From the Department of Internal Medicine and
¶ Comprehensive Cancer Center, The Ohio State University,
Columbus, Ohio 43210
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ABSTRACT |
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Fibroblast growth factors (FGF) elicit biological effects by binding to high affinity cell-surface receptors and activation of receptor tyrosine kinase. We previously reported that two NIH/3T3 derivatives, NR31 and NR33 (NR cells), express high levels of full-length FGF-1 and exhibit a complete spectrum of transformed phenotype. In the present study, we report that NR cells respond to the mitogenic stimulation of truncated FGF-1 but not to the full-length FGF-1. Incubation of the NR cells with either form of FGF-1 resulted in its binding to cell-surface FGF receptors, activation of mitogen-activated protein (MAP) kinase, and induction of c-fos and c-myc. These data demonstrate that the FGF receptor-mediated, MAP kinase-dependent signaling pathway is not defective in the NR cells. Our data further suggest that the activation of MAP kinase in response to full-length FGF-1 is not sufficient for mitogenesis. Subcellular distribution of exogenously added FGF-1 demonstrated that full-length FGF-1 fails to translocate to the nuclei of NR31 cells. Although the full-length FGF-1 was detected in the nuclear fractions of both NIH/3T3 and NR33 cells, its half-life is much shortened in NR33 than in NIH/3T3 cells. These observations suggest that non-responsiveness of the two NR cell lines may be due to defectiveness at different steps of nuclear translocation mechanism of FGF-1.
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INTRODUCTION |
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Fibroblast growth factors
(FGF)1 comprise a family of
14 structurally related polypeptide mitogens. They modulate cell
growth, proliferation, and differentiation. FGFs also regulate
angiogenesis, embryogenesis, and neurite outgrowth (1, 2). FGFs elicit these functions mainly by binding to high affinity cell-surface receptors and activating a family of receptor tyrosine kinases (3, 4).
FGF activation of FGFR1 triggers autophosphorylation on at least seven
tyrosine residues (5). One of these sites, Tyr-766 of FGFR1, serves as
a binding site for phospholipase C- (6). The mutation of this site
abrogates association and activation of phospholipase C-
and
phosphatidylinositol hydrolysis but not FGF-1-mediated mitogenesis in
L6 myoblast and BaF3 cells or differentiation of PC12 cells (7-9).
However, autophosphorylation of Tyr-766 may be required for efficient
endocytosis of FGF receptors (10). Autophosphorylation of Tyr-653 and
Tyr-654 is required for intrinsic tyrosine kinase activity. The
mutation of other autophosphorylation sites of FGFR1 did not affect
FGF-1-dependent cell proliferation in L6 myoblasts or
neuronal differentiation in PC12 cells. The mutation of these four
autophosphorylation sites together or even including Tyr-766 of FGFR1
also did not affect FGF-1-induced phosphorylation of Shc and a
Grb2-associated 90-kDa protein or activation of MAP kinase (MAPK/ERK;
see Ref. 5). FGFR1 triggers activation of at least two pathways, a
Ras-dependent and a phospholipase
C-
-dependent protein kinase C pathway. Both pathways
converge on a common signaling molecule Raf-1 and then activate the
downstream MAPK signaling cascade and mitogenesis (11).
Several reports have demonstrated that Ras-dependent MAPK signaling pathway is important for FGFR1-mediated biological responses (12-14). Ras-dependent activation of Raf-1 and MAPK is sufficient for FGFR-mediated mitogenesis in BaF3 cell line expressing FGFR1 (11). The use of interfering mutants of MAPK showed that MAPK is necessary for fibroblast proliferation (15). FGF-mediated activation of MAPK is necessary and sufficient for mesoderm induction in Xenopus (12). Activation of MAPK is also shown to be involved in cellular transformation and differentiation (16). Expression of the constitutively activated MAPK kinase (MEK) in NIH/3T3 cells results in transformation of NIH/3T3 cells that closely resemble Ras-transformed NIH/3T3 cells and results in differentiation of PC12 cells (16). However, other reports showed that the activation of MAPK pathway is insufficient to promote FGF- (17) or PDGF (18)-mediated differentiation of PC12 cells.
MAPK has been implicated as a critical component of the mitogenic signal transduction cascade and is likely to play a role in cellular pathways that control growth and differentiation (19-21). MAPK belongs to a family of serine/threonine protein kinases that are activated in response to a variety of stimuli and growth factors. MAPK is a potent pleiotropic regulator of biological responses, and it requires dual phosphorylation at tyrosine and threonine residues for full catalytic activity (22, 23). MAPK can be activated by both Ras-dependent and Ras-independent pathways (24). Phosphorylation and activation of MAPK is mediated by MEK. The activation of MEK is regulated by either Raf or MEK kinase (25); activated MEK in turn phosphorylates and activates MAPK (26). Activated MAPK translocates to the nucleus and is shown to activate and/or phosphorylate several important signaling elements such as p90Rsk, p62Tcf, c-jun, and c-myc (20, 27, 28). Thus, MAPK provides a link between the receptor tyrosine kinase cascade and the serine/threonine protein kinase cascade and also between cytoplasmic and nuclear signaling processes.
Three different forms of FGF-1 have been reported (29, 30). The two truncated forms have deletion of the first 14 or 20 amino acids (aa) from the full-length, 154-aa form based on the predicted amino acid sequence of the FGF-1 gene (31). There has not been any reported difference in the biological response between the full-length and truncated FGF-1 proteins (1, 32). Recent studies showed that translocation of FGF-1 to the nucleus is essential for FGF-induced mitogenesis. Deletion of nuclear localization sequence (NLS), from aa 21 to 27, abolished the mitogenic activity of FGF-1 without affecting receptor tyrosine phosphorylation and induction of c-fos expression (33). The synthetic peptide containing NLS of FGF-1 and cell membrane-permeable sequence is able to stimulate DNA synthesis in an FGFR-independent manner (34). A dual mode of signaling pathway for FGF-1 has thus been proposed (35, 36). FGF-1 in fusion with diphtheria toxin is able to translocate, through diphtheria toxin receptor, into cells lacking functional FGF-1 receptors and induces DNA synthesis without detectable increase in tyrosine phosphorylation. The translocation of the fusion protein into cytosol and subsequently to the nucleus is essential for DNA synthesis. The receptor-mediated transduction pathway is necessary for other cellular processes including cell division and proliferation (35).
We have previously reported the characterization of various transfectants of NIH/3T3 expressing full-length human FGF-1. The expression of high levels of FGF-1 resulted in transformation of NIH/3T3 cells (37). In this report, we demonstrate that the transfectants, NR31 and NR33 (NR cells) which express high levels of full-length FGF-1 (154 aa), respond to the mitogenic stimulation of truncated FGF-1 (deletion of aa 1-14) but fail to respond to full-length FGF-1. Significantly, MAPK can be activated in NR cells with either full-length or truncated FGF-1. The subcellular distribution of exogenously added FGF-1 indicated that the full-length FGF-1 is unable to translocate to the nucleus of NR31 cells. Although the full-length FGF-1 was detected in the nuclear fractions of both NIH/3T3 and NR33 cells, its half-life is much shortened in NR33 than in NIH/3T3 cells. These observations suggest that non-responsiveness of the two NR cell lines may be due to defectiveness at different steps of nuclear translocation mechanism of FGF-1.
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MATERIALS AND METHODS |
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Growth Factors--
Full-length recombinant human (full-length
rhFGF-1, aa 1-154) and truncated recombinant human FGF-1
(rhFGF-1, aa 15-154) were cloned in prokaryotic pET20b(+)
expression system (Novagen, Madison, WI) and purified by
heparin-Sepharose affinity chromatography as described (38).
Full-length rhFGF-1 from NR31 (NhFGF-1; aa 1-154) was purified as
described (37). PDGF was purchased from Upstate Biotechnology (Lake
Placid, NY), native bovine brain FGF-1 from R & D Systems (Minneapolis,
MN), and calf serum and heparin from Life Technologies, Inc.
Antibodies and Other Reagents--
Monoclonal
anti-phosphotyrosine antibody (4G10) was kindly provided by T. Roberts
and B. Druker (Dana Farber Cancer Institute, Boston). Monoclonal
anti-p42ERK2 antibody, alkaline phosphatase-conjugated
anti-mouse, or rabbit IgG and myelin basic protein (MBP) were obtained
from Upstate Biotechnology. Polyclonal anti-p44ERK1
antibody and monoclonal anti-p53 antibody (Pab 240) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-bovine FGF-1
antibody was from Promega (Madison, WI). Horseradish
peroxidase-conjugated anti-rabbit IgG and the enhanced
chemiluminescence (ECL) immunodetection system were from Amersham Corp.
Na125I was obtained from ICN (Costa Mesa, CA),
[-32P]ATP, [
-32P]dCTP, and
[3H]thymidine were from NEN Life Science Products.
Prehybridization and hybridization reagents for Northern blot analysis
were from 5 Prime
3 Prime (Boulder, CO).
Cell Culture-- The NIH/3T3-derived stable transfectants (NR and Tr cell lines) overexpressing full-length rhFGF-1 have been described (37). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. The stable transfectants were maintained in media supplemented with 400 µg/ml G418.
Mitogen Assay-- Near-confluent monolayer cells were starved in media containing 0.5% calf serum for 48 h. The DNA synthesis was initiated by addition of 5 ng/ml various mitogens, and cells were incubated for an additional 20 h. Cells were then pulsed with 1 µCi of [3H]thymidine for 6 h. The level of DNA synthesis was determined by measuring the incorporation of [3H]thymidine in trichloroacetic acid-precipitable material as described previously (37).
Stimulation of Cells by Growth Factors and Preparation of Cell
Lysates--
Near-confluent monolayer NIH/3T3, NR31 and NR33, cells
were starved in media containing 0.5% calf serum for 48 h. Cells
were washed once with phosphate-buffered saline (PBS) and once with serum-free media containing 0.2% bovine serum albumin (BSA), 10 µg/ml heparin, and 25 mM HEPES (pH 7.4). Stimulation was
performed by addition of 30 ng/ml full-length or rhFGF-1 at 37 °C
for 10 min. Control and FGF-stimulated cells were washed three times with ice-cold PBS and harvested by scraping in PBS. The cells were
pelleted and suspended in lysis buffer containing 10 mM
Tris (pH 7.4), 50 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM sodium pyrophosphate, 10%
glycerol, 0.1% sodium orthovanadate, 50 mM NaF, and
protease inhibitors. Lysates were clarified by centrifugation at 14,000 rpm for 20 min at 4 °C to remove nuclei and cell debris.
Northern Blotting Analysis--
For analysis of FGFR1 mRNA
levels, total RNA was isolated from cells grown in the presence of 10%
calf serum. For analysis of induction of immediate early genes,
near-confluent cells were starved and stimulated with 30 ng/ml
full-length or rhFGF-1 for 0-4 h as described above. Cells were
lysed in 4 M guanidinium thiocyanate, and total RNA was
isolated using 5.7 M CsCl density gradient essentially as
described (31). Samples containing 10 µg of total RNA were
electrophoresed on 1% formaldehyde gel and transferred onto
nitrocellulose membrane (Schleicher & Schuell). The RNA was immobilized
by baking the membrane at 80 °C for 2 h prior to hybridization
with cDNA probes for human FGFR1 and mouse c-fos or
c-myc. cDNA probe for cyclophilin gene was used as an
internal control. The probes were generated with
[
-32P]dCTP (~3000 Ci/mmol) using redi
prime random primer labeling kit as described by the manufacturer
(Amersham Corp.). The hybridization was carried out in 50% formamide
solution (5 Prime
3 Prime, Inc.) containing 32P-labeled
probes (1 × 106 cpm/ml) and 10% dextran sulfate at
42 °C for 20 h. At the end of the hybridization period, the
filter was washed twice with 2 × SSC and 0.1% SDS at room
temperature and once at 50 °C followed by an additional wash at room
temperature for 15 min each and subjected to autoradiography.
Radioiodination and Binding Assay-- FGF-1 was radioiodinated with Na125I using the chloramine-T method as described (39). Iodinated protein was purified on a heparin-agarose column. FGF-1 bound to heparin-agarose column was eluted from the column with elution buffer containing 20 mM HEPES, 0.2% BSA, and 2.0 M NaCl. When subjected to SDS-PAGE, iodinated as well as unlabeled full-length rhFGF-1 migrated as a single band. The biological activity of the labeled rhFGF-1 was determined by its ability to stimulate DNA synthesis of serum-starved NIH/3T3 cells. The specific activity of the different preparation of 125I-rhFGF-1 varied between 1 and 3 × 105 cpm/ng.
The binding assay was performed as follows. Cells were seeded at a density of 4 × 104 cells/well in 24-well plates. Cells were washed twice with cold serum-free Dulbecco's modified Eagle's medium 48 h later and incubated in 1 ml of binding buffer (Dulbecco's modified Eagle's medium containing 25 mM HEPES (pH 7.3), 0.2% BSA, and 10 µg/ml heparin) at 4 °C for 30 min. Subsequently, the cells were incubated in 0.2 ml of binding buffer containing serial dilution of full-length 125I-rhFGF-1 at 4 °C for 3 h. Nonspecific binding was obtained in a parallel assay using the same serial dilution of full-length 125I-rhFGF-1 but in the presence of 100-fold excess of non-radioactive rhFGF-1. At the end of the incubation period, cells were washed twice with cold binding buffer and once with cold PBS containing 1.0 M NaCl and then solubilized in 0.25 M NaOH. Bound radioactivity in duplicate samples was measured in a Beckman gamma counter, and data were subjected to Scatchard analysis (40).Western Blot Analysis-- To detect phosphorylated p44ERK1 and p42ERK2 in gel shift experiments, cell lysates containing 25 µg of protein were subjected to 7.5% SDS-PAGE, electrophoretically transferred to a nitrocellulose membrane, and subjected to immunoblot analysis. The nonspecific sites on the nitrocellulose membrane were blocked in Tris-buffered saline (TBS; 100 mM Tris (pH 7.5), 0.9% NaCl) containing 3% BSA for 2 h. The blot was then incubated with either anti-p44ERK1 or anti-p42ERK2 antibody at a concentration of 1 µg/ml in TBS containing 1% BSA at room temperature for 6 h. Following two washes in TBS at room temperature for 10 min each, the blot was incubated in 1:1000 dilution of alkaline phosphatase-conjugated anti-rabbit or anti-mouse IgG antibody at room temperature for 2 h. The blot was washed as above, and the immune complexes were detected by color reaction of alkaline phosphatase substrate reagents (Upstate Biotechnology). To detect p53 protein in the cytoplasmic and nuclear fractions, anti-p53 antibody was used as the primary antibody.
Immunoprecipitation of MAPK-- For detection of tyrosine phosphorylation of MAPK in NR and NIH/3T3 cells, antibodies to p42ERK2 and p44ERK1 (5 µg) were incubated with 50 µl of protein A-agarose (50% slurry) in 300 µl of PBS at 4 °C for 6 h. Antibody/protein A-agarose conjugates were washed twice with ice-cold PBS, and cell lysates containing 500 µg of protein in 300 µl of lysis buffer were then added to anti-p42ERK2 and p44ERK1 protein A-agarose conjugates and incubated overnight at 4 °C on a rotating wheel. Following incubation, immune complexes were recovered by centrifugation. The immune complexes were washed three times with lysis buffer containing 1 mM phenylmethylsulfonyl fluoride and 1 mM sodium orthovanadate and solubilized in SDS sample buffer, resolved on 10% SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody 4G10 as described above. Tyrosine-phosphorylated p42ERK2 and p44ERK1 were visualized by color reaction of alkaline phosphatase-conjugated secondary antibody. In a parallel experiment, immunoprecipitates of anti-p42ERK2 antibody were subjected to Western blot analysis using anti-p42ERK2 antibody followed by incubation with 1:2000 dilution of horseradish peroxidase-conjugated anti-mouse IgG antibody in TBS containing 0.05% Triton X-100 and ECL development to confirm the migration and abundance of the protein in immunoprecipitates.
In-gel Kinase Assay--
The in-gel kinase assay using MBP
as a substrate was performed as described (41). Briefly, cell lysates
containing 20 µg of protein were resolved in 10% SDS-PAGE containing
0.4 mg/ml MBP. The MBP/SDS-PAGE gel was washed with buffer containing
100 mM Tris (pH 5.5), 5 mM -mercaptoethanol,
and 20% isopropyl alcohol. The gel was denatured and renatured as
described (41). The renatured gel was then incubated in kinase buffer
(20 mM Tris (pH 7.2), 10 mM MgCl2,
15 mM
-glycerophosphate, and 0.3 mM sodium
orthovanadate) at 22 °C for 30 min. Kinase reaction was carried out
by incubating the gel in kinase buffer containing 50 µCi of
[
-32P]ATP and 50 µM ATP at 22 °C for
30 min. The gel was washed three times with 1% pyrophosphate in 5%
trichloroacetic acid, dried and subjected to autoradiography.
Cellular Fractionation-- Serum-starved cells grown in 35-mm Petri dishes were incubated in the presence of iodinated full-length or truncated rhFGF-1 at different temperatures and times. At the end of the incubation period, cells were washed three times with serum-free medium and three times with PBS. Lysis buffer was then added to the monolayer cells (PBS containing 10 mM EDTA, 1% Triton X-100, 15 mM NaCl, 1 mM sodium orthovanadate, 10 mM NaF, 200 units/ml aprotinin, 100 units/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and incubated at 4 °C for 10 min. The cells were harvested by scraping and incubated on ice for additional 15 min. The cell lysates were then fractionated into cytosol and nuclear fractions as described (35). Briefly, the lysates were centrifuged at 800 × g for 15 min, and the supernatant was designated as cytosolic fraction. The pellet was washed three times in lysis buffer containing 0.4 M sucrose and layered over lysis buffer containing 0.7 M sucrose and centrifuged as above. This fractionation step was repeated two more times. The pellet representing a nuclear fraction was resuspended in lysis buffer, sonicated, and freeze-thawed twice. The total protein content in cytosolic and nuclear fractions was determined using Bio-Rad protein assay dye reagent. Ten µg of total proteins from each sample were subjected to 15% SDS-PAGE and autoradiography. To monitor the purity of subcellular fractionation, the amount of p53 in the nuclear and cytoplasmic fractions was determined by Western blotting analysis.
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RESULTS |
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NR31 and NR33 Cells Are Non-responsive to Full-length FGF-1-- We previously described that four NIH/3T3 derivatives (Tr31-5-1, Tr33-1-2, Tr34-4-1, and Tr34-1-1) stably transfected with full-length human FGF-1 cDNA in a retroviral expression vector are able to form foci, acquire anchorage independence, and can induce tumor formation in nude mice (37). We further showed that these four transfectants, unlike the parental NIH/3T3, produce an FGF-1-like mitogen, which was eluted off of a heparin-Sepharose column at 1.2 M NaCl and can stimulate serum-starved NIH/3T3 to synthesize DNA (37). Among the four transfectants, only Tr31-5-1 and Tr33-1-2 produced high levels of FGF-1 as determined by RNase protection analysis, Western blotting analysis, and [3H]thymidine incorporation assay (37). Initially, we examined the ability of various mitogens to stimulate DNA synthesis in various transfectants and NIH/3T3 cells. All transfectants displayed stronger mitogenic response to calf serum and PDGF than parental NIH/3T3 cells did. Surprisingly, Tr31-5-1 and Tr33-1-2 responded to the FGF-1 purified from Tr31-5-1 (NhFGF-1) in stimulating DNA synthesis only at 1.7 and 7.9%, respectively, the level of NIH/3T3 cells (Fig. 1). Tr31-5-1 and Tr33-1-2 transfectants are hence designated as NR31 and NR33 cells, respectively. The lack of response of NR cells to NhFGF-1 cannot be attributed to the trivial explanation of a defective mitogen as NhFGF-1 is mitogenic for Swiss/3T3, NIH/3T3, Tr34-4-1, Tr34-1-1, and an FGF-1 antisense transfectant Tr31-11-1 (Fig. 1). The lack of response of NR31 and NR33 was specific to NhFGF-1 as these cells can still be mitogenically stimulated by calf serum and PDGF (Fig. 1). We noted that these cells are also responsive to native bovine FGF-1 (Fig. 1), an FGF-1 with deletion of the first 14 amino acids from the full-length protein (30). We have repeated this experiment at least 10 times with triplicate plates and obtained the same results.
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Expression of FGFR1 mRNA in NR Cells-- NIH/3T3 cells predominantly expressed FGFR1 as the FGF receptor. We compared mRNA levels of FGFR1 in NR cells with those in parental NIH/3T3 cells using Northern blot analysis. The results demonstrated that the levels of FGFR1 transcripts in NR31 and NR33 cells, after normalization against the levels of cyclophilin mRNA, were approximately 80% compared with NIH/3T3 (data not shown). Thus, the data show that NR cells express normal levels of FGFR1 mRNA.
Ligand Binding Properties of NR Cells-- It is possible then that the failure of full-length rhFGF-1 to stimulate DNA synthesis in NR cells could be due to the inability of full-length rhFGF-1 to bind its receptors. We examined binding properties of iodinated full-length rhFGF-1 to NR cells and compared with those of the parental NIH/3T3 cells. Iodinated full-length rhFGF-1 was biologically active as determined by [3H]thymidine incorporation in responsive cells. Binding of full-length rhFGF-1 was specific and saturable (Fig. 3). Scatchard analysis of saturable binding studies with full-length rhFGF-1 revealed that both NR31 and NR33 cells bound rhFGF-1 with high affinity and as efficiently as NIH/3T3 cells. The dissociation constant (Kd) for NIH/3T3 as well as NR cells was in the range of 350-610 pM. The numbers of FGF-1 receptors expressed in NR31 and NR33 were 21,000-23,000 sites per cell and were comparable with 28,000 sites/cell in NIH/3T3 cells (Fig. 3). These results demonstrate that the NR cells retained the binding activity of the parental NIH/3T3 cells toward full-length rhFGF-1 and that the expression of full-length rhFGF-1 in NR cells did not impair the function of receptors or down-modulate the expression of the receptors. Therefore, the difference in the mitogenic potential of NIH/3T3 and NR cells to full-length rhFGF-1 could be due to a difference in downstream signaling.
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Phosphorylation of MAPK in NR Cells--
It has been reported that
externally supplied FGF-1 relays its mitogenic signal to the nucleus
via activation of the MAPK/ERK cascade. We therefore, examined the
effects of full-length and rhFGF-1 on the activation of MAPK.
Phosphorylation of two isoforms of MAPK, p44ERK1 and
p42ERK2, causes a shift in the electrophoretic mobility of
these proteins. In serum-starved NR31 and NR33, a low level (20% of
total p42ERK2 protein) of constitutive phosphorylation of
p42ERK2 was detected (Fig. 4,
B and C, lanes 2 and 3).
The stimulation of cells with either form of rhFGF-1 resulted in
increased phosphorylation of both p44ERK1 and
p42ERK2 in NIH/3T3 as well as in NR31 and NR33 cells.
Furthermore, in all three cell types, the levels of phosphorylation of
p44ERK1 in response to full-length and truncated rhFGF-1
were similar (Fig. 4, A and B). The quantitative
analysis of the immunoblot showed that both full-length rhFGF-1 and
rhFGF-1 caused mobility shift of a significant amount of
p44ERK1 and p42ERK2, with p44ERK1
phosphorylated at higher levels than p42ERK2 in all three
cell lines (Fig. 4C, lanes 4-9).
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Stimulation of MAPK Activity in NR Cells--
We then determined
if the phosphorylated MAPK in NR cells are catalytically active by
measuring their ability to phosphorylate MBP using in-gel kinase assay.
Phosphorylation at both tyrosine and threonine residues is essential
for full catalytic activity of MAPK (42). Fig.
6 shows that both full-length rhFGF-1 and rhFGF-1 activated 42- and 44-kDa kinases in NR31, NR33, and NIH/3T3 cells. The size of these activated kinases corresponds to ERK2 and
ERK1, respectively. The kinase activity of both forms of ERK was higher
in the NR cells than in NIH/3T3 cells regardless of whether the
full-length rhFGF-1 or
rhFGF-1 was the source of mitogen (Fig. 6,
A and D; lanes 5, 6, 8, and 9). Also
stimulation of p44ERK1 activity was much greater
(4-6-fold) than p42ERK2 activity in all three cell types,
perhaps due to high basal levels of p42ERK2 activity in
serum-starved cells.
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Induction of Immediate Early Genes in NR Cells--
The immediate
early genes c-fos, c-jun, and c-myc
have been identified as downstream substrates of MAPK (21). Stimulation of three cell types with either full-length or rhFGF-1 caused a
rapid and transient induction of c-fos and c-myc
early response genes, with maximal expression of c-fos
within 30 min and c-myc between 1 and 2 h (Fig. 7,
A and B).
Expression of the c-myc gene was much more stable over a 4-h
period compared with the expression of c-fos. Low levels of
c-myc transcript but not c-fos were detected in
serum-starved cells (Fig. 7, A and B, lanes 1- 3). Thus, NR31 and NR33 cells are able to respond to the
stimulation by full-length rhFGF-1 with respect to receptor binding,
activation of MAPK, and induction of immediate early genes but are
unable to complete the mitogenesis.
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Full-length rhFGF-1 Fails to Translocate to the Nuclei of NR
Cells--
It has been shown that the transport of exogenous FGF-1 to
the nucleus is essential for DNA synthesis and cell growth (33-36). Since the receptor-mediated signaling of full-length FGF-1 in NR cells
appear to be intact, we decided to look at the nuclear localization of
FGF-1 in the NR cells. Iodinated full-length and rhFGF-1 were added
to serum-starved cells, and the subcellular distribution of
125I-FGF-1 was examined using SDS-PAGE. At 4 °C, nearly
equal amounts of full-length and
rhFGF-1 were detected in the
cytosolic fractions of NIH/3T3 and NR cells. As expected, the majority
of the rhFGF-1 was found in cytosolic fractions (Fig.
8, lanes 1, 3, and
5) with molecular mass of 17.3 kDa for full-length (Fig.
8A) and 15.8 kDa for
rhFGF-1 (Fig. 8B). When
cells were incubated with either form of 125I-rhFGF-1 for 3 or 20 h at 37 °C before lysis, significant amounts of
full-length as well as
rhFGF-1 were found in the nuclear fractions of NIH/3T3 and NR33 cells (>60%). In contrast, very little
full-length 125I-rhFGF-1 (3-14%) was detected in the
nuclear fractions of NR31 under the same conditions even though a
considerable amount of 125I-
rhFGF-1 was still detected
in the nuclear fractions of NR31 (33-60%). The cytosolic fractions of
internalized iodinated FGF-1 are degraded and appear to be equal in all
three cell types (Fig. 8, lanes 7, 9, and
11).
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[3H]Thymidine Incorporation during Post-washing
Conditions--
Fig. 8 shows that, under post-washing conditions
(37 °C for 3 h, wash; then 37 °C for 20 h), very little
of the full-length or rhFGF-1 was detected in the nuclear fractions
of NR cells (lanes 16 and 18), yet they were
readily detectable (42-76%) in NIH/3T3 cells (lane 14). We
examined [3H]thymidine incorporation under this same
condition to see if DNA synthesis correlates with the presence of FGF-1
in the nucleus. Indeed, a significant amount of
[3H]thymidine uptake was observed in NIH/3T3 but not in
either NR31 or NR33 cells (Fig. 10).
These findings agree well with our observations of subcellular
distribution of 125I-rhFGF-1 in nuclear fractions (Fig. 8).
Thus, stimulation of DNA synthesis requires the continuous presence of
FGF-1 in the nucleus. Collectively, the differences of subcellular
localization of full-length and
rhFGF-1 in NR cells suggest that
there may be mutation(s) in the mechanism of nuclear translocation of
full-length rhFGF-1 in NR31 cells, whereas the mutations in NR33 cells
may be further downstream in the nuclear import. Therefore, these two
NR cell lines are apparently defective at different steps of nuclearly
targeted signaling.
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DISCUSSION |
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In the present study, we have characterized the activation of
receptor-mediated signaling pathway and mitogenesis mediated by
full-length and rhFGF-1 in NIH/3T3 and in nonresponsive NR cells. We
have also examined the nuclear translocation of exogenous rhFGF-1 in NR
cells and compared with that in parental NIH/3T3 cells. Our results
demonstrate that the "classical" receptor-mediated signaling
pathway is not defective in NR cells and that the activation of MAPK is
not sufficient for the full-length rhFGF-1-mediated mitogenic signaling
cascade in NR cells. Furthermore, our data suggest that the
non-responsiveness of NR cells to mitogenic stimulation of the
full-length rhFGF-1 may be due to defective steps in the nuclear
translocation pathway.
The comparison of mitogenic activities of various growth factors
demonstrated that NR31 and NR33, unlike the parental NIH/3T3 cells,
could not be activated to initiate DNA synthesis in response to
full-length FGF-1 (Figs. 1 and 2). These results suggest that the NR
cells may have lost a signaling component(s) responsive to full-length
FGF-1, and the first 14 aa of full-length FGF-1 may have an inhibitory
role in eliciting mitogenic signaling. These observations prompted us
to evaluate the signaling cascade mediated by the full-length and
rhFGF-1. We initially speculated that overexpression of FGF-1 in
NR cells may down-modulate the expression of surface receptors in NR
cells. Northern blot analysis showed that all three cell lines
expressed comparable levels of FGFR1 transcripts (data not shown).
Scatchard analysis revealed that the binding properties of NR cells to
full-length rhFGF-1 were indistinguishable from those of NIH/3T3 cells.
Furthermore, NIH/3T3 as well as NR cells expressed similar number of
receptors per cell (Fig. 3). Thus, cell-surface receptors for FGF-1 in
NR cells are not down-modulated.
One of the FGF-1-stimulated mitogenic signaling pathways includes FGFR,
Ras, Raf-1, MEK, and MAPK, leading to transcriptional activation of
immediate early genes and DNA synthesis. It has been demonstrated that
the activation of MAPK cascade was essential for nerve growth factor-
and FGF-induced differentiation of PC12 and for proliferation of
NIH/3T3 (16). Ras-dependent activation of Raf and MAPK was
sufficient and necessary for transduction of FGFR mitogenic signal in
BaF3 cells (11). FGF also activated MAPK signaling cascade in cardiac
myocytes (44). On the other hand, activation of Ras/Raf/MAPK pathway
mediated by PDGF receptor was not sufficient for PC12
differentiation (18). Our data demonstrated that NR31 and NR33 achieved
full activation of MAPK catalytic activity in response to either form
of rhFGF-1 (Figs. 4-6). Thus, the biochemical characterization of
essential steps of FGF-1 signaling pathway revealed that the NR and
NIH/3T3 cells respond to FGF-1 in a similar manner.
The constitutive activation of MAPK in NR cells (Figs. 4-6) may contribute to the transforming properties of these cells. The constitutive activation of MAPK has been found in cell lines expressing Ras or Raf oncogenes (45, 46). Overexpression of constitutively activated MEK resulted in the transformation of NIH/3T3 cells (16). Thus, it appears that activation of MAPK cascade is a critical step for the FGF-1-mediated induction of various cellular responses. Our data demonstrate that the activation of MAPK cascade is required but not sufficient for full-length rhFGF-1 to initiate DNA synthesis in NR31 and NR33 cells.
In addition to the activation of MAPK pathway, we compared the pattern
of tyrosine-phosphorylated proteins in the cytosols of NIH/3T3 and NR
cells. In response to either full-length or rhFGF-1, the pattern of
tyrosine-phosphorylated proteins among the three cell lines was
similar. Thus, FGF-1 induced tyrosine phosphorylation of FGFR1, a
90-kDa protein and MAPK (data not shown). These results suggest that
the FGFR-mediated intracellular pathways involving the activation of
tyrosine phosphorylation could not explain the lack of mitogenic
response of NR cells to full-length rhFGF-1.
A nuclear event activated by growth factors is the induction of
immediate early gene transcription. Transcription of c-fos is enhanced upon stimulation with FGF-1, but its activation by FGF-1 is
not sufficient for DNA synthesis (33). We showed that the increased
expression of c-myc and c-fos follows the
stimulation of cells with either form of FGF-1 (Fig. 7, A
and B). However, the mitogenic signal elicited by the
full-length rhFGF-1 does not lead to DNA synthesis in NR cells. It
has been demonstrated that the nuclear localization of externally
supplied FGF-1 is essential for mitogenic response (33-35). Diphtheria
toxin fused with FGF-1 can translocate through the toxin receptor and
induce DNA synthesis without measurable increase in tyrosine
phosphorylation (35). Synthetic peptides containing NLS of FGF-1 were
able to stimulate DNA synthesis in an FGFR-independent manner (34). From our observations of subcellular distribution of exogenous full-length rhFGF-1 and rhFGF-1 in nuclear and cytosolic fractions of three cell lines, it appears that the full-length
125I-rhFGF-1 fails to translocate to the nucleus of NR31.
Although the full-length FGF-1 was found in the nuclear fraction of
NR33, it has a much shorter half-life in NR33 than in NIH/3T3,
correlating with the inability of NR33 cells to synthesize DNA in
response to full-length FGF-1. These findings agree with those reported by others (33-35).
The shorter half-life of the mitogens in the nuclei of NR cells (Fig.
8, A and B, lanes 16 and
18) may also explain the reduced level of mitogenic response
to rhFGF-1 and possibly to bovine FGF-1 (Figs. 1 and 2). Moreover,
both NR cells under post-washing condition did not respond not only to
full-length rhFGF-1 but also to
rhFGF-1 in stimulating DNA
synthesis, whereas NIH/3T3 cells responded to both forms of rhFGF-1
under the same post-washing condition (Fig. 10). These findings are
consistent with the assumption that the continuous presence of FGF-1 in
the nucleus (Fig. 8, A and B, lanes 14, 16, and 18) is essential for the mitogenic response.
Together, our data suggest that, in addition to the activation of MAPK
pathway, translocation of FGF-1 to the nucleus is required for DNA
synthesis and mitogenesis in NIH/3T3 and in NR cells.
A number of recent reports suggest that the nuclear targeting of most nuclear proteins is initiated by binding of cytosolic receptor (termed importin) to NLS. Importin mediates docking of the NLS-importin complex at the nuclear pore complex (47, 48), whereas GTPase Ran/TC4 (49, 50) and NTF2 (51, 52) mediate the translocation of NLS-importin complex through the nuclear pore complex. It is possible that NR31 and NR33 cells may have defects in separate steps of the two-step process of receptor-mediated nuclear protein import pathway. In NR31, importin protein may fail to bind to the NLS of FGF-1 and thus cannot transport it to the nucleus. The binding of cytosolic receptor to NLS may have been inhibited by yet another cytosolic factor(s). In NR33, the defect could be in the second step of nuclear import. Thus, the NLS-receptor complex may dock at the nuclear pore complex but fail to enter the nucleus through the nuclear pore. Alternatively, full-length rhFGF-1 imported into the nucleus of NR33 cells may have degraded rapidly or being transported back to the cytoplasm.
In summary, our data demonstrate that the activation of MAPK is insufficient for FGF-1-mediated mitogenesis in NR cells. Our data further suggest that (i) dual signal transduction pathways, including both Ras-dependent signaling pathway and nuclear localization of FGF-1, may be required for the necessary integration of specific and distinct signals to induce mitogenesis, and (ii) the first 14 aa of FGF-1 may play an interfering role in its nuclear translocation. Availability of NR31 and NR33 cell lines should facilitate the characterization of the nature of nuclear signaling elicited by FGF-1 as well as the nuclear translocation mechanism in general.
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ACKNOWLEDGEMENTS |
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We thank Drs. T. Roberts and B. Druker for providing anti-phosphotyrosine antibody 4G10; M. J. Botelho and S. Ihsanullah for technical assistance; and P. A. Swanson for preparation of the manuscript. We also thank members of the Chiu laboratory for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01CA45611 and R01DK47506 and by American Cancer Society institutional grants.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.
§ Present address: Dept. of Molecular Genetics, The Ohio State University, Columbus, OH 43210.
To whom correspondence should be addressed: Dept. of Internal
Medicine, The Ohio State University, 480 W. Ninth Ave., Columbus, OH
43210-1245. Tel.: 614 -293-4803; Fax: 614-293-5631; E-mail: chiu.1{at}osu.edu.
1
The abbreviations used are: FGF-1, fibroblast
growth factor 1; BSA, bovine serum albumin; ECL, enhanced
chemiluminescence; ERK, extracellular-signal regulated kinase; NhFGF-1,
full-length human FGF-1 isolated from NR31 cells; rhFGF-1, full-length
recombinant human FGF-1; rhFGF-1, truncated rhFGF-1; FGFR1, FGF
receptor 1; MAPK, mitogen-activated protein kinase; MBP, myelin basic
protein; NR cells, non-responsive cells; PBS, phosphate-buffered
saline; PDGF, platelet-derived growth factor; TBS, Tris-buffered
saline; aa, amino acid(s); PAGE, polyacrylamide gel electrophoresis;
NLS, nuclear localization sequence; MEK, MAPK kinase.
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