(Received for publication, December 13, 1995)
From the
Fibroblast growth factor-1 (FGF-1) is a potent mitogen for
mesoderm- and neuroectoderm-derived cell types in vitro.
However, a mutant FGF-1 with deletion in its nuclear localization
sequence (NLS, residues 21-27) is not mitogenic in
vitro. We demonstrated that synthetic peptides containing this NLS
were able to stimulate DNA synthesis in a FGF receptor-independent
manner after they were delivered into living NIH 3T3 cells by a
cell-permeable peptide import technique. The stimulation of maximal DNA
synthesis by these peptides required the presence of peptides during
the entire G phase of the cell cycle. The mitogenic effect
was specific for the NLS of FGF-1 because a peptide with double point
mutations at lysine residues was inactive in stimulating DNA synthesis.
Our results suggest that the NLS plays an important role in the
mitogenic pathway initiated by exogenous FGF-1 by its direct
involvement in the nuclear transport and signaling of internalized
FGF-1.
Mitogenic signaling for many growth factors is triggered by
their binding to the transmembrane receptor tyrosine kinases, for
example, those for epidermal growth factor and platelet-derived growth
factor. Upon ligand-receptor binding, these receptors are dimerized and
autophosphorylated. The activated receptors further phosphorylate the
receptor substrates to initiate intracellular kinase signaling cascades (1, 2, 3) . It is evident, however, that
there may be alternative signaling pathways for some growth factors
involving their nuclear transport and signaling (for review see (4) ). In this context, subsequent to receptor-ligand
internalization, growth factor ligands may translocate to the nucleus
and directly function in mitogenic processes(5, 6) .
FGF-1 ()is one of two prototype members of the fibroblast
growth factor family. It is a potent mitogen for many cell types and is
involved in embryogenesis, angiogenesis, and neurite
outgrowth(7, 8) . The mechanism by which FGF-1
transmits mitogenic signals is still not entirely clear. It has been
shown, however, that a mutant FGF-1 with deletion in its nuclear
localization sequence (NLS) Asn-Tyr-Lys-Lys-Pro-Lys-Leu (residues
21-27) failed to stimulate DNA synthesis and cell proliferation in vitro although it could still bind to the FGF receptor and
induce intracellular receptor-mediated tyrosine phosphorylation and
c-fos expression(5) . The fact that
FGF-1-(21-27) was able to direct
-galactosidase into the
nucleus, as well as the evidence of nuclear localization of
FGF-1(9, 10) , suggest that nuclear transport of FGF-1
following receptor-mediated internalization might be important for
stimulating DNA synthesis by FGF-1 in vitro.
To examine directly the functional role of the NLS in FGF-1-stimulated mitogenesis, we have delivered the peptide encompassing this sequence into living cells by using our recently developed cell-permeable peptide import method (CPPI) (see (11) ). We demonstrated in this report that cell membrane-permeable peptides containing this NLS sequence can stimulate DNA synthesis in NIH 3T3 cells in a FGF receptor-independent manner. Our results together suggest the nuclear transport of FGF-1 plays an important role in the mitogenic pathway initiated by exogenous FGF-1. This may represent an important signaling mechanism for certain growth factors.
Figure 1: Sequences of cell membrane-permeable peptides containing the NLS of FGF-1 and control peptides (single-letter amino acid code). The cell membrane-translocating sequence (11) is single underlined, the NLS of FGF-1 is double underlined, and the residues mutated in the NLS sequence are in boldface. These two regions are separated by a spacer region of A-A-A.
Figure 2:
A, demonstration of the intracellular
SA peptide in NIH 3T3 cells by fluorescence microscopy analysis.
Confluent NIH 3T3 cells were treated at 37 °C with 100 µg/ml of
SA
or diluent for 30 min. Intracellular SA
peptide deposits
were detected by indirect immunofluorescence assay using anti-SM
peptide IgG and rhodamine-labeled anti-rabbit antibody (11) and
analyzed in an Olympus fluorescence microscope using a
100 oil
immersion lens. B, [
H]thymidine
incorporation by NIH 3T3 cells stimulated by synthetic peptides (left panel) or by FGF-1 (right panel). Serum-starved
NIH 3T3 cells were incubated with various amounts of peptides or
FGF-1/heparin (5 units/ml). After 16 h,
[
H]thymidine was added, and 4 h later, the cells
were washed, solubilized, and the [
H]thymidine
incorporation was determined. Data are the mean ± S.D. of
triplicate samples and calculated as multiplicity of counts in the
tested sample over the control sample (untreated cells). The experiment
was repeated three times with similar results. C, cell cycle
distribution of NIH 3T3 cells treated with SA peptide or FGF-1.
Serum-starved cells are untreated (control) or treated with SA peptide
(100 µg/ml) or FGF-1 (15 ng/ml) for 20 h, and DNA content in each
phase of the cell cycle was determined by flow cytometric analysis
stained with propidium iodide. This is the analysis of a single
representative experiment. A statistical analysis of the results of six
experiments can be seen in Table 1.
The mitogenic effect of the SA peptide was verified by flow cytometric analysis of the DNA distribution in each phase of the cell cycle in the SA peptide-treated 3T3 cells. As shown in Table 1and Fig. 2C, the DNA content in the S-phase, which reflected the cell fractions in this phase, was significantly increased when the cells were treated for 20 h with the SA peptide at 100 µg/ml, which coincided with the fully effective concentration in the thymidine incorporation assay (Fig. 2B). A similar, but stronger, stimulation was observed in the cells treated with FGF-1 containing the same NLS ( Table 1and Fig. 2C). These results support the important role of the NLS region of FGF-1 in inducing DNA synthesis.
Figure 3:
A, the effect of NLS mutations on SA
peptide-stimulated [
H]thymidine incorporation by
NIH 3T3 cells. Serum-starved NIH 3T3 cells were treated with various
amounts of SA
or its mutant peptides. The rest of the procedure
was the same as described in the legend to Fig. 2B. Bars represent the mean ± S.D. of six samples and are
calculated as multiplicity of counts in the tested sample over the
control sample (untreated cells). The differences between SA
peptide and SA
M4 peptide and between SA
peptide and SA
M3
peptide at 75 and 100 µg/ml were significant (p < 0.01
and 0.05, respectively) by analysis of variance. The experiment was
repeated three times with similar results. B, long term
peptide exposure required for the SA peptide-stimulated
[
H]thymidine incorporation by NIH 3T3 cells.
Serum-starved cells were untreated (control) or treated with SA peptide
(100 µg/ml) or FGF-1 (15 ng/ml)/heparin (5 units/ml) for the
indicated time periods. The rest of the procedure was the same as
described in the legend to Fig. 2B. Bars represent the mean ± S.D. of triplicate samples and are
calculated as multiplicity of counts in the tested sample over the
control sample. The experiment was repeated three times with similar
results. C, the induction of tyrosine phosphorylation by SA
peptide or FGF-1 in NIH 3T3 cells. Serum-starved cells were untreated (lane C) or treated with SA peptide (100 µg/ml) or FGF-1
(15 ng/ml) for the indicated time periods. The immunoblot of the
equivalent amount of whole cell lysates was obtained with
anti-phosphotyrosine antibody. The tyrosine-phosphorylated 90-kDa
protein and 150-kDa PLC-
protein affirmed by anti-PLC
antibody were indicated.
In this report, we suggest a dissociation of FGF-1-stimulated
mitogenesis from its receptor tyrosine kinase activation in NIH 3T3
cells. We have demonstrated that the peptide containing the NLS of
FGF-1 can stimulate DNA synthesis after it is delivered into NIH 3T3
cells by using a cell-permeable peptide import method(11) . Our
finding is supported by a recent observation that a mutant FGF-1 with
deletion in its NLS is not mitogenic in vitro(5) . We
thus propose that the NLS of FGF-1 may play two functional roles in
exogenous FGF-1-stimulated mitogenesis. First, in mediating nuclear
translocation of FGF-1, internalization of FGF-1 following its receptor
binding may allow the association of the partitioned growth factor
through the NLS with the intracellular machinery that facilitates
nuclear transport of FGF-1. A number of cytosolic proteins have been
known to mediate nuclear translocation of various NLS-containing
proteins (for review see (23) ). This role of the NLS of FGF-1
may not be crucial (24) because internalized FGF-1 with a
molecular size of 16.5 kDa should enter the cell nucleus by free
diffusion. However, the NLS could be important if FGF-1 is transported
to the nucleus in the form of FGFFGF receptor complex. As
concerns the second role of the NLS of FGF-1, the functional ability of
SA peptides in stimulating DNA synthesis suggests that the NLS is
directly involved in the FGF-1-induced nuclear mitogenic signaling.
Such signaling may be initiated by the binding of the positively
charged NLS to specific molecules in the nucleus or on the nuclear
membrane. The importance of the basic residues as demonstrated by our
mutagenesis study is buttressed by our recent finding that
cell-permeable peptides containing the NLS of nuclear factor
B p50
protein or the NLS of v-Rel protein can also stimulate DNA synthesis in
NIH 3T3 cells in a manner similar to SA peptides (data not shown).
Because the basic cores of the two NLS sequences, KRQK (p50) and KRQR
(v-Rel), are similar to that of the NLS of FGF-1, KKPK (Fig. 1),
a 4-residue sequence motif, K-K(R)-X-K(R), may be functionally
important. It is expected that this proposed sequence motif can be
found in many intracellular NLS-containing proteins. However, it may
become physiologically relevant only when a significant amount of
molecules containing this motif is translocated into the nucleus, for
example by receptor-mediated FGF-1 internalization or by cell-permeable
peptides as shown in this study.
The mitogenic effect of SA peptides is not limited to NIH 3T3 cells. We found that SA peptides could also induce DNA synthesis in bovine hamster kidney-21 cells. However, the same peptides, unlike full-length FGF-1, were inactive in murine LE-II endothelial cells despite their good cell membrane permeability in this cell line (data not shown). These results thus suggest that different mechanisms may be involved in FGF-1-stimulated mitogenesis in various cell types.