Modulation of Mitogenic Activity of Fibroblast Growth Factors by Inorganic Polyphosphate*
Toshikazu Shiba
,
Daisuke Nishimura ¶,
Yumi Kawazoe ¶,
Yuichiro Onodera ¶,
Kaori Tsutsumi ¶,
Rie Nakamura ¶ and
Minako Ohshiro ¶
From the
Frontier Research Division, Fujirebio
Inc., 51, Komiya, Hachioji, Tokyo 192-0031, Japan and
¶Division of Molecular Chemistry, Graduate School
of Engineering, Hokkaido University, Sapporo 060-8628, Japan
Received for publication, April 3, 2003
, and in revised form, May 7, 2003.
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ABSTRACT
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The proliferation of normal human fibroblast cells was enhanced by the
addition of inorganic polyphosphate (poly(P)) into culture media. The
mitogenic activities of acidic fibroblast growth factor (FGF-1) and basic
fibroblast growth factor (FGF-2) were also enhanced by poly(P). A physical
interaction between poly(P) and FGF-2 was observed, and FGF-2 was both
physically and functionally stabilized by poly(P). Furthermore, poly(P)
facilitated the FGF-2 binding to its cell surface receptors. Because poly(P)
is widely distributed in mammalian tissues, it may be a spontaneous modulator
of FGFs.
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INTRODUCTION
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Inorganic polyphosphates
(poly(P))1 are linear
polymers of many tens or hundreds of orthophosphate residues linked by high
energy phosphoanhydride bonds that have been found in a wide range of
organisms including bacteria, fungi, algae, mosses, insects, and protozoa and
in the tissues of higher plants and animals
(14).
The biological functions of poly(P) have been investigated mostly in
microorganisms, and the following functions have been proposed: (i) storage
substance of energy or orthophosphate; (ii) chelator of metal cations; (iii)
donor for sugar and adenylate kinase; (iv) buffer against alkaline stress; (v)
structural element in competence for DNA entry and transformation; and (vi) a
regulatory factor of gene expression
(14).
Although the presence of poly(P) has been demonstrated in the rat brain, rat
liver, human peripheral blood mononuclear cells, human erythrocytes, human
gingival fibroblasts, human osteoblasts, and human plasma and intracellularly
in the nucleus, the mitochondria, lysosomes and plasma membrane
(5), little is known regarding
the functions of poly(P) and the effects of poly(P) on mammalian cells.
Recently, the involvement of poly(P) in apoptosis and in modulation of the
mineralization process in bone tissue
(5,
6) has been suggested.
Because there has been no report concerning the direct effect of poly(P) on
mammalian cells and because poly(P) is widely distributed in mammalian tissues
and plasma (5), we speculated
that poly(P) has some physiological effect on cells. Based on this idea, we
first studied in this report the effect of poly(P) on mammalian cell growth or
proliferation in vitro and revealed the novel poly(P) functions
concerning the modulation of mitogenic activity of fibroblast growth factors
(FGF)
(8).2
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EXPERIMENTAL PROCEDURES
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MaterialsNormal dermal fibroblasts (NHDF) isolated from
adult human were purchased from BioWhittaker, Inc. Normal human gingival
fibroblasts (HGF) isolated from adult human were provided by Dr. Nishimura
(Osaka Dental University). Balb/c 3T3 cells were from Riken Cell Bank
(Tsukuba, Japan). Poly(P) type 65 (sodium salts with average chain length of
65 phosphate residues) was purchased from Sigma. Concentrations of poly(P) are
given in terms of phosphate residues. As the control of poly(P),
NaPO4 buffer (orthophosphate) was used. The pH of the
NaPO4 buffer was adjusted to 7.0 by mixing the same concentrations
of Na2HPO4 and NaH2PO4 solution.
MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
cell proliferation assay kit was from Promega. Human recombinant acidic
fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2)
were from Toyobo (Osaka, Japan). Anti FGF-2 antibody was from Santa Cruz
Biotechnology, Inc.
Assay for Cell ProliferationCells were seeded to
96-multiwell plates at 5 x 103 cells/well (100 µl/well)
and cultured in Eagle's minimal essential medium (for NHDF) or Dulbecco's
modified Eagle's minimal essential medium (D-MEM) (for Balb/c 3T3 and HGF)
containing 10% FBS for 24 h. After cells had adhered, the medium was replaced
with Eagle's minimal essential medium or D-MEM without FBS and cells were
further incubated for 48 h. The medium was replaced again with an appropriate
medium described under the following sections and figure legends. After
incubation at 37 °C, cell number was directly counted using
hematocytometer after trypsinization or was evaluated by MTS assay
(8). For MTS assay, the medium
was replaced with 100 µl of Eagle's minimal essential medium (without
Phenol Red) and 25 µl of mixture of MTS (Promega) and phenazine
methosulfate solution (2 µg/ml MTS, 0.92 µg/ml phenazine methosulfate)
was added to each well. After incubation for 75 min at 37 °C, the
absorbance at 490 nm of each well was measured. The cell number was quantified
by means of the bioreduction activity of viable cells
Preparation of Short Chain [32P]poly(P)Long
chain [32P]poly(P) was synthesized by using purified
Escherichia coli polyphosphate kinase and purified as described
previously (9). To prepare
short chain [32P]poly(P) (average chain length of around 65
phosphate residues), the long chain [32P]poly(P) was hydrolyzed by
20 mM HCl for 2 min at 95 °C and the hydrolyzed poly(P) was
separated by 15% polyacrylamide gel electrophoresis together with poly(P) type
65 as a maker. The conditions of gel electrophoresis were the same as those
described in the legend of Fig.
3. The gel was stained by 0.05% toluidine blue containing 5%
glycerol. The portion of the gel corresponding to the position of poly(P) type
65 was cut, and short chain [32P]poly(P) was isolated from the gel.
Isolated gel was soaked in H2O and shaken at room temperature for 2
h. After brief centrifugation, the supernatant was collected and short chain
[32P]poly(P) was recovered from the gel.

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FIG. 3. Physical interaction between poly(P) and FGF-2. Various
concentrations of FGF-2 were mixed with [32P]poly(P) (16.3 pmol in
D-MEM, 10-ml reaction mixture). After the mixtures were incubated at 37 °C
for 10 min, 2 µl of gel loading solution (40% glycerol, 0.0025% bromphenol
blue) was added and samples were applied onto 15% polyacrylamide gel
(acrylamide:bis-acrylamide = 10:1). After electrophoresis was performed with
Tris borate EDTA buffer (23)
at 8 V/cm2, the gel was dried and [32P]poly(P) and
[32P]poly(P)-FGF-2 complexes were visualized and the intensity of
radioactivity was quantified by using a BAS2000 radioimage analyzer
(FUJIX).
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Assay for Binding between FGF-2 and High Affinity Surface
ReceptorsThe assay procedure was basically followed as described
previously (10). Confluent
Balb/c 3T3 cells were plated onto 60-mm dishes at 35,000 cells/cm2
in D-MEM with 10% FBS at 37 °C. After 24 h, the medium was changed to
D-MEM with 2% FBS with 50 mM sodium chlorate to remove heparin
sulfate proteoglycan (HSPG). Cells were further incubated for 72 h at 37
°C and were then washed once with a binding buffer (D-MEM and 25
mM HEPES) at 4 °C. A fresh binding buffer was added (4
ml/well), and cells were incubated at 4 °C for 10 min. FGF-2 was added at
the indicated concentrations, and cells were incubated at 4 °C for 2.5 h.
At the end of the binding period, cells were placed on ice and washed three
times with ice-cold binding buffer. Cell surface receptor-bound FGF-2 was
extracted with two washes (one 5-min wash and one rapid wash, 0.5 ml
each/dish) at room temperature using 2 M NaCl in 20 mM
sodium acetate, pH 4.0. Washed solutions (2 x 0.5 µl) containing the
extracted cell surface receptor-bound FGF-2 were collected, and 5 ml of 8%
bovine serum albumin was added to each sample as a carrier protein.
Trichloroacetic acid was added to the final concentration of 5%, and
precipitated proteins were pelleted by centrifugation. After removal of the
supernatant, the protein pellet was neutralized by 1 N KOH and
dissolved by SDS-PAGE sample buffer. FGF-2 was separated by 15% SDS-PAGE and
visualized by Western blotting.
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RESULTS
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Stimulation of Cell Growth by Adding Poly(P) into Culture
MediaIntriguingly, poly(P) stimulated the proliferation of both
NHDF and HGF cells in the absence of serum
(Fig. 1, A and
B). NHDF and HGF were grown continuously after
25110 h of incubation with poly(P), whereas there was almost no growth
of cells incubated with Na-PO4 buffer or with media only. To rule
out the possibility that poly(P) enhances the reduction activity of
mitochondria resulting in the higher value of absorbance as measured by the
MTS method (8) regardless of
the cell number, cell growth was monitored by counting the number of cells.
The number of viable cells after 3 days of incubation is shown in
Fig. 1C. In agreement
with the result of measurement by the MTS method, the number of cells
increased by
2.8-fold after 72 h of incubation with poly(P). However, no
proliferation of cells incubated with medium containing NaPO4
buffer was observed.

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FIG. 1. Enhancement of cell proliferation by poly(P). Cell culture and the
basic proliferation assay were performed as described under
"Experimental Procedures." After serum starvation, NHDF
(A and C) and HGF (B) cells were incubated with
serum-free media, media with 1.34 mM poly(P), or media with 1.34
mM NaPO4 buffer. The viable cell number was quantified
by the MTS method (8) in
A and B and was directly counted by the use of a
hematocytometer in C. Error bars represent the means ± S.D. of
six samples.
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Enhancement of Mitogenic Activity of FGFs by Poly(P)One
possible mechanism of growth stimulation by poly(P) is that poly(P) enhances
the activity of growth factors that were released into the culture medium from
the cultured cells themselves. Therefore, in this study, we focused on the
possibility of modulation of FGF activity by poly(P). Since FGFs have been
implicated as autocrine growth factors
(11), it is possible that
poly(P) somehow modulates the biological activity of FGFs.
If poly(P) does in fact modulate the mitogenic activity of FGF, cell
proliferation would be enhanced by co-treatment with poly(P) and FGF rather
than by single treatment with poly(P) or FGF. To examine whether the
co-treatment of poly(P) and FGF is effective in stimulating mitogenic activity
of FGFs, Balb/c 3T3, NHDF, and HGF whose growth could be dependent on FGF were
treated with FGF-1 or FGF-2 in combination with poly(P)
(12). As shown in
Fig. 2, cell growth was
slightly stimulated by both FGF-1 and FGF-2, and moreover, the levels of
growth stimulation by both FGFs became greater in the presence of poly(P).
Cell growth in the medium containing FGF-1 and poly(P) was 1.61.9 times
higher than that in the medium containing only FGF-1. Similarly, cell growth
in the medium containing FGF-2 and poly(P) was 1.31.7 times higher than
that in the medium containing only FGF-2. Because the levels of growth
stimulation by co-treatment of poly(P) and FGFs are greater than the sum of
stimulation levels of single treatment with poly(P) and FGFs in Balb/c 3T3 and
NHDF, poly(P) seems to augment the mitogenic activities of FGFs. However, in
HGF, the level of growth stimulation by co-treatment with FGF-2 and poly(P) is
not greater than the sum of stimulation levels of single treatment with
poly(P) and FGFs. From these results, it is difficult to rule out the
possibility that poly(P) and FGFs independently function on cell growth
stimulation. To show the direct evidence of FGF modulation by poly(P), the
physical and functional interactions between poly(P) and FGFs have been
examined.

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FIG. 2. Augmentation of mitogenic activity of FGF by poly(P). Cell culture
and MTS assay (8) were
performed as described under "Experimental Procedures." After
serum starvation, cells were incubated with serum-free D-MEM (None)
or serum-free D-MEM containing Poly(P), FGF-1, Poly(P) together with FGF-1,
FGF-2, Poly(P) together with FGF-2, 10% FBS, heparin, or poly(P) together with
heparin. Concentrations of Poly(P), both FGFs, and heparin are 1.34
mM, 10 ng/ml, and 50 µg/ml, respectively. After 30 h of
incubation at 37 °C, viable cell number was quantified by the MTS assay
(8). The level of proliferation
was calculated in terms of a relative value when the level of untreated cells
(None) was defined as 1. Error bars represent the means
± S.D of six samples.
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Physical Interaction Between FGF-2 and Poly(P)Physical
interaction between FGF-2 and poly(P) was examined by a gel shift assay as
described in the legend of Fig.
3. When FGF-2 was incubated with short chain
[32P]poly(P), FGF-2 bound to the poly(P) depending on its
concentration, poly(P) formed a complex with FGF-2, and the complex almost
remained at the origin of the gel (Fig.
3). This result shows that poly(P) does bind to FGF-2.
Furthermore, it may possible to roughly estimate the stoichiometry between
poly(P) and FGF-2. Since 8.93 pmol of FGF-2 completely trapped all of
[32P]poly(P) (16.3 pmol, estimated in terms of phosphate residue,
corresponds to 0.251 pmol of poly(P) calculated from the number of average
chain length of [32P]poly(P) as 65)
(Fig. 3, lane 3), one
molecule of poly(P) can bind to <35.6 molecules of FGF-2. It still would be
difficult to speculate the structures of poly(P)-FGF-2 complex only from this
result because some FGF-2 molecules may not contribute to the formation of the
complex under our experimental condition. However, it may also be possible
that poly(P) somehow facilitates oligomer formation of FGF-2 as in the case of
heparin-like glycosaminoglycans
(13) and a FGF-2 oligomer
requires only a few phosphate molecules of poly(P) for its binding. The
modulation of mitogenic activity of FGF-2 may result from the oligomer
formation of FGF-2 initiated by poly(P).
Stabilization of FGF-2 by Poly(P)To further examine the
effect of poly(P) on FGF-2, the stability of FGF-2 with or without poly(P) was
evaluated. Fig. 4A
shows the degradation of FGF-2 in culture medium in the presence or absence of
poly(P). Intact FGF-2 clearly remained after 24 h of incubation with poly(P)
(Fig. 4A, lane
17), whereas intact FGF-2 was not observed after 24 h of incubation
without poly(P) (Fig.
4A, lane 9) or with NaPO4 buffer
(Fig. 4A, lane
25). The half-life of FGF-2, which was calculated from the intensity of
the bands of Fig. 4A,
was 13.7 h when FGF-2 was incubated with poly(P), whereas it was only 4.7 h
when FGF-2 was incubated with NaPO4 buffer
(Fig. 4B).

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FIG. 4. Physical and functional stabilization of FGF-2 by poly(P).
A, physical stabilization of FGF-2 by poly(P). FGF-2 (200 ng/ml) was
incubated with D-MEM, D-MEM with 1.34 mM poly(P), or D-MEM with
1.34 mM NaPO4 buffer at 37 °C. Samples were taken
every 2 h after 12 h of incubation as indicated and mixed with SDS-PAGE gel
loading buffer (23). Samples
were separated by 14% SDS-PAGE, and FGF-2 was detected by Western blotting
using an anti-FGF-2 antibody (Santa Cruz Biotechnology, Inc.) and goat
anti-rabbit IgG alkaline phosphatase conjugate (Bio-Rad) as a second antibody.
B, elongation of FGF-2 half-life by poly(P). The intensity of the
bands that were visualized in panel A was quantified by using image
analyzing software (NIH image), and plotted. C, functional
stabilization of FGF-2 by poly(P). Cells were cultured as described under
"Experimental Procedures." After serum starvation, medium was
replaced with six kinds of new serum-free D-MEM that had been prepared as
follows: a medium containing 10 ng/ml FGF-2 in combination with 1.34
mM poly(P) (medium 1); a medium containing 10 ng/ml FGF-2
in combination with poly(P) that had been preincubated for 24 h at 37 °C
(medium 2); a medium containing 10 ng/ml FGF-2 (medium 3); a
medium containing 10 ng/ml FGF-2 that had been preincubated for 24 h at 37
°C (medium 4); a medium with no preincubation and no additives
(medium 5); and a medium that had been preincubated for 24 h at 37
°C (medium 6). After the indicated times, cell proliferation was
assayed by the MTS method (8).
The meanings of each symbol are summarized in the same panel.
Pre-in, 24-h preincubation with FGF-2. Error bars represent
the means ± S.D. of six samples.
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Since FGF-2 was physically stabilized by poly(P), stability of the
biological activity of FGF-2 was also examined using Balb/c 3T3 cells. Since
FGF-2 almost loses its mitogenic activity within 24 h of incubation at 37
°C, the residual activity after preincubation (24 h at 37 °C) of FGF-2
was examined in the presence and absence of poly(P). As shown in
Fig. 4C, cells
cultured in a medium containing FGF-2 that has been preincubated with poly(P)
(medium 2) maintained the same population levels as that of cells cultured in
a medium containing FGF-2 without preincubation (medium 3). On the other hand,
a medium containing FGF-2 that had been preincubated without poly(P) (medium
4) showed slight proliferation activity but the activity decreased to the same
level as that of media without FGF-2 (media 5 and 6) after 83 h of incubation.
This means that FGF-2 that had been preincubated at 37 °C was stabilized
by poly(P) and maintained its proliferation activity at the same level as that
of FGF-2 that had not been preincubated. In addition, the highest
proliferation activity was observed in the medium containing FGF-2 and poly(P)
without preincubation. This finding is consistent with the result shown in
Fig. 2, indicating the
enhancement of mitogenic activity of FGF-2 by poly(P). These results indicate
that poly(P) stabilizes the biological activity of FGF-2.
Facilitation of FGF-2 Binding to Its Receptors by Poly(P)To
further examine the effect of poly(P) as an FGF-2 modulator, we analyzed
whether poly(P) not only stabilizes FGF-2 but facilitates FGF-2 binding to FGF
receptors. The biological activity of FGF-2 is mediated by interaction with
high affinity cell surface receptors
(1416).
In addition to binding to receptors, FGF-2 binds to HSPG on the cell surface.
Because many studies have indicated that the binding to HSPG facilitates FGF-2
receptor binding and activation
(1720),
we removed the cell surface HSPG by sodium chlorate treatment in order to
observe the direct effect of poly(P) on FGF-2 and its receptor binding
(10). Sodium chlorate-treated
cells were incubated with FGF-2, and the receptor-bound FGF-2 was collected
and analyzed by Western blotting (Fig.
5A) and intensities of the bands were quantified and
plotted in Fig. 5B.
The amount of FGF-2 that bound to FGF receptors in the presence of poly(P) was
more than twice that in the absence of poly(P). This clearly indicates that
poly(P) facilitates the binding between FGF-2 and FGF receptors. Poly(P),
FGF-2, and FGF receptors may form a trimolecular complex on the cell surface.
There may also be direct interactions between poly(P) and FGF receptor that
facilitate FGF-2 binding and receptor dimerization as in the case of heparin
(1720).

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FIG. 5. Effect of poly(P) on the binding of FGF-2 to high affinity surface
receptors. A, visualization of FGF-2 bound to the receptor by
Western blotting. Various concentrations of FGF-2 (10, 20, and 40 ng/ml) was
exposed to cells with 1.34 mM poly(P) or NaPO4 buffer
(control). B, quantification of receptor-bound FGF-2. The
intensities of FGF-2 bands visualized in panel A were quantified by
using image-analyzing software (NIH image). C, stability of FGF-2
during the binding assay. FGF-2 (40 ng/ml) was incubated with poly(P) (1.34
mM) or NaPO4 buffer (1.34 mM) at 4 °C in
the binding buffer for 2.5 or 5.5 h or in the elution buffer for 30 min. After
incubation, FGF-2 was precipitated with trichloroacetic acid and the intact
protein was visualized by Western blotting following the same procedure as
described under "Experimental Procedures."
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To rule out the possibility that stabilization of FGF-2 by poly(P) occur
during incubation with binding buffer or elution buffer, stability of FGF-2
during binding assay was evaluated. FGF-2 was incubated with poly(P) or
NaPO4 buffer in the binding buffer or in the elution buffer at 4
°C. The amount of FGF-2 remaining in the buffer was shown in
Fig. 5C. Since almost
the same amount of FGF-2 was detected in buffers with poly(P) or
NaPO4 up to 5.5-h exposures of the binding buffer
(Fig. 5C, lanes
14) and up to 30-min exposures of the elution buffer
(Fig. 5C, lanes
5 and 6), there is no stabilization effect of poly(P) on FGF-2
during this binding assay.
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DISCUSSION
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With regard to the mechanism of growth stimulation by poly(P), a similar
effect has also been reported by adding heparin to culture media. Heparin and
heparin-like glycosaminoglycans are well known potent modulators of FGF-1 and
FGF-2, and they potentiate the mitogenic activity of both FGFs
(12,
1720).
Heparin sulfate stabilizes FGFs and binds to a site on the receptor and at
least one site on the growth factor. Several models propose an important role
for heparin sulfate not only in facilitating FGF-2 binding to its receptor
tyrosine kinase but also in promoting signaling via the formation of receptor
dimers. Such dimers are capable of trans-phosphorylation of the cytoplasmic
domain of the receptor, leading to the generation of phosphotyrosine, that is
an important initiator of the intracellular signaling pathway
(1720).
Poly(P) may also facilitate FGF-2 binding to its receptors and promote
signaling through the same binding sites of heparin sulfate, FGF-2, and its
receptors. However, poly(P) and heparin have completely different chemical
structures, besides both molecules are negatively charged cellular polymers.
It is probable that the mechanism for modulation of FGF activity by poly(P) is
different from that of heparin. Based on our results, the level of growth
stimulation by poly(P) is higher than that by heparin whose concentration is
enough for maximum growth stimulation (Fig.
2) (21). This also
suggests that the binding sites between poly(P) and FGFs could be different.
Further analyses are needed to elucidate the detailed mechanism of interaction
between poly(P) and FGF-2.
One hypothesis is that poly(P) has biological functions for controlling the
activity of FGF in vivo. Since poly(P) is widely distributed in
mammalian tissues (5), it is
possible that degradation of FGF in tissues that have been injured is
prevented if there was a mechanism for local regulation of poly(P)
concentration. Furthermore, it is also possible that poly(P) interacts with
other proteins or polypeptides, including growth factors, cytokines, hormones,
and other physiologically active factors. Recently, Kuroda et al.
(22) showed that poly(P)
modulate the activity of Lon protease in E. coli depending on its
nutrient condition. This also supports the possibility that poly(P) is a
protein modulator in a wide variety of organisms to regulate the functions of
proteins after translation. Although there have been few reports describing
the functions of poly(P) in eucaryotes, we believe that our present findings
provide a key to elucidate the importance of poly(P) in higher organisms.
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FOOTNOTES
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* This work was supported by a grant-in-aid for innovations through
Business-Academic-Public Sector Cooperation and a grant-in-aid for Scientific
Research on Priority Areas (B) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan. The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Tel./Fax: 81-426-45-4755; E-mail:
tz-shiba{at}fujirebio.co.jp.
1 The abbreviations used are: poly(P), inorganic polyphosphates; FGF,
fibroblast growth factor; FGF-1, acidic FGF; FGF-2, basic FGF; NHDF, normal
dermal fibroblasts; HGF, human gingival fibroblasts; D-MEM, Dulbecco's
modified Eagle's minimal essential medium; FBS, fetal bovine serum; HSPG,
heparin sulfate proteoglycan. 
2 Shiba, T. (August 28, 1998) Japan Patent Application 10-242416,
pending. 
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REFERENCES
|
---|
- Kulaev, I. S. (1979) The Biochemistry of
Inorganic Polyphosphates, John Wiley & Sons, Inc., New
York
- Wood, H. G., and Clark, J. E. (1988) Annu.
Rev. Biochem. 57,
235260[CrossRef][Medline]
[Order article via Infotrieve]
- Kornberg, A. (1995) J.
Bacteriol. 177,
491496[Abstract]
- Kulaev, I. S., Vogabov, V. M., and Kulakovskaya, T.
(1999) J. Biosci. Bioeng.
88,
111129[CrossRef]
- Schröder, H. C. (1999) Prog. Mol.
Subcell. Biol. 23,
4581[Medline]
[Order article via Infotrieve]
- Leyhausen, G., Lorenz, B., Zhu, H., Geurtsen, W., Bohnensack, R.,
Muller, W. E. G., and Schröder, H. C. (1998) J.
Bone Miner. Res. 13,
803812[Medline]
[Order article via Infotrieve]
- Shiba, T. (December 25, 2001) U. S. Patent
6,333,193B1
- Marshall, N. J., Goodwin, C. J., and Holt, S. J.
(1995) Growth Regul.
5,
6984[Medline]
[Order article via Infotrieve]
- Ahn, K., and Kornberg, A. (1990) J. Biol.
Chem. 265,
1173411739[Abstract/Free Full Text]
- Fannon, M., and Nugent, M. A. (1996) J.
Biol. Chem. 271,
1794917956[Abstract/Free Full Text]
- Souttou, B., Hamelin, R., and Crepin, M. (1994)
Cell Growth Differ. 5,
615623[Abstract]
- Naski, M. C., and Ornitz, D. M. (1998)
Front. Biosci. 3,
D781D794[Medline]
[Order article via Infotrieve]
- Venkataraman, G., Sheiver, Z., Davis, J. C., Sasisekharan, R.
(1999) Proc. Natl. Acad. Sci. U. S. A.
96,
18921897[Abstract/Free Full Text]
- Johnson, D. E., and William, L. T. (1993)
Adv. Cancer Res. 60,
141[Medline]
[Order article via Infotrieve]
- Givol, D., and Yayon, A. (1992) FASEB
J. 6,
33623369[Abstract/Free Full Text]
- Jaye, M., Schlessinger, J., and Dionne, C. A. (1992)
Biochim. Biothys. Acta.
1135,
185199
- Kiefer, M. C., Baird, A., Nguyen, T., George-Nascimento, C., Mason,
O. B., Boley, L. J., Valenzuela, P., and Barr, P. J. (1991)
Growth Factors 5,
115127[Medline]
[Order article via Infotrieve]
- Kan, M., Wang, F., Xu, J, Crabb, J. W., Hou, J., and McKeehan, W. L
(1993) Science
259,
19181921[Medline]
[Order article via Infotrieve]
- Roghani, M., Mansukhani, A., Dell'Era, P., Bellosta, C., Rifkin, D.
B., and Moscatelli, D. (1994) J. Biol.
Chem. 269,
39763984[Abstract/Free Full Text]
- Pantoliano, N. W., Horlick, R. A., Springer, B. A., VanDyk, D. E.,
Tobery, T., Wetmore, D. R., Lear, J. D., Nahapetian, A. T., Bredley, J. D.,
and Sisk, W. P. (1994) Biochemistry
33,
1022910248[Medline]
[Order article via Infotrieve]
- Gospodarowicz, D., and Cheng, J. (1986) J.
Cell. Physiol. 128,
475484[Medline]
[Order article via Infotrieve]
- Kuroda, A., Nomura, K., Ohtomo, R., Kato, J., Ikeda, T., Takiguchi,
N., Ohtake, H., and Kornberg, A. (2001)
Science 293,
705708[Abstract/Free Full Text]
- Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY