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J. Biol. Chem., Vol. 275, Issue 42, 32753-32762, October 20, 2000
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From the Center for Molecular Medicine, Maine Medical Center
Research Institute, South Portland, Maine 04106
Received for publication, March 20, 2000, and in revised form, July 26, 2000
Amlexanox binds S100A13 and inhibits
the release of fibroblast growth factor 1 (FGF1). Because members of
the S100 gene family are known to be involved with the function of the
cytoskeleton, we examined the ability of amlexanox to modify the
cytoskeleton and report that amlexanox induces a dramatic reduction in
the presence of actin stress fibers and the appearance of a random, non-oriented distribution of focal adhesion sites. Correspondingly, amlexanox induces the complete and reversible non-apoptotic inhibition of cell migration and proliferation, and although amlexanox does not
induce either the down-regulation of F-actin levels or the depolymerization of actin filaments, it does induce the tyrosine phosphorylation of cortactin, a Src substrate known to regulate actin
bundling. In addition, a dominant negative form of Src is able to
partially rescue cells from the effect of amlexanox on both the actin
cytoskeleton and cell migration. In contrast, the inhibition of cell
proliferation by amlexanox correlates with the inhibition of cyclin D1
expression without interference of the receptor tyrosine
kinase/mitogen-activated protein kinase signaling pathway. Last, the
ability of amlexanox to inhibit FGF1 release is reversible and
correlates with the restoration of the actin cytoskeleton, suggesting a
role for the actin cytoskeleton in the FGF1 release pathway.
Amlexanox, an anti-allergic drug that binds S100A13, a relatively
new member of the S100 gene family, is able to inhibit the heat
shock-induced release of fibroblast growth factor
(FGF1)1 (1). FGF1 and FGF2
are the prototype members of a large family of heparin binding growth
factor genes that regulate numerous biological processes, including
mesoderm formation, neurogenesis, and angiogenesis in vivo
(2). Since the FGF prototypes are characterized by the lack of a
classical signal peptide sequence to provide access to the conventional
endoplasmic reticulum-Golgi secretion pathway, it has been suggested
that the release of both FGF1 and FGF2 may proceed through a novel
release pathway (3). Our laboratory recently demonstrated that FGF1 is
released as a reducing agent- and denaturant-sensitive complex,
containing the p40 extravesicular domain of p65 Synaptotagmin (Syt) 1 in vitro (4, 5) and that FGF1, p40 Syt1, and S100A13
are components of a heparin binding complex in vivo (1).
It is well established that several members of the S100 gene family are
associated with the cytoskeleton (6, 7) and that the actin cytoskeleton
is essential in transmembrane signaling, endocytosis, and secretion
(8). There is also increasing evidence that actin microfilaments and
the subplasmalemmal cytoskeleton are involved in several aspects of
vesicle transport (9). Indeed, in yeast, actin cytoskeleton mutants
accumulate large secretory vesicles and exhibit defects in endocytosis
that correlate with changes in actin-polarized organization (10). The
actin cytoskeleton is also required by mammalian cells for cell
proliferation, motility, and morphological changes (8).
The Src pathway also plays a central role in the modulation of the
organization of the actin cytoskeleton in response to extracellular stimuli through the phosphorylation of several actin-binding proteins including cortactin (11, 12). Indeed, the activation of the Src pathway
correlates with the induction of cell migration and the redistribution
of cortactin and F-actin, in response to FGF1 (13).
Since (i) amlexanox is an inhibitor of FGF1 and p40 Syt1 release
in vitro (1), (ii) this reagent binds S100A13, a member of
the heparin binding complex containing p40 Syt1 and FGF1 (1, 14), and
(iii) members of the S100 gene family are involved in the regulation of
cytoskeletal function (6, 7), we examined the effect of amlexanox on
cell morphology, migration, proliferation, and cytoskeletal
organization. We report that amlexanox induces a
Src-dependent phosphorylation of cortactin that may be
responsible for the reversible inhibition of cell migration and
organization of actin stress fibers and induces a Src-independent
reversible inhibition of cell proliferation that correlates with cyclin
D1 down-regulation.
Cell Culture--
Human umbilical vein endothelial cell (HUVEC),
NIH 3T3, Swiss 3T3, and L6 cells were grown as described previously
(13, 15). Newborn rat aorta smooth muscle cells were grown in a 1:1 mixture of DMEM (Cellgro) and Ham's F-12 (Cellgro) supplemented with
5% (v/v) fetal bovine serum (Hyclone) (16) and human fibroblasts (IMR
90 strain; ATCC) were grown in DMEM supplemented with 10% (v/v) fetal
bovine serum. Glioma cell lines, U-251Mg and U-563Mg, a gift of B. Westermark (University of Uppsala, Uppsala, Sweden), were grown in DMEM
supplemented with 10% (v/v) bovine calf serum (BCS; Hyclone) (17). NIH
3T3 cells were transfected with a mutated form of Xenopus
laevis Src in which lysine 294 was replaced by alanine and
tyrosine 526 was replaced by phenylalanine (18). The Src mutant, kindly
provided by R. Friesel was cloned into pcDNA 3.1/Hygro vector
(Invitrogen) using the XhoI and XbaI restriction sites. The transfection was performed using a multi-component lipid-based reagent (FuGENE 6, Roche Molecular Biochemicals). Swiss 3T3
or NIH 3T3 cells were made quiescent by incubating the confluent
monolayer in serum-free hormone-defined medium (DMI) as described (19).
Amlexanox (AA673) was a generous gift of Takeda Chemical Industries,
Osaka, Japan and was solubilized in equimolar NaOH. Latrunculin and
jasplakinolide were purchased from Biomol and Molecular Probes, respectively.
Fluorescence Microscopy and F-actin
Expression--
Immunofluorescence and F-actin staining were performed
as described previously (13) Focal adhesion sites, vinculin and
tubulin, were stained using, respectively, a monoclonal
anti-phosphotyrosine (Upstate Biotechnology), a monoclonal
anti-vinculin (Sigma), and a monoclonal anti-tubulin (Sigma) antibody.
To examine F-actin expression, cells were fixed in 4% (v/v)
formaldehyde in buffer A (5 mM Pipes, pH 7.2, containing 5 mM KCl, 138 mM NaCl, 4 mM NaHCO3, 0.4 mM KH2PO4,
2 mM MgCl2, and 2 mM EGTA) and
permeabilized with 0.5% (v/v) Triton X-100 in buffer A for 20 min. The
monolayers were rinsed in 0.1 M glycine in buffer A for 10 min and washed 5 times with buffer A. The cells were incubated in 1 µM NBD-phallacidin (Molecular Probes) in buffer A for
1 h and washed 5 times for 5 min with buffer A, and the
F-actin-bound NBD-phallacidin was extracted with methanol at 4 °C
for 90 min. Simultaneously, cells plated at the same density and
treated as described above were used to quantitate cell number using a
hematocytometer. Fluorescence of the methanol extraction
solution was measured at 465-nm excitation and 535-nm emission and
normalized against cell number (21).
Immunoprecipitation and Immunoblot Analysis--
Cells were
grown to confluence and incubated for 48 h in DMI, washed with
cold phosphate-buffered saline, scraped in cold phosphate-buffered
saline containing 1 mM sodium orthovanadate, and collected
by centrifugation (1.000 × g, 10 min). Cell pellets were lysed for 20 min in 0.5 ml of cold lysis buffer (20 mM
Tris, pH 7.5, containing 300 mM sucrose, 60 mM
KCl, 15 mM NaCl, 5% (v/v) glycerol, 2 mM EDTA,
1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride,
2 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.2% (w/v) deoxycholate)
containing 1 mM sodium orthovanadate, and cell lysates were
clarified by centrifugation (2.000 × g, 10 min). For
actin immunoblot analysis, cells were treated as described above except the cells were not pretreated with DMI and were lysed in cold lysis
buffer not containing sodium orthovanadate. Actin levels were evaluated
using an anti-actin monoclonal antibody from Sigma. For cyclin D1
analysis, quiescent Swiss 3T3 cells were treated for 6 h with
either 1 mM amlexanox or with 10 ng/ml FGF1 or with both
and lysed as described above. Protein concentration was determined using the Pierce BCA protein assay kit. To examine protein tyrosine phosphorylation, immunoprecipitation was performed using a rabbit antibody against cortactin (12), a rabbit antibody against focal adhesion kinase (Sigma), two rabbit antibodies against ERK1 and ERK2
(Santa Cruz), a rabbit antibody against PDGFR type B (Upstate Biotechnology), a rabbit antibody against FGFR1 (13), and a rabbit
antibody against the insulin receptor Analysis of Cell Migration, Cell Proliferation, and
Apoptosis--
Cell proliferation was assessed as described previously
(15), and cell migration was evaluated using an in vitro
model of wound repair as described (22). To examine apoptosis,
confluent monolayers of HUVEC and NIH 3T3 cells were incubated with or
without 0.375 M or 1 mM amlexanox. To prevent
the loss of floating apoptotic cells from the cell population, fresh
medium was added every 2 days to the culture dish without removing the
old medium. After 3 days, the cells were harvested by trypsin
digestion, and the floating cells were independently collected by
centrifugation and combined with the cells obtained from the monolayer.
The cells were washed with phosphate-buffered saline, and smears were
prepared and fixed in 70% (v/v) ethanol. The cells were stained with
100 ng/ml Hoechst #33258 (Sigma) for 1 min, and apoptotic nuclei with fragmented chromatin were quantitated using a fluorescence microscope. Five hundred cells were counted per treatment, and apoptotic cells were
expressed as a percentage of total cells counted. The experiment was
repeated in triplicate using two wells for each condition.
Analysis of FGF1 Release--
The reversibility of FGF1 release
after amlexanox pretreatment was performed using NIH 3T3 cells stably
transfected with FGF1 (23). Cells were grown until 70% confluence and
incubated overnight in the presence or absence of 1 mM
amlexanox. The monolayers were washed with DMEM containing 5 units/ml
heparin (The Upjohn Co.). Cells previously treated with amlexanox were
also incubated in DMEM containing 5 units/ml heparin but in the
presence or absence of 0.375 mM amlexanox, and the cells,
previously not treated with amlexanox, were further incubated under
amlexanox-free conditions. The cell populations were subjected to heat
shock (42 °C for 110 min) as previously reported (5, 23). To
evaluate the effect of latrunculin on FGF1 and p40 Syt1 release, NIH
3T3 cells transfected with FGF1 and p65 Syt1 were grown until 70%
confluent and were subjected to heat shock (42 °C, 110 min) in the
presence and absence of 400 nM latrunculin (Biomol). Media
conditioned by this response were processed and analyzed by FGF1 and/or
Syt1 immunoblot analysis as described previously (5, 23).
Amlexanox Reversibly Modifies Cell Morphology--
We have
previously demonstrated that the S100A13 binding compound, amlexanox,
inhibits the release of FGF1 and p40 Syt1 from NIH 3T3 cells in
response to heat shock (1). Since several members of the S100A13 family
are cytoskeleton-associated proteins (6, 7), we questioned whether
amlexanox could modify cell morphology. After exposure for 24 h to
amlexanox, NIH 3T3 cells exhibited a dramatic change in their
morphology that included a larger and more flattened phenotype and
often displayed long processes that are stable for at least 10 days
(Fig. 1, A and B).
Interestingly, the amlexanox-induced morphology of NIH 3T3 cells
completely reverted to the normal phenotype 2 days after the removal of
amlexanox.
Amlexanox was also able to produce similar, reversible effects on cell
morphology in a large variety of cell lines, including HUVEC (Fig. 1,
C and D), Swiss 3T3 cells, human IMR90
fibroblasts, newborn rat aorta smooth muscle cells, rat L6 myoblasts,
and the murine glioma cell lines U251Mg and U563Mg (data not shown).
Interestingly, the phenotype exhibited by HUVEC populations resembled
the phase contrast morphology of the senescent HUVEC phenotype
resulting from extended serial propagation in vitro (24). In
addition, serum starvation accelerated the effects of amlexanox upon
cell morphology. NIH and Swiss 3T3 cells serum-starved for 2 days and then treated with 1 mM amlexanox exhibited a more dramatic
and rapid morphological change than cells maintained in complete cell culture medium (data not shown).
Amlexanox Induces Changes in the Actin Cytoskeleton--
The
induction and reversion of morphological changes produced by amlexanox
prompted us to examine the organization of the cytoskeleton using HUVEC
populations which are quite sensitive to agents that promote apoptosis.
Interestingly, we observed no differences in the level of apoptosis
between control (2.5% ± 1.0) and amlexanox-treated (2.6% ± 1.1)
HUVEC populations as well as NIH and Swiss 3T3 cells (data not shown),
using an assessment of chromatin fragmentation by Hoechst staining. In
addition, fluorescein isothiocyanate-phalloidin staining of HUVEC
populations treated for 4 days with amlexanox revealed a dramatic
attenuation of the F-actin cytoskeleton (Fig. 1, E and
F). Indeed, the amlexanox-treated cells exhibited either a
complete absence or strong down-regulation of phalloidin-positive
F-actin stress fibers. However, the subplasmalemmal F-actin cortex
remained readily apparent in the amlexanox-treated cells. This change
in the appearance of the F-actin cytoskeleton became visible as early
as 4 to 8 h after the addition of amlexanox and was fully
prominent after 24 h (data not shown). Similar changes in the
F-actin cytoskeleton were induced by 1 mM amlexanox in Swiss (data not shown) and NIH 3T3 cells (Fig. 3, A and
B). In addition, the changes in the F-actin cytoskeleton
induced by amlexanox were reversible. Indeed, 2 hours after the removal
of amlexanox, the majority of the HUVEC population previously treated
for 4 days with amlexanox exhibited the presence of prominent F-actin stress fibers in vitro (Fig. 1G). In contrast,
however, despite the dramatic attenuation of the F-actin cytoskeleton,
the amlexanox-treated HUVEC population did not exhibit any significant
change in the organization of their tubulin-containing microtubule and
vimentin-containing intermediate filament networks (data not shown).
Fluorescein isothiocyanate-phalloidin staining of amlexanox-treated,
serum-deprived NIH and Swiss 3T3 cells exhibited a very rapid (30 min
to 1 h) disappearance of actin stress fibers and the fragmentation
of the actin cytoskeleton (data not shown).
The strong attenuation of F-actin stress fibers after treatment with
amlexanox prompted us to evaluate the total level of actin and the
level of F-actin in HUVEC populations before and after exposure to
amlexanox for 48 h. As shown in Fig. 1H, actin immunoblot analysis revealed no difference in the levels of actin expression between amlexanox-treated and control HUVEC populations. Similarly, we did not observe a difference in the content of
polymerized actin as measured by the fluorescence of methanol-extracted
NBD phallacidin between the control (0.045 ± 0.003 relative
units/cell) and amlexanox-treated (0.056 ± 0.004 relative
units/cell) HUVEC populations. The presence of 1 mM
amlexanox did not alter the steady state levels of the actin transcript
in Swiss 3T3 cells as assessed by reverse transcriptase-polymerase
chain reaction (data not shown). In addition, jasplakinolide, an agent
that binds and stabilizes filamentous actin (20), prevented the
amlexanox-induced changes in cell morphology and attenuation of F-actin
stress fibers (data not shown).
Amlexanox Induces Cortactin Tyrosine Phosphorylation--
Because
(i) amlexanox was able to affect the organization of actin stress
fibers without affecting the expression of actin and its polymerization
and (ii) serum deprivation potentiated the effect of amlexanox, we
evaluated the level of tyrosine phosphorylation of cortactin, an
actin-binding protein whose phosphorylation by Src has been implicated
in the rearrangement of the actin cytoskeleton after growth factor
stimulation (25). Since Swiss 3T3 cells under conditions of serum
deprivation display a very low level of cortactin tyrosine
phosphorylation, we examined this cell culture system for the tyrosine
phosphorylation of cortactin in response to amlexanox. As shown in Fig.
2, we observed that the amlexanox was
able to stimulate the tyrosine phosphorylation of cortactin in a rapid
and sustained manner. Interestingly, the intensity of cortactin
tyrosine phosphorylation stimulated by amlexanox (Fig. 2) appeared to
be greater than that achieved by FGF1 after a 6-h incubation period.
Phosphotyrosine analysis of cortactin immunoprecipitates also revealed
the presence of other tyrosine-phosphorylated proteins including a p60
protein that was phosphorylated in parallel with cortactin in
amlexanox-treated cells as well as in cells stimulated with FGF1 for
6 h (Fig. 2). Two additional proteins, p110 and p140, were also
phosphorylated on tyrosine residues in an
amlexanox-dependent manner 30 min and 2 h after
amlexanox treatment but not in the presence of FGF1 (Fig. 2).
A Dominant Negative Form of Src Prevents the Amlexanox-induced
Collapse of Actin Cytoskeleton and Down-regulates Cortactin Tyrosine
Phosphorylation in Amlexanox-treated Cells--
Since (i) cortactin is
a protein phosphorylated by Src in response to extracellular growth
factors stimulation (12), (ii) phosphotyrosine analysis of cortactin
immunoprecipitates after amlexanox treatment revealed the presence of a
p60 protein (Fig. 2), and (iii) cortactin is involved in the regulation
of actin bundling (25), we investigated the involvement of the Src
phosphorylation in the amlexanox-dependent collapse of the
actin cytoskeleton using a dominant negative (dn) mutant of Src (18).
Stable dnSrc NIH 3T3 cell transfectants were obtained, and these
displayed enhanced levels of the Src transcript and protein, as
determined by reverse transcriptase-polymerase chain reaction and
immunoblot analysis, respectively (data not shown). Interestingly,
treatment with 1 mM amlexanox for 48 h in the presence
of 10% (w/v) BCS resulted in the disappearance of actin stress fibers
in NIH 3T3 cells (Fig. 3, A
and B) but failed to completely destroy stress fibers in
dnSrc NIH 3T3 cell transfectants (Fig. 3, C and
D). Correspondingly, amlexanox also induced a less dramatic
morphological change in dnSrc NIH 3T3 cell transfectants (data not
shown).
Although NIH 3T3 cells displayed high levels of cortactin tyrosine
phosphorylation under conditions of cellular quiescence and these
levels were not significantly altered by the addition of either
amlexanox or FGF1, cortactin tyrosine phosphorylation was significantly
reduced in the dnSrc NIH 3T3 cell transfectants treated for 6 h
with 1 mM amlexanox (Fig.
4A). This decrease in cortactin tyrosine phosphorylation also correlated with the
conservation of the actin cytoskeleton observed in the
amlexanox-treated dnSrc NIH 3T3 cell transfectants (Fig. 3).
Amlexanox Reversibly Inhibits Cell Migration--
Since (i)
amlexanox modifies the reorganization of the actin cytoskeleton through
a Src-related mechanism and (ii) Src is involved in the regulation of
cell migration (13), we examined the ability of amlexanox to interfere
with cell motility (22). Indeed, the migratory ability of NIH 3T3 cells
was significantly diminished by the presence of amlexanox in a
dose-dependent manner, with a half-maximum value of
approximately 100 µM amlexanox (Fig. 5A). The vehicle (1 mM NaOH) used to deliver amlexanox did not modify the
migratory ability of NIH 3T3 cell population (Fig. 5A).
Similar results were obtained using Swiss 3T3 cells and HUVEC (data not
shown). This repression of cell migration was reversible (data not
shown) and occurred at concentrations of amlexanox similar to those
that were able to alter both cell morphology and actin cytoskeleton.
Since dnSrc NIH 3T3 cell transfectants exhibited an attenuation of the
effect of amlexanox on the actin cytoskeleton, we evaluated the
capacity of amlexanox to suppress the motility of these cells. As shown
in Fig. 5B, amlexanox exhibited a strong inhibition of NIH
3T3 cell migration, whereas the inhibition of cell motility was less
dramatic in two clones of the dnSrc NIH 3T3 cell transfectants treated
with amlexanox.
Amlexanox Induces Changes in Focal Adhesion Site Distribution and
Modifies the Tyrosine Phosphorylation of Focal Adhesion
Kinase--
Since actin stress fibers are known to be structurally
linked to focal adhesion sites through
Since Src has been demonstrated to regulate cell migration by forming a
complex with focal adhesion kinase (FAK) in focal adhesion sites (29),
we examined whether the amlexanox-induced redistribution of focal
adhesion sites in NIH 3T3 cells correlated with a change in the
tyrosine phosphorylation of FAK. Phosphotyrosine analysis of FAK
immunoprecipitates exhibited similar levels of tyrosine phosphorylation
in control NIH 3T3 and dnSrc NIH 3T3 cell transfectants (Fig.
4B). After treatment with 1 mM amlexanox for
6 h under serum-free conditions, cells displayed a moderate down-regulation of FAK phosphorylation in both control NIH 3T3 and
dnSrc NIH 3T3 cell transfectants (Fig. 4B). This was also observed in Swiss 3T3 cells (data not shown).
Amlexanox Reversibly Inhibits Cell Proliferation--
Since (i)
amlexanox inhibits the release of FGF1 from NIH 3T3 cells in response
to heat shock (1), (ii) the ability of FGF1 to induce biologic
responses requires its presence in the extracellular compartment to
mediate receptor-dependent signaling (2, 3), and (iii)
other reagents that affect the integrity of actin cytoskeleton also
inhibit cell proliferation (30), we examined the ability of amlexanox
to modify in vitro the growth of HUVEC, cells that are
dependent upon the presence of a source of extracellular FGF for growth
(15). Amlexanox was able to inhibit the growth of HUVEC (Fig.
6A) in a
dose-dependent manner, with a half-maximum value of
approximately 30 µM amlexanox (data not shown). The
vehicle (0.375 mM NaOH) used to deliver amlexanox did not
modify the proliferative ability of the HUVEC population (Fig.
6A). The ability of amlexanox to repress HUVEC growth was time-dependent, with significant inhibition of HUVEC growth
observed after 72 h of treatment with the drug (data not shown).
We also examined the ability of amlexanox to inhibit DNA synthesis.
HUVEC populations were treated for 2 or 4 days with amlexanox in a
concentration-dependent manner, and their ability to
incorporate [3H]thymidine in the nucleus was assessed by
autoradiography. We observed a dose-dependent inhibition of
DNA synthesis in cells treated with amlexanox with a half-maximal
effect at 30 µM amlexanox (data not shown). Similar
results were observed with human IMR90 fibroblasts, newborn rat aorta
smooth muscle cells, rat L6 myoblasts, NIH 3T3 cells, Swiss 3T3 cells,
and the murine glioma cell lines U251Mg and U563Mg. Although the Swiss
3T3 and NIH 3T3 cells were less responsive to amlexanox with
half-maximal inhibition of cell growth at 300 µM, the
remainder of the cells exhibited growth inhibition at concentrations of
amlexanox similar to that observed with HUVEC populations (data not
shown).
We also examined whether the inhibition of cell growth was reversible.
Confluent populations of HUVEC were exposed to 0.375 mM
amlexanox, and after 4 days, the cells were harvested by trypsin digestion and replated, and the amlexanox-pretreated cells were incubated with or without 0.375 mM amlexanox. As shown in
Fig. 6A, the HUVEC population previously exposed to
amlexanox initially exhibited a decrease in their proliferative ability
but after 3 days exhibited a population doubling time similar to the
control, amlexanox-free population. In contrast, the HUVEC population
previously exposed to amlexanox was not able to proliferate if
amlexanox remained present in the cell culture medium (Fig.
6A). Flow cytometry analysis of HUVEC treated with amlexanox
and Swiss 3T3 cells synchronized by incubation in the presence of
either DMI and/or 4 mM thymidine and stimulated with serum
in the presence and absence of amlexanox demonstrated that amlexanox
inhibits the cell cycle in G1 and G2 phases
(data not shown).
Amlexanox Down-regulates Cyclin D1 Expression without Affecting the
FGF Receptor Signaling--
Because amlexanox was able to reversibly
inhibit the cell proliferation blocking cell cycle in both
G1 and G2 phases, we evaluated the expression
of cyclin D1, a protein that plays a key role in the progression from
G0 through G1 into the S phase and is
up-regulated in G1 phase of cell cycle (24). As shown in
Fig. 6C, quiescent Swiss 3T3 cells did not express
detectable levels of cyclin D1, and amlexanox was not able to induce
cyclin D1 expression. However, cells stimulated for 6 h with FGF-1
demonstrated an induction of cyclin D1 protein expression, and this
induction was inhibited by the simultaneous exposure of cells to 1 mM amlexanox (Fig. 6C). We also evaluated cyclin
D1 protein levels during Swiss 3T3 cell proliferation in response to
serum in the presence and absence of 1 mM amlexanox. As
shown in Fig. 6C, proliferating populations of Swiss 3T3
cells exhibited high levels of cyclin D1, but after exposure to
amlexanox, the levels of cyclin D1 were significantly and rapidly decreased.
To understand if the amlexanox-dependent inhibition of
cyclin D1 induction was the result of its ability to interfere with polypeptide growth factor/receptor/Ras/mitogen-activated protein kinase
signaling pathways, we evaluated the capacity of FGF1, PDGF-BB, and
insulin to activate their receptors in the presence of amlexanox.
Quiescent Swiss 3T3 cells were treated for 6 h with 1 mM amlexanox or incubated for an additional 6 h in
serum-free medium without amlexanox and then were stimulated for 30 min
with either 10 ng/ml FGF1, 5 ng/ml PDGF-BB, or 1 µg/ml insulin in the presence or absence of amlexanox. Phosphotyrosine immunoblot analysis following FGFR-1, PDGFR type B, and insulin receptor Amlexanox Inhibition of FGF1 Secretion Is Reversible--
Since
the effects of amlexanox on the actin cytoskeleton, cell morphology,
and cell migration are reversible, we examined whether the inhibition
of FGF1 release by amlexanox was also reversible. FGF1 NIH 3T3 cell
transfectants were treated for 18 h with 1 mM amlexanox in the presence of 10% (v/v) BCS to produce a collapse of
actin cytoskeleton. Amlexanox was removed from the medium, and the
cells were subjected to heat shock (42 °C, 110 min) in the presence
or absence of 0.375 mM amlexanox. As shown in Fig. 8A, amlexanox treatment
resulted in a significant inhibition of FGF1 release, but FGF1 release
was completely restored if amlexanox was removed from the cell culture
medium before the temperature stress.
To further investigate the role of the actin cytoskeleton in FGF1
release, we evaluated the effect of latrunculin, a drug known to
depolymerize F-actin (30), on FGF1 and p40 Syt1 release. NIH 3T3 cells
stably transfected with either FGF1 (23) or with FGF1 and p65 Syt1 (4,
5) were subjected to temperature stress in the presence and absence of
400 nM latrunculin. As shown in Fig. 8, B and
C, a significant inhibition of both FGF1 and p40 Syt1
release in response to heat shock was observed in the
latrunculin-treated cells.
Amlexanox is a particularly efficient inhibitor of mammalian cell
migration and proliferation in vitro. Cells exposed to
amlexanox exhibited a dose-dependent inhibition of cell
motility and growth without an increase in apoptotic cell death.
Consistent with the inability of amlexanox to induce apoptosis, at
least in concentrations used in these experiments, was the observation
that the inhibition of cell migration and proliferation by amlexanox
was reversible.
In association with the suppression of cell migration and
proliferation, prominent changes in cell morphology were also observed. Indeed, in most situations these morphologic changes were rapid and
were readily visible after approximately 8 to 12 h. These morphologic alterations included an increase in cell size, the formation of a more flattened appearance, and the generation of long
dendrite-like processes. Interestingly, the changes correlated with a
strong attenuation of the presence of actin stress fibers in cells
treated with amlexanox at concentrations that not only inhibit cell
migration and proliferation but also alter and disorient the
distribution of focal adhesion sites. In addition, we also observed
more rapid and prominent morphological changes induced by amlexanox
with a disappearance of actin stress fibers and subplasmalemmal cortex
under serum-free conditions.
It is clear that the effects of amlexanox on the cytoskeleton are not
due to the down-regulation of intracellular levels of actin or to the
depolymerization of actin filaments. Also, using an in vitro
system of actin polymerization, we observed that amlexanox was unable
to inhibit this process.2 We
therefore suggest that the process of actin microfilament bundling may
be involved in mediating the effects of amlexanox. Supportive of this
interpretation is the observation that jasplakinolide, an agent known
to stabilize actin stress fibers (20), is able to prevent the effects
of amlexanox on cell morphology and actin cytoskeleton.
The mechanism utilized by amlexanox appears to involve the function of
cortactin and Src. Cortactin is a Src substrate and an F-actin-binding
protein whose tyrosine phosphorylation results in a dramatic reduction
in F-actin cross-linking activity and actin bundling (25). Indeed,
amlexanox is able to rapidly induce and sustain the tyrosine
phosphorylation of cortactin, and in response to amlexanox, cortactin
is able to associate with a variety of phosphotyrosine-containing
proteins including a p60 polypeptide. Since it has been demonstrated
that cortactin is phosphorylated by Src in response to FGF1 and other
growth factors as a relatively late event in the G1 phase
of cell cycle (12, 31), we evaluated the role of Src pathway in
mediating the effect of amlexanox. The overexpression of a dominant
negative form of Src, known to act as an inhibitor of endogenous Src
activity (18), attenuated the effect of amlexanox on both cell
morphology and actin stress fibers. In addition, amlexanox was also
able to significantly reduce the levels of cortactin tyrosine
phosphorylation in the dnSrc NIH 3T3 cell transfectants. The dnSrc NIH
3T3 cell transfectants were also less sensitive to amlexanox-induced
inhibition of cell motility and to the redistribution of focal adhesion
sites, suggesting that these effects of amlexanox may also be mediated,
in part, by Src.
Since it is well established that FAK is localized at focal adhesion
sites (29) and is involved in the regulation of cell migration in
response to extracellular stimuli (27), we evaluated whether amlexanox
was able to affect FAK phosphorylation. Interestingly, amlexanox was
only able to produce a moderate down-regulation of FAK phosphorylation
in both NIH 3T3 and the dnSrc NIH 3T3 cell transfectants, and this
effect required a prolonged exposure (3-6 h) to amlexanox. These
results suggest that the redistribution of focal adhesion sites in
amlexanox-treated cells may be a consequence of the collapse of the
actin cytoskeleton. This interpretation is consistent with the
observation that a 1-h treatment with amlexanox under conditions of
serum deprivation was able to affect the intensity and the distribution
of vinculin-positive focal adhesion sites in NIH 3T3 cells but not
dramatically alter the tyrosine phosphorylation of FAK (data not shown).
Because the treatment of cells with amlexanox was able to prevent the
FGF1-induced up-regulation of cyclin D1 in both quiescent and
proliferating populations of Swiss 3T3 cells, we evaluated the capacity
of FGF1, PDGF-BB, and insulin to activate their cell surface receptors
in presence of amlexanox. Interestingly, the exposure of cells to
amlexanox for 6 h did not prevent the ability of FGF1, PDGF-BB,
and insulin to induce the auto-phosphorylation of their receptors. In
addition, amlexanox treatment did not alter the ability of FGF1 and
PDGF-BB to induce ERK-1 and ERK-2 phosphorylation in quiescent Swiss
3T3 cells and did not prevent the FGF1-induced migration of ERK-1 and
ERK-2 to the nucleus (data not shown). We also evaluated the capacity
of amlexanox to interfere with FGFR1 activation as a function of
exposure time to amlexanox using quiescent populations of Swiss 3T3
cells, and we observed a decrease in the ability of FGF1 to induce the
autophosphorylation of FGFR1 only after long term exposure (18 h) of
the cells to amlexanox. Shorter time periods of exposure to amlexanox,
which were able to induce the disaggregation of actin cytoskeleton and
prevent the up-regulation of cyclin D1, failed to interfere with
receptor signaling. Since amlexanox was able to inhibit the
proliferation of dnSrc NIH 3T3 cells and the induction of cyclin D1 in
dnSrc NIH 3T3 cells (data not shown), we suggest that the
anti-proliferative effect of amlexanox is dissociated from its effect
on the Src pathway. This could be explained by the ability of
amlexanox to interfere with cyclin D1 induction and the molecular
events that are responsible for the G1 transition, yet
these appear to be independent of the induction of the polypeptide
growth factor/receptor/Ras/mitogen-activated protein kinase signaling
pathway. Our data may also suggest the importance of the actin
cytoskeleton during pre-replicative events of the G1 phase.
Other biochemical agents such as the cytochalasines and latrunculins
also induce the disorganization of actin cytoskeleton as well as the
inhibition of cell proliferation (30). However, unlike amlexanox, these
agents induce apoptosis, and their effects on the actin cytoskeleton
are more rapid and include the attenuation of stress fibers, a
prominent decrease in F-actin protein levels, and a reduction in the
subplasmalemmal F-actin cortex. In addition, the latrunculins and
cytochalasines induce prominent cell rounding and the formation of
cytoplasmic blebs, and these effects were not observed with amlexanox
in the presence of serum (30). Indeed, to our knowledge, this is the
first study to identify a reagent that is able to induce the reversible
disassembly of actin bundles without influencing actin polymerization
and inducing apoptosis. The biological activities of amlexanox may also
offer novel opportunities for its use as a reagent for studies in the
fields of cell biology and experimental medicine. The reversible
suppression of cell growth may potentially be used to synchronize
cells, and its effect on actin stress fibers may prove useful for
studies of the organization and functions of the actin cytoskeleton. In
addition, amlexanox may also prove useful as a reagent to assess the
role of actin stress fibers in the cellular trafficking of organelles
and macromolecules. Lastly, the ability of amlexanox to reversibly
inhibit human endothelial cell migration and proliferation suggest that
its anti-inflammatory activities in vivo may possess an
anti-angiogenic component. However, the poor solubility of amlexanox
will require the development of novel methods for the efficient
in vivo delivery of this rather interesting biological antagonist.
The reason for studying the mechanism of amlexanox activity was the
observation that amlexanox is able to bind S100A13 (14), a protein that
together with Syt1 is a component of a heparin binding multiprotein
complex involved in the mechanism of FGF1 release (1). Little is known
about the biological functions and intracellular localization of
S100A13, and it is a relatively novel member of the S100 gene family of
calcium-binding proteins (32). However, it is well described that S100
gene family members, S100A1 and S100A4 (6, 7) co-localize with actin
stress fibers. In addition, S100B is able to associate with the
actin-capping protein, CapZ (33), and S100A2 is interactive with
tropomyosins (34), which are known to regulate the bundling of actin
filaments (35). Thus, the ability of amlexanox to induce cortactin
tyrosine phosphorylation and to modify actin stress fibers in
vitro may reflect either the potential co-localization of S100A13
with the actin filaments or a potential role of S100A13 to cooperate in the organization of actin cytoskeleton.
The effect of amlexanox on the Src pathway and actin stress fiber
disassembly could underlie the mechanism used by this agent to
interfere with the function of proteins involved in the release of FGF1
as well as its ability to suppress the release of histamine by mast
cells, an important allergenic mediator (36). We observed that the
effect of amlexanox on FGF1 release is reversible and correlates with
the restoration of the actin cytoskeleton. Moreover, latrunculin,
another reagent that affects the integrity of the actin cytoskeleton
through a different mechanism that involves the depolymerization of
F-actin (30), is also able to inhibit the release of FGF1 and p40 Syt1
in response to temperature stress. Thus it is possible that the
function of the Src pathway and the actin cytoskeleton may be involved
in the regulation of the intracellular trafficking responsible for FGF1
and Syt1 release. Since (i) p65 Syt1, a transmembrane component of
intracellular vesicles (37), is involved in the regulation of
exocytotic and endocytotic organelle trafficking (38), including the
docking of synaptic vesicles to the plasma membrane (39), and (ii) the
function of intracellular p65 Syt1 is required for FGF1 release (4), we
suggest that Syt1-positive intracellular vesicles containing the
extravesicular FGF1 homodimer may traffic along actin stress fibers and
that the function of S100A13 or other members of the S100 gene family may be involved in the regulation of this trafficking.
We thank the officers of Takeda
Pharmaceuticals, Ltd. for their generosity in supplying amlexanox, A. Mandinova and U. Aebi (University of Basel), for their assessment of
the ability of amlexanox to block actin polymerization in
vitro, and R. Friesel (Maine Medical Center) for the dnSrc mutant.
*
This work was supported in part by National Institutes of
Health Grants HL35627, HL32348, and AG07450 (to T. M.).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: Massachusetts General Hospital, Harvard Medical
School, 100 Blossom St., Boston, MA 02114.
¶
Present address: Dept. of Geriatric Medicine, University of
Florence, School of Medicine, Florence, Italy.
Published, JBC Papers in Press, July 31, 2000, DOI 10.1074/jbc.M002336200
2
A. Mandinova, U. Aebi, M. Landriscina, I. Prudovsky, and T. Maciag, unpublished observation.
The abbreviations used are:
FGF, fibroblast
growth factor;
BCS, bovine calf serum;
DMI, defined medium with
insulin;
dn, dominant negative;
FAK, focal adhesion kinase;
Syt, synaptotagmin;
NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl));
HUVEC, human umbilical vein endothelial cells;
DMEM, Dulbecco's
modified Eagle's medium;
Pipes, 1,4-piperazinediethanesulfonic acid;
ERK, extracellular signal-regulated kinase;
PDGF, platelet-derived
growth factor;
PDGFR, PDGF receptor;
PAGE, polyacrylamide gel
electrophoresis.
Amlexanox Reversibly Inhibits Cell Migration and
Proliferation and Induces the Src-dependent Disassembly
of Actin Stress Fibers in Vitro*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit (Upstate Biotechnology) as described previously (12).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The effect of amlexanox on cell morphology
and changes in the appearance of the actin cytoskeleton. NIH 3T3
cells (A and B) and HUVEC (C and
D) were grown for 4 days as described under "Materials and
Methods" in the absence (A and C) or in the
presence of 1 mM (B) or 0.375 mM
(D) amlexanox. Phase contrast microscopy (A-C,
10×; D, 20×) was used to record the phenotype.
Fluorescence microscopy of actin distribution (E-G) is
shown in HUVEC populations incubated in the presence (F) or
in the absence (E) of 0.375 mM amlexanox for 4 days. Actin distribution in HUVEC populations treated with 0.375 mM amlexanox for 4 days and further incubated for 2 h
in normal growth medium without amlexanox (G) is shown.
Fluorescent photomicrographs were taken with a 100× objective.
H, immunoblot analysis of total actin levels in HUVEC
populations exposed for 2 days in the presence or absence of 0.375 mM amlexanox.
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Fig. 2.
Tyrosine phosphorylation of cortactin in
Swiss 3T3 cells treated with amlexanox. Swiss 3T3 cells were grown
to confluence and incubated for 48 h in DMI. Quiescent cells were
further incubated with either 1 mM amlexanox for the times
indicated or with 10 ng/ml FGF1 for 6 h. Cell lysates were
immunoprecipitated with rabbit anti-cortactin antibody 2719 (12),
resolved by 8% (w/v) acrylamide SDS-PAGE, and subjected to immunoblot
analysis with an antiphosphotyrosine antibody (Upstate Biotechnology)
as described under "Materials and Methods." The arrows
indicate the position of cortactin and three other
phosphotyrosine-containing proteins with approximate masses of 60, 110, and 140 kDa.
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Fig. 3.
Changes in the actin cytoskeleton and
immunofluorescence microscopy of focal adhesion sites in NIH 3T3 and
dnSrc NIH 3T3 cells transfectants treated with amlexanox.
Fluorescence microscopy is shown of actin distribution in NIH 3T3
(A and B) and dnSrc NIH 3T3 cell transfectants
(C and D) incubated in the presence (B
and D) and absence (A and C) of 1 mM amlexanox for 2 days. Immunofluorescent microscopy of
focal adhesion sites is shown in NIH 3T3 (E and
F) and dnSrc NIH 3T3 cell transfectants (G and
H) incubated in the presence (F and H)
or absence (E and G) of 1 mM
amlexanox for 2 days. Fluorescent photomicrographs were taken with a
100× objective.
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Fig. 4.
Tyrosine phosphorylation of cortactin and FAK
in NIH 3T3 and dnSrc NIH 3T3 cell transfectants treated with
amlexanox. A, cortactin phosphotyrosine analysis. DnSrc
NIH 3T3 cell transfectants were grown to confluence and incubated for
48 h in DMI. Quiescent cells were further incubated in DMI for
6 h or were incubated for 6 h in DMI containing 1 mM amlexanox or 10 ng/ml FGF1. Cortactin
immunoprecipitation and immunoblot were performed as described under
"Materials and Methods." B, FAK phosphotyrosine
analysis. Quiescent NIH 3T3 and dnSrc NIH 3T3 cell transfectants were
incubated in DMI or in DMI containing 1 mM amlexanox for
6 h. Cell lysates were immunoprecipitated with a rabbit anti-FAK
antibody, resolved by 7.5% (w/v) acrylamide SDS-PAGE, and subjected to
immunoblot analysis with an antiphosphotyrosine antibody as described
under "Materials and Methods."
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Fig. 5.
The effect of amlexanox on cell
migration. A, the concentration dependence of amlexanox
on cell migration. NIH 3T3 cells were grown to confluence, monolayers
were scraped with a razor blade, and cells were incubated for 24 h
with different concentrations of amlexanox as indicated. Serum-induced
cell migration into the denuded area was expressed as the number of
cells present beyond the wound edge (the average of 10 microscopic
fields). B, an assessment of the ability of amlexanox to
inhibit cell migration in dnSrc NIH 3T3 cell transfectants. NIH 3T3 and
dnSrc NIH 3T3 cell transfectants (clones 5 and 7) were grown to
confluence, and cell migration was evaluated in the presence of 1 mM amlexanox as described previously (22). The data are
reported as percent inhibition relative to the positive
control.
-actinin (26) and are
involved in the regulation of cell migration and adhesion (27), we
questioned whether amlexanox treatment was able to modify the
organization of focal adhesion sites in NIH 3T3 cells and dnSrc NIH 3T3
cell transfectants. Immunohistochemical staining for vinculin, a
component of focal adhesion sites (28), demonstrated that amlexanox
treatment in the presence of serum resulted in a significant decrease
in the intensity and the redistribution of focal adhesion sites in NIH
3T3 cells. Instead of the parallel and well oriented focal adhesion
sites that are often concentrated at the leading edge of the cell and
usually observed in cells undergoing migration (24), we observed a
radial or random distribution of the vinculin-positive focal adhesion
sites in the amlexanox-treated cells (Fig. 3, E and
F). This phenotype is usually associated with non-migratory and "sedentary" populations of cells (24). The effect of amlexanox on focal adhesion site orientation in the dnSrc NIH 3T3 transfectants was not as dramatic as that observed with control NIH 3T3 cells (Fig.
3, G and H), and this is consistent with the
resistance of dnSrc NIH 3T3 cell transfectants to amlexanox-induced
inhibition of cell migration. In addition, under short term conditions
of serum deprivation, amlexanox produced a more dramatic and faster disappearance of focal adhesion sites in NIH 3T3 cells, whereas amlexanox treatment produced only a partial redistribution of focal
adhesion sites in the dnSrc NIH 3T3 transfectants (data not shown).
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Fig. 6.
The effect of amlexanox on HUVEC
proliferation and the ability of amlexanox to down-regulate cyclin D1
expression without interference with ERK1 and ERK2 tyrosine
phosphorylation. A, the reversibility of the inhibition
of HUVEC proliferation by amlexanox; cells were grown in the presence
or absence of 0.375 mM amlexanox or in the presence of
0.375 mM NaOH for 4 days, harvested by trypsin digestion,
counted, and replated at a density of 6 × 104
cells/well. Cells pretreated with amlexanox were further grown in the
presence or absence of the same concentration of amlexanox, and the
cells pretreated with 0.375 mM NaOH were further grown in
presence of 0.375 mM NaOH. Cells were harvested by trypsin
digestion at 4, 6, 8, and 10 days after replating and counted in a
hematocytometer. B, tyrosine phosphorylation of ERK1 and
ERK2 in Swiss 3T3 cells treated with amlexanox. Swiss 3T3 cells were
grown to confluency and incubated for 48 h in DMI. Quiescent cells
were further incubated for 6 to 18 h in DMI in the presence of 1 mM amlexanox and were stimulated with 10 ng/ml FGF1 for
1 h in the presence or absence of amlexanox. Cell lysates were
immunoprecipitated with polyclonal anti-ERK1 and ERK2 antibodies as
described under "Materials and Methods." Immunoprecipitated
proteins were resolved by 10% (w/v) SDS-PAGE and probed with an
anti-phosphotyrosine antibody (upper panel) or with
anti-ERK1 and ERK2 antibodies (lower panel). Lane
1, quiescent Swiss 3T3 cells; lane 2, 6 h with
amlexanox; lane 3, 18 h with amlexanox; lane
4, FGF1 alone for 1 h; lane 5, 6 h with
amlexanox and 1 h with FGF1; lane 6, 18 h with
amlexanox and 1 h with FGF1. C, cyclin D1 levels in
Swiss 3T3 treated with amlexanox. Quiescent Swiss 3T3 cells were
stimulated for 6 h with 10 ng/ml FGF1 in either the presence or
absence of 1 mM amlexanox. In a parallel experiment, Swiss
3T3 cells were grown in medium containing 10% BCS until 50%
confluency and treated with 1 mM amlexanox for 6, 15, and
24 h. Cells were harvested, and cell lysates were prepared as
described under "Material and Methods." Lysates were analyzed by
12% (w/v) acrylamide SDS-PAGE and probed with a mouse monoclonal
anti-cyclin D1 antibody (Sigma). Lane 1, quiescent Swiss 3T3
cells; lane 2, 6 h with amlexanox; lane 3,
6 h with FGF1; lane 4, 6 h with amlexanox and
FGF1.
subunit immunoprecipitation demonstrated that exposure to amlexanox was not
able to induce the tyrosine phosphorylation of these receptors and was
unable to prevent their tyrosine phosphorylation in response to their
respective ligands (Fig. 7). In addition,
ERK1 and ERK2 phosphorylation in response to either FGF1 (Fig.
6B) or PDGF-BB (data not shown) was not affected by exposure
of quiescent Swiss 3T3 cells to amlexanox. These data suggest that the
ability of polypeptide growth factors to induce the receptor tyrosine
kinase activation of the mitogen-activated protein kinase pathway is independent of the presence of the F-actin cytoskeleton.
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Fig. 7.
Tyrosine phosphorylation of FGFR1, PDGFR type
B, and insulin receptor subunit in Swiss 3T3
cells treated with amlexanox. Swiss 3T3 cells were grown to
confluency and then incubated in DMI for 48 h. Cells that were
destined for stimulation with insulin were incubated for 48 h in
insulin-free DMI. Quiescent cells were further incubated for 6 h
in either serum-free medium or in the presence of 1 mM
amlexanox. The cells were stimulated for 30 min with either 10 ng/ml
FGF1, 5 ng/ml PDGF-BB, or 1 µg/ml insulin in the presence or absence
of 1 mM amlexanox. Cell lysates were prepared and
immunoprecipitated (i.p.) with either a polyclonal
anti-FGFR1 antibody, a polyclonal anti-PDGFR type B antibody, or with a
polyclonal anti-insulin receptor (Rec.) subunit antibody
as described under "Material and Methods." Immunoprecipitates were
resolved by 7.5% (w/v) acrylamide SDS-PAGE and probed with an
anti-phosphotyrosine antibody. The arrows indicate FGFR1,
FGFR1, p90, PDGFR type B, and insulin receptor subunit.
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Fig. 8.
The reversible effect of amlexanox on FGF1
release and the ability of latrunculin to inhibit FGF1 and p40 Syt1
release. A, NIH 3T3 cells transfected with FGF1 were
grown until 70% confluence and treated for 18 h with 1 mM amlexanox to obtain a complete collapse of actin
cytoskeleton. Amlexanox was removed, and the cells were incubated for
110 min at 42 °C in DMEM containing 5 units/ml heparin with and
without 0.375 mM amlexanox. Conditioned media were prepared
as described under "Materials and Methods," resolved by 15% (w/v)
acrylamide SDS-PAGE, and subjected to immunoblot analysis with a rabbit
anti-FGF1 antibody. B and C, NIH 3T3 cells
transfected with FGF1 (B) or cotransfected with FGF1 and p65
Syt1 (C) were grown until 70% confluent and incubated for
110 min at 42 °C in DMEM containing 5 units/ml heparin in the
presence or absence of 400 nM latrunculin. Conditioned
media were prepared as described under "Materials and Methods,"
resolved by 12% (w/v) acrylamide SDS-PAGE, and subjected to immunoblot
analysis with a rabbit anti-FGF1 (B) and a rabbit anti-Syt1
(C) antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by a fellowship from the Catholic University of Rome.
To whom correspondence should be addressed: Center for
Molecular Medicine, Maine Medical Center Research Institute, 125 John Roberts Rd., South Portland, Maine 04106. Tel.: 207-761-9783; Fax:
207-828-8071; E-mail: maciat@mail.mmc.org.
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DISCUSSION
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