Considering that in vitro cultured Sk-Hep1 hepatocarcinoma cells release FGF-2 and shed membrane vesicles only when cultured in the pres-ence of serum, we added serum to starved cells an
Trang 1Simona Taverna, Salvatrice Rigogliuso, Monica Salamone and Maria Letizia Vittorelli
Dipartimento di Biologia Cellulare e dello Sviluppo, Universita` di Palermo, Italy
Most proteins destined for secretion into the
extracellu-lar matrix are targeted by the presence of N-terminal
signal peptides, which direct their translocation into
the rough endoplasmic reticulum (rER) They are
sub-sequently transferred to the Golgi apparatus and then
secreted into the extracellular space However, a
grow-ing number of secreted proteins have been identified
that lack N-terminal signal peptides These proteins do
not enter the rER and their secretion is not influenced
by drugs such as brefeldin A and monensin, which
block secretion by classical mechanisms [1] Therefore,
they are secreted by alternative, unconventional
pro-cesses; these proteins include the inflammatory cytokine interleukin-b1, galectins, macrophage migration inhibi-tory factor, acid and basic fibroblast growth factors (FGF-1, FGF-2) and sphingosine kinase 1 The mecha-nisms of their secretion are the subject of numerous studies and different pathways have been described [2]
An unconventional secretion mechanism common
to several signalling proteins devoid of the typical signalling sequence appears to be mediated by the shedding of vesicles into the extracellular matrix These vesicles, also called exovesicles, are observed to bud from the cell membranes and to be released into the
Keywords
FGF-2; microfilaments; microtubules;
secretion of leaderless proteins; shed
vesicles
Correspondence
M L Vittorelli, Dipartimento di Biologia
Cellulare e dello Sviluppo, Universita` di
Palermo, Viale delle Scienze ed 16,
90128 Palermo, Italy
Fax: +39 0 9165 77430
Tel: +39 0 9165 77407
E-mail: mlvitt@unipa.it
(Received 2 August 2007, revised 18
January 2008, accepted 31 January 2008)
doi:10.1111/j.1742-4658.2008.06316.x
We have previously reported how the release of fibroblast growth factor-2 (FGF-2) is mediated by shed vesicles In the present study, we address the question of how newly synthesized FGF-2 is targeted to the budding vesicles Considering that in vitro cultured Sk-Hep1 hepatocarcinoma cells release FGF-2 and shed membrane vesicles only when cultured in the pres-ence of serum, we added serum to starved cells and monitored intracellular movements of the growth factor FGF-2 was targeted both to the cell periphery and to the nucleus and nucleolus Movements toward the cell periphery were not influenced by drugs affecting microtubules, but were inhibited by cytocalasin B Involvement of actin in FGF-2 trafficking toward the cell periphery was supported by coimmunoprecipitation and immune localization experiments Colocalization of FGF-2 granules mov-ing to the cell periphery and FM4-64-labelled intracellular lipids were not observed Ouabain and methylamine, two inhibitors of FGF-2 release, were analyzed for their effects on FGF-2 intracellular localization and on vesicle shedding Ouabain inhibited FGF-2 movements toward the cell periphery The FGF-2 content of shed vesicles was therefore reduced Methylamine inhibited vesicle shedding; in its presence, FGF-2 clustered at the cell periphery, but the rate of its release decreased FGF-2 targeting to the nucleus and nucleolus was not affected by cytocalasin B, whereas it was inhibited by drugs that modify microtubule dynamics Neither ouabain, nor methylamine interfered with FGF-2 translocation to the nucleus and nucleolus FGF-2 targeting to the budding vesicles and to the nucleus and nucleolus is therefore mediated by fundamentally different mechanisms
Abbreviations
CM, conditioned media; FGF-2, fibroblast growth factor-2; FITC, fluorescein isothiocyanate; NLS, nuclear localization sequence;
rER, rough endoplasmic reticulum; uPA, urokinase type of plasminogen activator.
Trang 2extracellular medium The vesicle diameters are in the
range 100–1000 nm; the vesicle composition and
func-tion depend on the type of cells from which they have
been produced Vesicles are found to be involved in
cell motility and tumour progression mechanisms as
well as in bone formation [3] Involvement of shed
ves-icles in cell–cell and cell–matrix interactions is partially
mediated by the presence of several
membrane-associ-ated proteolytic enzymes [4–6] and signalling molecules
[7–9] However, shed vesicles also channel the secretion
of several leaderless proteins that apparently
accumu-late in their internal lumen For example, Cooper and
Barondes [10] reported that lectin 14, a signalling
pro-tein involved in muscle differentiation, is released as a
component of budding vesicles; MacKenzie et al [11]
and Bianco et al [12] reported that interleukin-b1 is
secreted as a component of shed vesicles in response to
ATP acting on P2X receptors
Pathways targeting specific molecules to the budding
vesicles have not been clarified In a previous study,
we investigated the mechanism of FGF-2 secretion and
reported that FGF-2 is released from Sk-Hep1 cells
and from NIH 3T3 cells transfected with FGF-2
cDNA through vesicle shedding [13] FGF-2, also
known as basic fibroblast growth factor, belongs to
the fibroblast growth factor superfamily FGFs are
structurally related heparin-binding growth factors that
exhibit almost ubiquitous involvement in vertebrate
embryonic and fetal development [14], as well as in
many physio-pathological processes occurring in adult
organisms [15] FGF-1 and FGF-2 are secreted from
the cell into the extracellular matrix, although they
lack the classical secretion sequence Unlike FGF-1,
which appears to be released in response to stress
con-ditions as a component of a multiprotein complex
[16,17], FGF-2 is secreted constitutively; however, its
release by in vitro cultured cells is inhibited by serum deprivation [18] FGF-2 transmits pro-angiogenic [19,20] and pro-lymphangiogenic [21] signals It is also involved in inducing smooth muscle and endothelial cell growth and in regulating early development stages
of various organs [22], including the brain [23,24] FGF-2 is also one of the most significant regulators of human embryonic stem cell self-renewal and of cancer tumourigenesis [25]
As a signalling protein, FGF-2 acts both directly at the nuclear level and after secretion Indeed, after its synthesis, FGF-2 can be secreted [1] or transferred to the nucleus and to the nucleolus where it behaves like
a transcriptional factor, inducing cell growth and rRNA synthesis [26]
Five FGF-2 isoforms (18, 22, 22.5, 24 and 34 kDa) have been identified in humans, all of which present some nuclear localization sequences However, although the 34 kDa isoform presents an arginine-rich repeat domain similar to the nuclear localization sequence (NLS) present in HIV Rev protein, this sequence is absent in isoforms with lower molecular weight; the 34,
24, 22.5 and 22 isoforms present several glycine-arginine repeats identified as an NLS and a nonclassical bipartite NLS in the C-terminus [27] The 18 kDa isoform only presents the bipartite NLS A portion of the bipartite NLS regulates localization of the factor in nucleoli [28]
As a secreted molecule, FGF-2 interacts both with matrix and membrane-bound proteoglycans and with five members of a family of high-affinity tyrosine kinase FGF receptors [29] Receptor-mediated signalling patterns appear to be utilized not only in paracrine, but also in autocrine mechanisms [30] According to Bossard et al [31], receptor-bound FGF-2 is endocytosed and can be transferred to the nucleus along with associated proteoglycans; nuclear
Fig 1 Targeting of endogenous FGF-2 to shed vesicles and nuclei (A) Colocalization of FGF-2 and b1 integrin in Sk-Hep1 cells cultured in 3D type I collagen gels Immunostaining of (a) FGF-2, (b) b1 integrin and (c) merging FGF-2 was detected using Texas red-conjugated sec-ondary antibodies; b1 integrin was detected using FITC-conjugated antibodies Arrows indicate shed vesicles Scale bar = 10 lm (B) Time-course of serum-induced FGF-2 intracellular movements observed by immunolocalization experiments FGF-2 immunolocalization at 0, 15,
30, 45, 60 min after serum addition (a–e) Top: sections 1 lm from the surface; thin arrows indicate FGF-2 granules Bottom: sections 3 lm from the surface; thick arrows indicate area of the nuclei in which large variations of FGF-2 concentration are observed FGF-2 was detected using Texas red-conjugated secondary antibodies Scale bar = 10 lm (C) Number of FGF-2 positive granules (diameters in the range 0.01–
1 lm) in immunostained sections 1 lm from the surface of cells fixed 0, 15, 30, 45 and 60 min after serum addition Granules were counted
by IMAGEJ software Asterisks indicate a significant difference between each time compared to the 30-min value (D) FGF-2 concentration in nuclei FGF-2 concentration (absorbance) was evaluated by IMAGEJ software in sections 3 lm from the surface of cells fixed 0, 15, 30, 45 and 60 min after serum addition Asterisks indicate a significant difference between each time compared to the 30-min value Values are mean ± SD of 15 measurements from five independent experiments in (B) to (D) (E) Induction of uPA activity in GM7373 cells by SK-Hep1 vesicles (a) Casein ⁄ plasminogen zymographies for detection of uPA activity, performed as described in the Experimental procedures on extracts of GM7373 cells that had been incubated in 5 mL of media for 16 h with 0, 25, 50 and 100 lg of SK-Hep1 vesicles (lanes 1–4) Asterisks indicate a significant difference between each amount compared to the previous one (b) Densitometric analysis of lysis bands due
to uPA activity Arbitrary units represent densitometric values of lysis bands, with 100 considered as the basal uPA activity of GM7373 untreated cells Values are the mean ± SD of 15 measurements from five independent experiments.
Trang 3accumulation of the endocytosed molecule requires
interaction with a protein called ‘translokin’ and
depends on the tubulin cytoskeleton
In the present study, we analyzed mechanisms
allow-ing translocation of FGF-2 from cytoplasmatic sites to
the budding vesicles and⁄ or to other cell districts
Foetal bovine serum was reported to induce both
vesicle shedding [6,32] and FGF-2 secretion [1,18] and,
in our previous studies, we observed that the two phe-nomena were clearly associated Approximately 30 min after the serum was added to starved cells, granules containing FGF-2 appeared in the proximity of the cell membrane and colocalization between FGF-2 and b1 integrin, a molecule known to be clustered in vesicle membranes [6], became evident Shortly after that, FGF-2 was detected in shed vesicles [13]
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Trang 4We therefore aimed to follow the FGF-2
transloca-tion pathway to the cell membrane by analyzing events
following serum addition To check whether
cytoskele-ton components were mediating FGF-2 trafficking, we
analyzed the effects of cytoskeleton perturbation on
FGF-2 intracellular movements To improve our
anal-ysis of FGF-2 intracellular movements and targeting,
we also tested the effects of known FGF-2 secretion
inhibitors on both FGF-2 intracellular movements and
vesicle shedding
Results
Localization of FGF-2 in SK-Hep1 cells and their
shed vesicles
Figure 1A shows vesicle shedding by SK-Hep1 cells
cultured in 3D collagen gels As previously reported
[13], when SK-Hep1 cells are grown in a complete
medium, FGF-2 is observed to be localized in cell
pro-trusions in association with b1 integrin, a molecule
that is also specifically clustered in shed vesicles [6] In
3D gels, shed vesicles remain trapped in the collagen
and can be observed using confocal microscopy Shed vesicles, testing positive for both FGF-2 and b1 inte-grin antigens, are visible in Fig 1A (arrows)
As previously reported [13], the shedding of FGF-2 positive vesicles occurs in cells showing no signs of apoptosis or necrosis Cell cultures used for the present experiments were kept in serum-free media for 72 h in most instances and then stimulated by serum addition However, the percentage of apoptotic⁄ necrotic cells was always negligible (data not shown)
Immunolocalization of FGF-2 in starved cells (i.e cells kept for 72 h in a serum-free medium) and in cells that, after starvation, were cultured in the presence of 10% fetal bovine serum for varying lengths of time, showed that serum addition resulted in FGF-2 positive granules, which were almost entirely absent in starved cells (Fig 1B), appearing at the cell periphery During the first 45 min of cell growth in the complete media, the number of granules localized at the cell periphery progressively increased; however, their number dropped (Fig 1B,C) after 1 h, indicating that FGF-2 had been released As previously reported [13], 1 h after adding serum, FGF-2 rich vesicles can be
Fig 2 Involvement of cytoskeleton elements in FGF-2 intracellular trafficking (A) Effects of nocodazol treatment on tubulin organization and
on serum induced FGF-2 movements (a, b) Immunolocalization of tubulin, respectively, in control cells (a) and in cells fixed 30 min after the addition of 10 l M nocodazol (b) (c, d) FGF-2 immunolocalization in control cells fixed 30 min after serum addition (c) and in cells fixed
30 min after serum and 10 l M nocodazol addition (d) Sections 2 lm from the surface Tubulin was detected using TRITC-labelled secondary antibodies; FGF-2 was detected using Texas red-conjugated secondary antibodies Thin arrows indicate FGF-2 granules; thick arrows indicate nucleoli Scale bar = 10 lm (B) Effects of cytocalasin B treatment on serum induced FGF-2 movements (a, b) Immunolocalization of actin respectively in control cells (a) and in cells fixed 30 min after the addition of 1 l M cytochalasin B (b) Sections 2 lm from the surface Thin arrows indicate FGF-2 granules; thick arrows indicate nucleoli Actin was labelled with FITC-phalloidin FGF-2 was detected using Texas red-conjugated secondary antibodies Scale bar = 10 lm (C) FGF-2 concentration in nuclei 30 min after serum addition, in control cells and in cells treated with drugs affecting the cytoskeleton organization FGF-2 concentration (absorbance) was evaluated by IMAGEJ software, as described in the Experimental procedures, in sections 3 lm from the surface, 30 min after serum (control) or serum and the following drugs had been added to starved cells: 10 l M nocodazol (Nocadazol), 1 l M paclitaxel (Paclitaxel) 1 l M colchicine (Colchicine) and 1 l M cytochala-sin B (Cytochalacytochala-sin) Asterisks indicate a significant difference between the treated cells and controls (D) Numbers of FGF-2 positive gran-ules near the cell surface 30 min after serum addition in control cells and in cells treated with drugs affecting the cytoskeleton organization FGF-2 positive granules (diameters in the range 0.01–1 lm) were counted in immunostained sections 1 lm from the surface by IMAGEJ soft-ware, as described in the Experimental procedures Control cells (Controls); cells treated with 10 l M nocodazol (Nocadazol); 1 l M paclitaxel (Paclitaxel); 1 l M colchicine (Colchicine); 1 l M Cytochalasin B (Cytochalasin) Drugs and serum were added together (E) Amount of vesicles recovered from conditioned media of control cells and of cells treated with drugs affecting the cytoskeleton organization Media were condi-tioned by 3–6 h of growth of 4 · 10 7
control cells grown in complete medium (Control) and by cells treated for the same time with complete medium to which the following drugs had been added: 10 l M nocodazole (Nocadazole); 1 l M paclitaxel (Paclitaxel); 1 l M colchicine (Colchi-cine) or 1 l M cytochalasin B (Cytochalasin) Vesicle amount was evaluated by Bradford microassay method in at least three different experi-ments (F) Induction of uPA activity in GM7373 cells by vesicles shed by controls and by cells treated with drugs affecting the cytoskeleton organization Casein ⁄ plasminogen zymographies for detection of uPA activity were performed as described in the Experimental procedures
on GM7373 cells that had been incubated for 16 h with vesicles shed by control cells and by cells treated with drugs affecting the cytoskel-eton organization Vesicles had been obtained from ml of conditioned media uPA activity of GM7373 cells incubated without vesicles (Med-ium), with SK-Hep1 vesicles shed in media conditioned by 3–6 h of growth of 4 · 10 7 nontreated cells grown in complete medium (Control) and by cells treated for the same length of time with complete medium to which the following drugs had been added: 10 l M nocodazol (Nocadazol); 1 l M paclitaxel (Paclitaxel); 1 l M colchicine (Colchicine) and 1 l M cytochalasin B (Cytochalasin) With respect to cytochalasin B,
a comparison between the specific stimulatory activity of control vesicles and of vesicles shed by treated cells is also shown (Cytochalasin
sp ac.) Arbitrary units represent densitometric values of lysis bands with 100 considered as the basal uPA activity of GM7373 cells Values are the mean ± SD of 15 measurements from five independent experiments.
Trang 5recovered from cell-conditioned media (CM), whereas
FGF-2 is still undetected in vesicle-free CM Therefore,
we suggest that, after 1 h of cell culture in the
com-plete medium, FGF-2 positive granules are released as
vesicle components
Immunolocalization studies also showed that, when
cells were kept in the presence of serum for 30 min or
more, the average concentration of FGF-2 also
increased in the nucleus; moreover, the factor
accumu-lated in specific areas of the cell nucleus that were
FGF-2 negative (Fig 1B,D) in starved cells Because
FGF-2 is known to stimulate rRNA synthesis [26,27],
these nuclear areas are considered to correspond to
nucleoli
FGF-2 can induce expression of the urokinase type
of plasminogen activator (uPA) in endothelial cells
[33,34] We therefore performed assays of
vesicle-asso-ciated FGF-2 by testing the ability of vesicles to
induce uPA production in GM7373 endothelial cells
We analyzed induction by incubating GM7373 cells with vesicles for 16 h; uPA activity was then measured
by casein⁄ plasminogen zymography of endothelial cell extracts Induction was dose-dependent (Fig 1E); moreover, we already knew that the stimulatory effect
of SK-Hep1 vesicles on uPA production by GM7373 cells was completely abolished by anti-FGF-2 serum [13] We therefore considered the specific stimulatory effect of vesicles on uPA production as an indirect method to evaluate their FGF-2 content
Involvement of cytoskeleton in FGF-2 intracellular trafficking
To analyze the involvement of cytoskeleton elements in targeting endogenous FGF-2 to the cell nucleus and to the cell periphery, Sk-Hep1 cells were treated with drugs
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Control Nocodazol Paclitaxel Colchicine Cytochalasin
Trang 6affecting tubulin or actin organization and FGF-2
im-munolocalization was analyzed by confocal microscopy
The molecules tested were nocodazol and colchicine,
which cause microtubule disassembly; paclitaxel, an
inhibitor of tubulin depolymerization; and
cytochala-sin B, a drug caucytochala-sing actin depolymerization
The serum-induced increase of FGF-2 concentration
in the nucleus was less marked in cells treated with the
three drugs affecting microtubular organization These
drugs also had clear inhibitory effects on FGF-2
locali-zation in nuclear areas apparently corresponding to
nucleoli (Fig 2A, panel d, thick arrows) On the other
hand, FGF-2 export toward the cell membrane was
not modified by treatment with drugs affecting
micro-tubular organization; in fact, granules at the cell
periphery were also clearly visible in treated cells
(Fig 2A, panel d, thin arrows)
The effects of cytochalasin B treatment were the
reverse As shown in Fig 2B, FGF-2
immunolocaliza-tion experiments demonstrated that actin
depolymer-ization did not modify nuclear localdepolymer-ization of FGF-2
(Fig 2B, panel d, thick arrows); however,
cytochala-sin B treatment blocked FGF-2 movements toward the
cell membrane
To obtain a more quantitative picture detailing the
effects of drug treatments on cytoskeleton organization
and on FGF-2 movements toward the nucleus and the
cell periphery, the optical density in cell nuclei and the
number of FGF-2 granules at the cell periphery were
measured As shown in Fig 2C, nocodazol, colchicine
and paclitaxel had a significant inhibitory effect on
the increased concentration of FGF-2 induced by the
serum in the nucleus, whereas cytochalasin B had no
effect
By contrast, as shown in Fig 2D, the number of
FGF-2 positive granules observed at the cell periphery
30 min after the serum was added, was not modified
by treatment with drugs affecting microtubule
organi-zation, whereas it strongly decreased in cytochalasin B
treated cells
In brief, and in accordance with the results
pre-viously reported by Bossard et al [31] concerning
nuclear and nucleolar localization of exogenous
FGF-2, our data suggest that microtubule integrity is
needed for nuclear localization of endogenous FGF-2
Actin filament integrity was not shown to be required
for targeting the factor to the cell nucleus By contrast,
FGF-2 targeting to the cell periphery was not
influenced by treatment with drugs affecting tubulin
organization, whereas it was blocked by cytocalasin b
treatment
We also tested the effects of drugs that influence the
cytoskeleton on cell attitude to shed membrane vesicles
and on vesicle capability to induce uPA production by GM7373 cells None of the three drugs affecting microtubule organization had significant effects on the amount of vesicles shed by treated cells (Fig 2E), nor
on their stimulation of uPA production (Fig 2F) There is therefore no evidence of microtubule involve-ment in the FGF-2 release mechanism By contrast, the amount of vesicles shed by cytochalasin B-treated cells (Fig 2E) was greatly reduced Moreover, not only the total, but also the specific stimulatory activity of vesicles shed by cytochalasin B treated cells on uPA production decreased (Fig 2F)
Therefore, microfilament organization appears to be required for both FGF-2 targeting to the budding vesi-cles and for vesicle shedding
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Fig 3 Immunolocalization of FGF-2 in comparison with actin fila-ments and lipidic structures (A) Double staining for FGF-2 and actin, 30 min after serum addition FGF-2 was detected using Texas red-conjugated secondary antibodies Actin was labelled with (a) phalloidin-labelled actin; (b) monoclonal antibody-labelled FGF-2; and (c) merging Arrow indicates FGF-2 granules on actin filaments Scale bar = 5 lm Sections 1 lm from the surface (B) Immunopre-cipitation with anti-FGF-2 serum Immunoprecipitate was immuno-stained with either monoclonal antibodies against actin or monoclonal antibodies against FGF-2 Lane 1, immunoprecipitation from cells cultured in complete medium; lane 2, immunoprecipita-tion from starved cells (C) Double staining for FGF-2 and lipids FGF-2 was detected using FITC-conjugated secondary antibodies (green fluorescence); lipids were stained with FM4-64 (red fluores-cence) (a) Cells in serum-free media (b) Cells 30 min after serum addition Sections 1 lm from the surface Scale bar = 10 lm.
Trang 7Further proof of the interaction between FGF-2
granules and the actin cytoskeleton was obtained by
double fluorescence experiments Actin filaments were
labelled with phalloidin and FGF-2 with antibodies
As shown in Fig 3A, 30 min after serum addition,
FGF-2 positive granules were seen to localize on actin
filaments (Fig 3A, arrow)
Actin involvement in FGF-2 release was confirmed
by immunoprecipitation experiments with anti-FGF-2
serum However, as shown in Fig 3B, actin was found
to be present only in immunoprecipitates obtained
from cells cultured in complete medium, and was
absent in immunoprecipitates obtained from starved
cells, thus indicating that FGF-2⁄ actin association occurs when serum induces FGF-2 intracellular traf-ficking toward the cell periphery
Analysis of the intracellular localization of FGF-2 and of lipidic structures
To verify whether FGF-2 intracellular granules are included in lipidic vesicles and whether they are targeted
to endolysosomal districts before being secreted, we performed double fluorescence experiments labelling lipids with FM4-64 (a red fluorescent molecule) and FGF-2 with monoclonal antibodies recognized by fluorescein isothiocyanate (FITC)-conjugated second-ary antibodies After being endocytosed (15 min of cell culture in the presence of the dye), FM4-64 stains intracellular lipidic structures [35] As shown in Fig 3C, FGF-2 granules do not colocalize with FM4-64-labelled intracellular lipidic vesicles in the absence (Fig 3C, panel a) or in the presence of serum (Fig 3C, panel b) Therefore, FGF-2 granules appear to move
on actin filaments without being enclosed in vesicles
α sub Na-K-ATPasi actin
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Fig 4 Effects of ouabain on FGF-2 intracellular trafficking (A)
Im-munolocalization of FGF-2 in controls and in ouabain treated cells.
(a) Cells fixed in serum-free media; (b) cells fixed 30 min after
serum addition; (c) cells fixed 30 min after addition of serum and
100 l M ouabain Sections 1 lm from the surface FGF-2 was
detected using Texas red-conjugated secondary antibodies Scale
bar = 10 lm (B) FGF-2 concentration (absorbance) in nuclei,
30 min after serum addition, in control cells and in cells treated
with ouabain FGF-2 concentration (absorbance) was evaluated by
IMAGEJ software in sections 3 lm from the surface, 30 min after
serum (Control) or serum and 100 l M ouabain (Ouabain) addition.
(C) Numbers of FGF-2 granules at the cell surface 30 min after
serum addition, in controls and in ouabain treated cells FGF-2
posi-tive granules (diameters in the range 0.01–1 lm) were counted in
immunostained sections 1 lm from the surface by IMAGEJ software,
as described in the Experimental procedures Controls, control cells
to which only serum was added; Ouabain, cells to which serum
and 100 l M ouabain were added Asterisks indicate a significant
dif-ference between ouabain treated cells and controls (D) Amount of
vesicle recovered by control and ouabain treated cells and their
stimulatory effect on uPA expression by endothelial cells (1)
Amount of vesicles recovered from complete medium conditioned
by 3 h of growth of 4 · 10 7 control cells (Control), and by 3 h of
growth of 4 · 10 7 cells in complete medium to which 100 l M
oua-bain had been added (Ouaoua-bain) (2) Induction of uPA activity in
GM7373 cells Casein ⁄ plasminogen zymographies for detection of
uPA activity performed as described in the Experimental
proce-dures on GM7373 cells that had been incubated for 16 h with
vesi-cles obtained from 5 mL of media conditioned by 3 h of growth of
4 · 10 7 control cells grown in complete medium (Control) and by
3 h of growth of 4 · 10 7 cells in complete medium to which
100 l M ouabain had been added (Ouabain) Arbitrary units
repre-sent densitometric values of uPA activity, where 100 is considered
as the basal uPA activity of GM7373 cells Asterisks indicate a
sig-nificant difference between vesicles shed by Ouabain treated cells
and controls (E) Detection of Na + ⁄ K + -ATPase in shed vesicles.
Western blot analysis of Na + ⁄ K + -ATPase in vesicles shed by
untreated (Control) and 100 l M ouabain treated cells Lane 1,
vesi-cles shed by untreated cells (30 lg of protein); lane 2, vesivesi-cles
shed by ouabain treated cells (30 lg of protein) Values are the
mean ± SD of 15 measurements from five independent
experi-ments.
Trang 8This result contradicts the hypothesis that shed vesicles,
such as exosomes [36], originate from
multivesi-cular bodies
Effects of drugs inhibiting FGF-2 release
In another group of experiments, we tested two
well-known inhibitors of FGF-2 secretion, ouabain and
methylamine, for their effects on intracellular FGF-2
trafficking and on vesicle shedding
Effects of ouabain
As shown in Fig 4A, FGF-2 immunolocalization
experiments demonstrated that ouabain interferes with
FGF-2 intracellular trafficking toward the cell
peri-phery whereas it does not interfere with trafficking
toward the nucleus (Fig 4A,B) FGF-2 positive
gran-ules, observed at the cell periphery 30 min after serum
addition, were virtually absent in cells treated with
both ouabain and serum (Fig 4A,C)
Ouabain treatment did not modify the amount of shed vesicles recovered from CM (Fig 4D, bars marked 1); however, when the stimulatory effect of vesicles on uPA production by GM7373 cells was tested, we observed that vesicles shed by ouabain-trea-ted cells had a decreased specific activity on uPA induction (Fig 4D, bars marked 2) Therefore, oua-bain interferes with FGF-2 intracellular trafficking toward the cell periphery
As ouabain is known to bind to a subunit of
Na+⁄ K+-ATPase [37,38], we looked for the presence
of this protein in vesicles As shown in Fig 4E, a molecule of the expected apparent mass (100 kDa) was marked by a monoclonal antibody against the a ATP-ase subunit The concentration of the recognized anti-gen decreased in vesicles shed by ouabain-treated cells
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Fig 5 Effects of methylamine on FGF-2 intracellular trafficking (A) Immunolocalization of FGF-2 in controls and methylamine treated cells (a) Cells fixed in serum-free media; (b) cells fixed 30 min after serum addition; (c) cells fixed 30 min after serum and 10 m M
methylamine addition Sections 1 lm from the surface FGF-2 was detected using Texas red-conjugated secondary antibodies Scale bar = 10 lm (B) FGF-2 concentration (absorbance) in nuclei,
30 min after serum or serum and methylamine addition FGF-2 con-centration (absorbance) was evaluated by IMAGEJ software in sec-tions 1 lm from the surface, 30 min after serum (Control) or serum and 10 m M methylamine (Methylamine) addition (C) FGF-2 concen-tration (absorbance) at the cell periphery, 30 min after serum and methylamine addition FGF-2 concentration (absorbance) was evalu-ated by IMAGEJ software in sections 1 lm from the surface, 30 min after serum (Control) or serum and methylamine addition (10 squares of length 2 lm were analyzed in each field) (D) Amount of recovered vesicles and uPA activity induction (1) Amount of vesi-cles recovered from medium conditioned by 4 · 10 7
cells grown for 3 h in complete medium and by cells treated for 3 h with com-plete medium and 10 m M methylamine (2) uPA induction by 20 lg
of vesicles shed by cells grown in complete medium and by cells treated for 3 h with complete medium and 10 m M methylamine (3) uPA induction by vesicles obtained from 5 mL of media conditioned
by 4 · 10 7 cells grown in complete medium and by cells treated for 3 h with complete medium and 10 m M methylamine (4) uPA induction by 5 mL of vesicle-free media conditioned by
4 · 10 7 cells grown in complete medium and by cells treated for
3 h with complete medium and 10 m M methylamine Control, untreated cells; Methylamine, cells treated for 3 h with 10 m M
methylamine Casein ⁄ plasminogen zymographies for detection of uPA activity were performed as described in the Experimental pro-cedures on GM7373 cells that had been incubated for 16 h with vesicles obtained from media conditioned by 3 h of growth of
4 · 10 7 control cells grown in complete medium and by cells trea-ted for 3 h with complete medium and 10 m M methylamine Arbi-trary units represent densitometric values of uPA activity, where
100 is considered as the basal uPA activity of GM7373 cells Val-ues are the mean ± SD of 15 measurements each of 10 squares of length 2 lm from three independent experiments.
Trang 9Effects of methylamine
Treatment with methylamine did not affect FGF-2
movements toward the cell nucleus (Fig 5A,B) As a
consequence of methylamine treatment, FGF-2
positive granules were observed to accumulate at the
cell periphery and to coalesce with the plasma
mem-brane, which became intensely positive for FGF-2
immunoreactive material (Fig 5A) In
methylamine-treated cells, FGF-2 positive granules were not easily
counted; therefore, the main immunofluorescence at
the cell periphery was evaluated Namely, in each
microscopic field, we selected 10 squares of length
2 lm at the cell periphery and analyzed them for
absorbance Fluorescence at the cell periphery was not
significantly modified by methylamine treatment
(Fig 5C) However, the amount of released vesicles
diminished (Fig 5D, bars marked 1) The specific
stim-ulatory effect of vesicles on uPA production by
GM7373 cells was not modified (Fig 5D, bars
marked 2); however, we observed a decrease in uPA
induction caused by adding the total amount of
vesi-cles recovered from a fixed volume of CM (Fig 5D,
bars marked 3) to GM7373 cells A proportional
decrease was also observed in uPA induction caused
by adding the same volume of vesicle-free CM
(Fig 5D, bars marked 4) to the cells Therefore, we
can conclude that the inhibitory effect of methylamine
on FGF-2 secretion is due to this molecule’s ability to
inhibit vesicle shedding
Discussion
We previously reported that the release of FGF-2, a
pro-angiogenic growth factor that lacks the signalling
sequence typical of most secreted proteins, is mediated
by vesicle-shedding Neither FGF-2 release, nor vesicle
shedding occurred when cells were kept in serum-free
media Thirty minutes after serum addition, FGF-2
granules were seen at the cell periphery and, after 1 h,
FGF-2 rich vesicles were recovered from CM; FGF-2
was detected in vesicle-free CM only 2 h later [13]
The present study aimed to analyze intracellular
trafficking of endogenous FGF-2, targeting the
mole-cule to the budding vesicles and⁄ or to different cell
districts Our strategy was to follow the changes in
FGF-2 intracellular localization shortly after adding
serum, both in controls and in cells treated with drugs
affecting cytoskeleton organization or FGF-2 release
To make a comparison of the FGF-2 content of
vesi-cles shed by control and treated cells, we performed
analyses of uPA activity in extracts of GM7373
endo-thelial cells treated with vesicles Induction of uPA
activity in endothelial cells is a typical response elicited
by FGF-2 [33,34], and we previously reported that the stimulatory effect of SK-Hep1 vesicles on uPA produc-tion by GM7373 cells was dose-dependent and fully neutralized by monoclonal anti-FGF-2 serum [13] Assays of cell vitality were performed in control and treated cells and data were collected using cell cultures
in which the number of apoptotic or necrotic cells, evaluated by acridine orange and trypan blue staining, was found to be negligible (i.e less than 3%)
When using untreated cells, we observed that some FGF-2 positive granules were detected at the cell periphery as early as 15 min after serum addition; their number then grew for approximately 45 min However, after 1 h of culturing in complete medium, the number
of FGF-2 granules abruptly dropped The decrease in the number of FGF-2 granules at the cell periphery was coupled with the appearance of FGF-2 rich vesi-cles in CM Therefore, the results were in agreement with the previously described [13] vesicle-mediated mechanism of FGF-2 release
FGF-2 is a molecule with autocrine, paracrine and intracrine signalling mechanisms With respect to intra-crine mechanisms, the factor has to be localized in the nucleus A detectable amount of FGF-2 was also found to be present in the nucleus of starved cells; however, when serum was added, the nuclear concen-tration of the molecule increased considerably We also noticed that, in starved cells, FGF-2 was undetectable
in specific areas of the nucleus apparently correspond-ing to nucleoli After serum addition, these areas of the cell nucleus became highly positive for FGF-2 immunostaining
To analyze the involvement of cytoskeletal elements
in FGF-2 targeting, we tested drugs that alter either tubulin or actin dynamism Treatment with drugs affecting the organization of microtubules (nocodazol, colchicine and paclitaxel) did not interfere with FGF-2 granule trafficking toward the cell membrane, nor with the amount of vesicles shed into the extracellular med-ium No differences were observed in the stimulatory effects of vesicles shed by control and treated cells on the uPA activity of endothelial cells Therefore, the results of these experiments show that FGF-2 export toward the cell periphery is mediated by mechanisms that do not require microtubule organization
By contrast, treatment with drugs that affect micro-tubule dynamics neither allowed for the serum-induced increase of FGF-2 concentration in the cell nucleus, nor the specific localization of FGF-2 in nucleoli Therefore, nuclear and nucleolar localization of endog-enous FGF-2 appears to follow a pathway requiring maintenance of the microtubule organization As
Trang 10reported by Bossard et al [31], tubulin organization
is also needed for nuclear targeting of endocytosed
FGF-2
Treatment with cytochalasin B showed that
endo-genous FGF-2 export toward the cell periphery
requires the integrity of the actin cytoskeleton These
treatments decreased FGF-2 transportation to the cell
periphery and to the budding vesicles Consequently,
the specific stimulatory activity of vesicles on GM7373
uPA activity decreased Interaction between FGF-2
and actin filaments was also demonstrated by
coimmu-noprecipitation and coimmunolocalization of the two
molecules Moreover coimmunoprecipitation
experi-ments showed that interactions between FGF-2 and
actin were induced by serum addition Cytochalasin B
considerably decreased the rate of vesicle shedding On
the other hand, cytochalasin B treatment did not affect
FGF-2 movements toward the nucleus and nucleolus
Therefore, the results indicate that FGF-2 movements
toward the cell periphery or the nucleus utilize totally
different mechanisms
We also determined whether FGF-2 positive
gran-ules, observed at the cell periphery, were enclosed in
lipidic vesicles However, colocalization experiments
performed using the lipidic colorant FM4-64 and
anti-FGF-2 serum showed that anti-FGF-2 granules were not
associated with lipidic structures These results indicate
a secretion mechanism that, unlike the production of
exosomes [36], does not involve multivescicular bodies
In a different group of experiments, we analyzed
FGF-2 intracellular localization, the amount of shed
vesicles and the stimulatory effect of vesicles on uPA
production, using SK-Hep1 cells treated with drugs
known to inhibit FGF-2 secretion The drugs tested
were ouabain and methylamine
Ouabain is a well-known inhibitor of Na+⁄ K+
-ATPase which binds to the a subunit of Na+⁄ K+
-ATPase However, the molecule was also reported to
inhibit FGF-2 secretion [37] and FGF-2 release was
reported to be ouabain-insensitive in cells expressing
an ouabain resistant ATPase a subunit A possible
explanation of the inhibitory effect of ouabain on
FGF-2 secretion may be that the electrochemical
gra-dient created by the Na+⁄ K+-ATPase is a prerequisite
for intracellular FGF-2 movements toward the cell
periphery or for its secretion However, FGF-2 and
ATPase a subunit were shown to
coimmunoprecipi-tate, and it was therefore suggested that, to allow
FGF-2 secretion, FGF-2 and ATPase a subunit had to
interact [38] The reasons why this interaction was
needed were not clarified Our results show that
oua-bain treatment blocks intracellular movements of
FGF-2 positive granules In treated cells, FGF-2
gran-ules were not observed to accumulate at the cell periphery The drug did not modify the rate of vesicle shedding; nevertheless, vesicles shed by ouabain-treated cells had a decreased specific stimulatory effect on GM7373 cells, indicating a decreased FGF-2 content The results obtained by treating cells with ouabain are in agreement with the vesicle-mediated mechanism
of FGF-2 secretion that we described; they show that ouabain-mediated inhibition of FGF-2 secretion is associated with a decrease in FGF-2 targeting to budding vesicles However, the mechanisms by which ouabain affects FGF-2 movements toward the cell periphery require further analysis
A hypothetical explanation of the drug effect is that direct interaction of the ATPase a subunit with FGF-2, inhibited by ouabain, is needed for the two molecules to
be transferred to the budding vesicles This hypothesis
is validated by our observation that ouabain treatment caused a decrease in the vesicle concentration of both FGF-2 and of the ATPase a subunit However, it is dif-ficult to explain how the two molecules come across and are allowed to run into each other We hypothesized that interaction could occur at the level of multivesicu-lar bodies or in other membrane-bound intracellumultivesicu-lar districts However, this hypothesis was contradicted by the results of lipidic staining, which failed to show any colocalization between intracellular FGF-2 granules and lipidic structures The site of interaction between the ATPase a subunit and FGF-2 therefore remains unknown To explain how such interaction could occur
in the cell cytoplasm, we are led to suspect the existence
of a splice variant of the ATPase a subunit lacking the N-terminal signalling sequence that drives the complete protein to the rER For the Na+⁄ K+-ATPase a sub-unit, such a variant is not known; however, it was described for an ouabain-sensitive H+⁄ K+-ATPase a2 subunit It was suggested that the alternative splicing of this molecule dictates isoform-specific differences in membrane targeting or cytoskeletal association [39] Different explanations of the effects of ouabain are also possible For example, FGF-2 trafficking to the budding vesicles might be inhibited by ouabain in response to one of the several signal transduction path-ways modulated by the alkaloid interaction with the
Na+⁄ K+-ATPase a subunits [40]
The results obtained after methylamine treatment clearly support the prominent role of shed vesicles in FGF-2 secretion Methylamine has been described to inhibit both FGF-2 release [1] and vesicle shedding [41] The results contained herein indicate that the methylamine inhibitory effect on FGF-2 release was due to its action on vesicle shedding FGF-2 granules coalesced with the cell membrane but were not