylation in
cells producing the FGF-2 of HMW (Fig. 9). To confirm the specific role
of the
isotype, cells were transfected with cDNA encoding
either normal or mutated PKC
. The normal
isotype induced an
increased phosphorylation of ERKs whatever the level of the HMW FGF-2
(results not shown), whereas cell transfection with increasing
concentrations of cDNA encoding the nonfunctional PKC
resulted
in a concentration-dependent decrease of ERK-1 and -2 phosphorylation only in cells expressing the HMW FGF-2 (Fig.
10; 0 ng doxycycline). Furthermore, at
the maximal concentration of the mutated PKC
(9 µg), the
phosphorylation level was comparable with that of control cells (Fig.
10; 2000 ng of doxycycline). These data clearly indicate that ERK
activation was PKC
-dependent and that this PKC isotype
must be in the active state in HMW FGF-2 expressing cells.
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Fig. 9.
Effect of PKC inhibitors on ERK-1 and -2 phosphorylation. After transfection CAT cells were cultured for
24 h either in the absence or in the presence of doxycycline and
different PKC inhibitors. GF109203X was used at the concentration of 1 µM to inhibit PKC ,
, and
; Gö 6976 was
used at the concentration of 50 nM to inhibit PKC
, and
rottlerin, which specifically inhibits PKC
, was used at the
concentration of 6 nM. Cell lysates were loaded on SDS-PAGE
and probed with the anti-phosphorylated ERK-1/2 and then reprobed with
the anti-ERK-1/2 antibody to assess equivalent protein loading. Data
from a single experiment are shown for phosphorylated ERK-1/2. Similar
results were obtained in three independent experiments. The relative
intensities of bands were determined with a laser densitometer. *,
p < 0.05; **, p < 0.01, compared with
values obtained in untreated cells (T).
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Fig. 10.
Effect of the dominant negative PKC
on ERK-1 and -2 phosphorylation. CAT cells
were transfected with the tet-off HMW FGF-2 expression system and
cotransfected with increasing amounts of the PKC
mutant (PKC
). After 24 h cells were lysed, and 20 µg of
protein were fractionated on SDS-polyacrylamide gels. Upper
panel, anti-ERK-1 and -2 were used to detect ERK protein levels.
Lower panel, detection of phosphorylated ERK-1 and -2 with
an anti-active ERK antibody. A representative SDS-PAGE is presented.
Levels of nonphosphorylated ERK-1/2 were comparable in the absence or
presence of doxycycline. Quantifications of phosphorylated ERKs were
done with a laser densitometer. Values are the means ± S.D. of
three separate experiments. **, p < 0.01 compared with
values obtained in the absence of PKC
.
activation (42), we analyzed the effect of the HMW FGF-2 on both
parameters. In CAT cells transfected with the void tet-off vectors,
doxycycline did not affect PKC
levels and phosphorylation (Fig.
11A). By contrast, in cells
expressing the HMW FGF-2, PKC
phosphorylation was found increased
by 1.65-fold in total cell homogenate (Fig. 11B). The kinase
translocated to the particulate fraction was about 75% of the total
PKC
against about 30% in control cells, and the particulate
fraction contained 2.5-fold more PKC
(Fig. 11C). These
data clearly indicate that the kinase C
was activated by the large
form of FGF-2. In addition, the present results show that
overexpression of normal or mutated PKC
did not modify the level of
ERK-1/2 proteins (Fig. 10); thus PKC
regulates ERK-1 and -2 activation without affecting their expressions.
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Fig. 11.
Phosphorylation and translocation of
PKC by the HMW FGF-2. CAT cells were
transfected with the tet-off HMW FGF-2 expression system and grown
24 h with (2000 ng) or without (0 ng) doxycycline before cells
lysis. Samples of 20 µg of total protein were immunoprecipitated with
the anti-PKC
antibody and then fractionated on SDS-polyacrylamide
gels. An antibody directed against the anti-phosphorylated serine was
used to detect the phosphorylated PKC
. A, control
experiments performed on cells transfected with the void tet-off
vectors. B, experiments performed as in A with
the tet-off vectors containing the HMW FGF-2 cDNA leading to the
expression of the HMF FGF-2 (0 doxycycline). C, PKC
activation by the HMW FGF-2. After cell transfection as in
B, the particulate fraction was prepared as
described under "Experimental Procedures," and equal amounts of
proteins were loaded in each lane (20 µg). After SDS-PAGE and
eletrotransfer, membranes were revealed with the anti-PKC
antibody.
The particulate fraction contained about 15% of total protein in both
doxycycline treated and untreated cells. Levels of translocated PKC
were increased by the HMW FGF-2 (0 ng/ml doxycycline). Quantifications
were performed with a laser densitometer. Values are the means ± S.D. of three separate experiments. **, p < 0.01.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
levels and ERK-1 and -2 activation. By comparing cells stably producing
the HMW FGF-2 to control CAT and LMW FGF-2 producing cells, differences
in PKC levels were observed. Indeed, the HMW FGF-2 of 210 aa
significantly modified the PKC amounts by increasing the
isotype by
a factor of 1.7 and by decreasing the
one by a factor of 3. Transient transfection of control CAT cells with the cDNA encoding
the HMW FGF-2 under the control of the
tetracycline-dependent promoter confirmed these opposite
regulations of PKC
and
and also showed the correlation between
HMW FGF-2 concentration and PKC levels. Otherwise, as mRNA levels
paralleled those of proteins, the modifications observed should take
place at the pretranslational level. Our data also indicate that the
two FGF-2 forms exert opposite effects on PKC expression, strongly
suggesting the involvement of different regulatory mechanisms.
and
as other PKC isotypes activate various signal
transduction pathways, and some of these signaling pathways converge on
ERK-1/2 (38). The functional relevance of the modified levels of PKCs
was investigated by analyzing ERK-1/2 activation in cells producing the
HMW FGF-2. The results obtained either with stably transfected A3 cells
or transiently transfected CAT cells expressing variable amounts of HMW
FGF-2 show an increased ERK-1 and -2 phosphorylation, correlated to
increased ERK-1 and -2 activities. Therefore, our findings indicate
that constitutive expression of HMW FGF-2 induces ERK activation.
in ERK
activation. First, rottlerin, a specific inhibitor of the
subtype,
reduced ERK-1 and -2 activation. Second, overexpression of functional
PKC
in control cells increased ERK-1 and -2 phosphorylation, indicating that this PKC isotype acts upstream from ERK-1 and -2 in
these cells. Finally, in cells expressing the HMW FGF-2, the
inactivated PKC
dose-dependently decreased ERK-1 and -2 phosphorylation. Thus, when overexpressed PKC
activates the signaling cascade leading to ERK phosphorylation in cells producing the
large form of FGF-2. Although the mechanism by which PKC
regulates
MEK-1 activation remains to be elucidated, recent studies show a PKC
-mediated activation of MEK-1/ERK but not of c-Raf, suggesting that PKC
acts downstream of c-Raf (44, 45). The HMW
peptide enhanced ERK activity 2-3-fold, which is low compared with the
increase of ERK activity observed early after cell stimulation by
rFGF-2. However, the activation evoked by the HMW peptide was not
short-lived because it was observed for at least 24 h in the doxycycline-regulated system and at any time in stably transfected A3
cells. Moreover, we found that MEK inhibition by PD98059 also decreased
the growth promoting effect of the HMW FGF-2, indicating that the
hyperphosphorylated ERK was functional and involved in the mitogenic
effect of the HMW FGF-2.2
and
protein expressions were modified by the HMW FGF-2 at a
pretranslational step, indicating that regulation occurred either at
the transcriptional or at the mRNA half-life levels. We previously
showed that the HMW FGF-2 regulated the expression of FGFR1 and laminin
B1 by modifying the half-life of their transcripts (33, 8). A similar
mechanism could be evoked for the PKCs. By contrast, ERK-1/2 proteins
remained constant either after short term transfection with inducible
HMW FGF-2 or in stably transfected cells, suggesting that the PKC
controlled ERK activity but not ERK biosynthesis. However, an increased
turnover rate of ERK-1/2 proteins cannot be excluded.
and
levels is an important finding in the understanding of the
intracrine regulation because these PKCs have been shown to exert
opposite effects according to the cell type (46, 47). Recent data
demonstrate the mitogenic action of PKC
and the growth inhibitory
effect of PKC
(48). Opposite regulations have also been reported on
the serum-responsive element, which plays an important role in the
control of gene expression (49) and on the heat shock protein-27
phosphorylation which is involved in multiple cell functions (50).
These observations suggest that the different expression patterns of
PKC
and
in cells producing the HMW FGF-2 might facilitate the
mitogenic response induced by this growth factor.
and
. Altogether these results give
new insights on the broad effects of the HMW FGF-2 on the intracellular
signaling pathways, which can be involved in growth promotion, one of
the well established biological effects of this FGF-2 isoform (1).
Analysis of the nuclear targets and gene promoters regulated by the HMW
FGF-2 may lead to the characterization of the genes that are thereby
activated and to further progress in the understanding of the
mechanisms involved in the intracrine regulation.
![]() |
FOOTNOTES |
---|
* This work was supported by Association pour la Recherche sur le Cancer Grant 6165 (to F. C.).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.
§ These authors contributed equally to this work.
¶ Supported by a long term fellowship from the Ligue Contre le Cancer des Hautes Alpes.
Supported by a long term fellowship from the Ligue Contre le
Cancer du Tarn and the Fondation pour la Recherche Médicale.
To whom correspondence should be addressed: INSERM U 531, ILB,
CHU Rangueil Bat L3 31054 Toulouse Cédex, France. Tel.:
33-05-61-32-24-04; Fax: 33-05-61-32-24-03; E-mail:
Francois.Clemente@rangueil.inserm.fr.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M001184200
2 A. Estival and F. Escaffit, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: FGF, fibroblast growth factor; aa, amino acids; ERK, extracellular signal-regulated kinases; rFGF, recombinant FGF (155 amino acids); FGFR1, fibroblast growth factor receptor type 1; HMW, high molecular weight; LMW, low molecular weight; MBP, myelin basic protein; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; tTA, tetracycline-controlled transactivator; PAGE, polyacrylamide gel electrophoresis; tet-off expression system, cotransfection of repressor tTA vector and TRE vector containing HMW FGF-2 cDNA.
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