1 Department of Pediatrics, Division of Clinical Chemistry and Biochemistry,
University of Zurich, Steinwiesstr. 75, CH-8032 Zurich, Switzerland
2 Department of Biochemistry, 30, quai Ernest Ansermet, CH-1211 Genève 4,
Switzerland
* Author for correspondence (e-mail: Claus.Heizmann{at}kispi.unizh.ch )
Accepted 13 May 2002
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Summary |
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Key words: S100A13, S100A6, Protein translocation, Calcium, Angiotensin II, ER-Golgi, Actin filament
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Introduction |
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Among the 20 members reported in the S100 calcium-binding protein family,
S100A13 is the only member that exhibits a ubiquitous expression in a broad
range of tissues (Ridinger et al.,
2000). S100A13 is one of the latest member of the S100 family and
was originally identified by screening the EST database
(Wicki et al., 1996
). It is
similar to most other S100 proteins homodimeric S100A13 possesses two
high- and two low-affinity sites for calcium
(Ridinger et al., 2000
).
However, it does not show a calcium-dependent exposure of the hydrophobic
surface, which is essential for the interaction of S100 proteins with their
target proteins. S100A13 is associated with the secretion of brain-derived
fibroblast growth factor-1 (FGF-1) and p40-synaptotagmin in response to heat
shock (Jackson et al., 1992
;
Landriscina et al., 2001
). In
addition, the anti-allergic and anti-inflammatory drug amlexanox which binds
to S100A12 and S100A13, represses the secretion of FGF-1, S100A13 and
p40-synaptotagmin 1 multi-aggregates
(Carreira et al., 1998
;
Shishibori et al., 1999
).
Therefore, S100A13 was postulated to play an important role in the release of
FGF-1, as the growth factor lacks a classical signal sequence for
secretion.
Calcium plays an important role as a second messenger in various signaling
pathways. A sudden increase in the intracellular calcium level alters
physiological cellular functions such as cell cycle progression,
differentiation and muscle contraction
(Schäfer and Heizmann,
1996). One of the most commonly used factors to increase the
intracellular calcium levels is angiotensin II. Angiotensin II is a potent
smooth muscle constrictor, which increases cytosolic calcium levels by
interacting with angiotensin receptors
(Schiffrin, 1998
;
Watts et al., 1998
;
Purdy and Arendshorst, 1999
;
Takeuchi, 1999
;
Rossier and Capponi, 2000
).
Through a GTP-binding protein, angiotensin II activates phospholipase C, which
generates inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and
releases calcium from intracellular stores
(Marks, 1992
;
Capponi, 1996
).
Translocation involves protein relocation to different intracellular
compartments, and it has been implicated in association with signal
transduction cascades. Protein translocation in response to intracellular
calcium change has long been observed with protein kinase C (PKC)
(Damron et al., 1998;
Oancea and Meyer, 1998
;
Marsigliante et al., 2001
) and
recently with some S100 proteins (Goebeler
et al., 1995
; Guignard et al.,
1996
; van den Bos et al.,
1996
; Brett et al.,
2001
). S100A8 and S100A9 show calcium-dependent translocation in
myelomonocytic cells and in epithelial cells
(Goebeler et al., 1995
;
van den Bos et al., 1996
).
S100A2, S100A4 and S100A6 translocate in response to ionophore A23187, cyclic
ADP-ribose and thapsigargin in some tumor cells
(Mueller et al., 1999
), and
translocation of S100A11 and S100B has been described in human glioblastoma
cell lines (Davey et al., 2000
;
Davey et al., 2001
). Since S100
proteins lack the classical signal sequence for secretion, it is of great
interest to investigate the translocation pathways of this group of proteins.
Novel pathways such as tubulin-filament-dependent translocation have been
reported in secretion and translocation of S100A8, S100A9 and S100A11
(Rammes et al., 1997
;
Davey et al., 2000
).
In an attempt to study the cellular function of S100A13, we investigated translocation in response to intracellular calcium rise by stimulating ECV (a human umbilical vein-derived endothelial cell line) with angiotensin II and immunohistochemical staining. The translocation pathways of S100A13 and S100A6 were studied by using various inhibitors to block the distinct pathways. We report here a classic translocation pathway for S100A13 in contrast to the novel pathways utilized by other members of the S100 family. By contrast, translocation of S100A6 in response to angiotensin-II-stimulated calcium rise seems to be an actin-filament-dependent process in endothelial cells, suggesting that two S100 proteins utilize different translocation pathways in the same cellular environment.
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Materials and Methods |
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Calcium and magnesium binding
Calcium binding was measured at 25°C by the flow dialysis method in
buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl). Protein concentrations were
20-30 µM. Treatment of the raw data and evaluation of the intrinsic
metal-binding constants was performed as described previously
(Cox, 1996). The data were
analyzed with the equation of Adair for four binding sites:
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SDS PAGE and western blotting
SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and
western blotting were used throughout the purification of recombinant human
S100A13 to monitor the presence of the protein in certain fractions. Either a
pre-cast 4-20% Tris-glycine gradient (BioWhittaker Molecular Applications,
Rockland, USA) or a 14% Tris SDS PAGE gel was used for both Coomassie G-250
(Pierco, Illinois, USA) staining and western blotting onto 0.45 µm
nitrocellulose membrane (BIO-RAD, Hercules, USA). ECV cell extract was
prepared by resuspending 5x106 cells in lysis buffer
containing 50 mM Tris, 250 mM NaCl, 2 mM EDTA and 1% nonidet P40 at pH 8.0.
Protein denaturation was carried out in loading buffer (16% SDS, 48% glycerin,
0.2 M Tris, pH 6.8, 2% ß-mercaptoethanol and 0.01% bromophenol blue) at
95°C for 10 minutes. Primary antibodies against the human recombinant
proteins (rabbit anti-S100A1, anti-S100A3, anti-S100A4, anti-S100A5,
anti-S100A6, anti-S100A13 and anti-S100B) used for western blotting were
diluted 1:1,000; and horseradish-peroxidase-conjugated goat anti-rabbit
secondary antibody (Sigma, Steinheim, Germany) was used at a dilution of
1:10,000. The specificity and the crossreactivity of the antibodies were
described previously (Ilg et al.,
1996; Ridinger et al.,
2000
; Schafer et al.,
2000
). Visualization of the protein was achieved by ECL (enhanced
chemiluminescence) (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Cell culture
ECV, a kind gift from T. Maciag (ECV-304), was maintained in 10% FCS-M199
medium with antibiotics (Invitrogen, Basel, Switzerland) at 37°C and 5%
CO2. For immunofluorescence stainings, 5x104 cells
were plated on the fibronectin-coated coverslips (2 µg/ml) (Sigma,
Steinheim, Germany) one day prior to the experiment in a 24-well plate.
Addition of various drugs was performed in M199 medium without FCS (fetal calf
serum), the working concentrations and incubation times used are as follows:
45 minutes with 5 µg/ml brefeldin A (Calbiochem), 120 minutes with 1 µM
amlexanox (Takeda Chemical Industries Ltd., Japan), 30 minutes with 1 µM
demecolcine (Sigma, Steinheim, Germany), 180 minutes with 1 mM bafilomycin A1
(Sigma, Steinheim, Germany). Cells were then stimulated by 0.1 µM
angiotensin II (Sigma, Steinheim, Germany) in stimulation buffer containing
140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM glucose, 1 mM HEPES, and 1
mM CaCl2 for 10 minutes before fixation.
Immunofluorescence staining
Cells were fixed after angiotensin II stimulation with 3.7% formaldehyde in
M199 medium for 15 minutes at 37°C. Permeabilization was carried out using
cold methanol at room temperature for 10 minutes, and cells were washed with
5% horse serum M199. The primary antibodies [rabbit anti-S100 antibodies and
mouse anti-tubulin antibody (Sigma, Steinheim, Germany)] were diluted 1:200
and incubated with the cells at 37°C for 1 hour. Mouse monoclonal anti
ß-COP antibody (Sigma, Steinheim, Germany) was used at a dilution of
1:80. Various S100 antibodies were pre-absorbed by mixing with 10 µg
antigen overnight at 4°C followed by centrifugation at 16,060 g.
The supernatant was then used instead of the primary antibody as a negative
control. Cells were washed twice with 5% horse serum M199 medium and incubated
with goat Cy3-conjugated anti-rabbit IgG or goat Cy3-conjugated anti-mouse IgG
secondary antibody (1:200) (Jackson Immuno-Research Laboratories, Inc.) for 1
hour at 37°C. For double staining, a goat anti-human S100A6 antibody and a
rabbit anti-human S100A13, antibody were used. Phalloidin-TRITC (Sigma,
Steinheim, Germany) was used to locate actin stress fibers in the cells, and
the standard staining protocol provided by the manufacturer was followed using
labeled phalloidin at 0.5 µM and incubation for 1 hour. To visualize the
protein distribution, a Cy3-conjugated anti-goat IgG secondary antibody
(1:200) and a Cy2-conjugated anti-rabbit IgG secondary antibody (1:200) were
used in the double staining experiment. After incubation with the secondary
antibody, the cells were again washed twice in 5% horse serum M199 medium
followed by a PBS (pH 9.0) (phosphate-buffered saline) wash. Slide mounting
was performed using Mowiol as mounting medium (Hoechst, Frankfurt, Germany),
and the slides were stored at 4°C in the dark.
Microscopy and confocal laser-scanning microscopy
Immunostained cells were analyzed by a Zeiss Axioskop microscope using a
40x oil objective lens. Images were taken by an AxioCam and processed in
Axio Vision 2.05 and Adobe Photoshop 5.5. For confocal microscopy, a Zeiss
Axioplan fluorescence microscope equipped with a confocal scanning unit
MRC-600 (Biorad) was used. The slides were visualized using a 40x oil
objective lens and an argon krypton laser with an excitation wavelength of 568
nm for Cy3 labeling. The images were enlarged by 2.5x and processed
using Adobe Photoshop.
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Results |
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Metal-binding studies of S100A13
Calcium binding to S100A13 was monitored by flow dialysis in the absence
and presence of 100 µM zinc (Fig.
2, Table 1). In the
absence of zinc, the isotherm strongly resembles the one reported previously
(Ridinger et al., 2000), with
two high- and two low-affinity sites ([Ca2+]0.5 of 10
µM and 500 µM, respectively) per dimer and positive cooperativity within
each group of sites (nH of 1.4 to 1.5). In the presence of
zinc, the calcium-binding parameters of the high-affinity set were not
changed, but a global 1.8-fold decrease in [Ca2+]0.5 was
observed for the low-affinity sites. Close inspection reveals that zinc
decreased merely the value of K'4 and thus disturbed the
positive cooperativity in the set of low calcium-affinity sites. With the
competition equation, [Zn2+]0.5 of 150 µM was
calculated for this pair of sites, although the zinc-calcium antagonism is a
priori not caused by direct competition
(Heizmann and Cox, 1998
). A
direct equilibrium gel filtration experiment at room temperature in buffer A
containing 50 µM free zinc yielded 0.6 protein-bound zinc per dimer, thus
confirming the distinct but weak interaction of zince with this protein. The
binding of zinc induces only small conformational changes in the environment
of Trp77, as shown by fluorescence and difference spectrophotometry
(Ridinger et al., 2000
).
Metal-free S100A13, which has a molten globule characteristic, strongly
enhances the fluorescence of the hydrophobic probe TNS
(2-p-toluidinylnaphthalene-6-sulfonate)
(Ridinger et al., 2000
), and
calcium binding to S100A13 strongly reduces the interaction and fluorescence
of the hydrophobic probe TNS. However, addition of 0.15 mM zinc to the
apoprotein has no effect at all, suggesting that hydrophobic properties are
not influenced by zinc binding. We also measured the calcium-binding profiles
in the presence of a five-fold excess (125 µM) of the anti-allergic drug
amlexanox over S100A13. In these flow dialysis experiments the perfusion
buffer contained also 1 µM of the drug, and the loss of drug in the
protein-containing compartment was reduced to 10%. This calcium-binding
profile (data not shown) was not significantly different from the one in the
absence of zinc shown in Fig.
2. Thus we can conclude by virtue of the rule of linked functions
(Weber, 1975
) that if the drug
binds directly to S100A13, the binding is independent of calcium.
|
|
Subcellular localization of S100 proteins in human endothelial
cells
Several S100 proteins have different subcellular distribution patterns in
various cell types in relation to their physiological functions
(Mandinova et al., 1998;
Mueller et al., 1999
;
Stradal and Gimona, 1999
). In
an attempt to understand the function of S100A13, we examined the subcellular
localization of endogenous S100A13 in ECV cells by immunostaining using a
rabbit polyclonal anti-human S100A13 antibody. As shown in
Fig. 3A, S100A13 is located
predominantly in the cell nucleus, which is different from its perinuclear
localization in human umbilical vein endothelial cells (HUVECs)
(Ridinger et al., 2000
). To
directly compare the subcellular localization of S100 proteins, we also
identified S100A1, S100A4, S100A6 and S100B in ECV. Nuclear staining was
observed with S100A4 (Fig. 3E)
and S100A5 (Fig. 3F)
antibodies; partial nuclear staining was observed with the S100A1
(Fig. 3C) antibody the
protein is located only in certain areas within the nucleus. S100A6
(Fig. 3B) and S100B
(Fig. 3G) were mainly found in
the cytoplasm and perinuclear area of the cells. Only background staining was
observed with an S100A3 antibody, which implies that the hair-specific S100A3
is not expressed at significant levels in this particular cell line
(Fig. 3D). Pre-absorbed S100
antibodies were used as negative controls, and only background staining was
observed (Fig. 3H).
|
The presence of several S100 proteins in ECV cells was confirmed by western blotting using specific S100 antibodies as shown in Fig. 4. S100A3 is also absent in this blot, whereas other S100 proteins appeared as monomers and various polymers. Similar protein separation and intensity was observed in each lane by Ponceau staining (data not shown) prior to western blotting.
|
Translocation pathway of endogenous S100A13 in endothelial cells
We observed S100 protein translocation in response to an increase in
intracellular calcium levels by angiotensin II stimulation. As shown in
Fig. 5A, endogenous S100A13 is
mainly distributed in the nuclei of ECV cells. Upon angiotensin II
stimulation, S100A13 translocated within small vesicles in the cytoplasm
(Fig. 5B). Since no vesicles
were observed with unstimulated cells, the redistribution of the small S100
protein is not caused by the fixation process
(Fig. 5A). Protein
translocation was observed by stimulating cells with angiotensin II for 5
minutes, but the phenomenon was more pronounced after 10 minutes of
stimulation. However, vesicles remained for up to 1 hour after the
stimulation. To further investigate the mechanism of protein translocation,
subcellular localization of endogenously expressed S100A13 was determined
after treatment with inhibitors of various secretion pathways. Brefeldin A was
used to impede protein transport from the ER to the Golgi complex
(Misumi et al., 1986). The
drug was added to the cells and incubated for 45 minutes before stimulation
with angiotensin II. Significantly fewer cells with vesicles translocating in
the cytoplasm were observed (Fig.
5C) compared with treatment with angiotensin II alone
(Fig. 5B). This implies an
ER-Golgi-dependent translocation pathway of S100A13 in ECV, which is unique
for the S100 family since S100 proteins lack the classic signal sequence for
secretion. Other secretion pathways such as the actin filament pathway, which
can be blocked by amlexanox (Tarantini et
al., 2001
), and the tubulin-dependent translocation, which is
inhibited by demecolcine (Davey et al.,
2000
), have therefore been investigated in this study.
Translocation in response to angiotensin II was not disturbed by adding either
amlexanox (Fig. 5D) or
demecolcine (Fig. 5F), which
indicates that the translocation of S100A13 is independent of the actin and
tubulin filament pathways. In an attempt to identify the type of vesicles that
translocate S100A13 in response to an intracellular calcium rise, bafilomycin
A was used to inhibit the formation of late endosomes. Bafilomycin A inhibits
vacuolar proton ATPase, which is known to generate the acidic lumernal
environment of endosomes and lysosomes
(Palokangas et al., 1998
).
Inhibition of acidification by bafilomycin A affects a number of other
cellular translocation pathways such as protein recycling from the plasma
membrane to the trans-Golgi and transport between the early and late endosomes
(the endosomal carrier vesicles) (Clague et
al., 1994
; Reaves and Banting,
1994
; van Weert et al.,
1995
; Gustafson et al.,
2000
). Indeed, fewer cells responding to angiotensin II
stimulation were observed with bafilomycin A treatment
(Fig. 5E). This set of data was
also investigated quantitatively by counting the fraction of cells with
vesicle appearance. Fig. 8A displays the fraction of cells with vesicular translocation of S100A13 in
response to angiotensin II and various secretion inhibitors. Significantly
fewer cells with translocating S100A13-containing vesicles were observed after
treatment with either brefeldin A (approximately a 50% decrease compared with
cells stimulated with angiotensin II) or bafilomycin A (approximately a 75%
decrease compared with cells stimulated with angiotensin II alone) in response
to angiotensin II; addition of amlexanox or demecolcine had no significant
effect on the translocation of S100A13 elicited by angiotensin II.
|
|
Translocation pathway of endogenous S100A6 in endothelial cells
To examine whether the unique translocation pathway observed with S100A13
in ECV is an universal phenomenon in this particular cell line, we
investigated the translocation pathway of S100A6 by immunohistochemistry
(Fig. 6). Endogenous S100A6 is
localized in the cytoplasm and perinuclear area of the cells
(Fig. 6A) and transported in
slightly larger vesicles than the ones observed with S100A13 in response to
angiotensin II stimulation (Fig.
6C). Addition of brefeldin A
(Fig. 6D), demecolcine
(Fig. 6E) or bafilomycin A
(Fig. 6F) had no effect on the
translocation of S100A6 in response to angiotensin II. However, treatment with
amlexanox effectively inhibited the translocation process
(Fig. 6B). Cell counting
reveals a nearly 50% decrease in the number of cells producing vesicles with
S100A6 when cells were treated with amlexanox before angiotensin II
stimulation (Fig. 8B). This
implies an actin-stress-fiber-dependent translocation pathway of S100A6 in
contrast to the ER-Golgi-dependent S100A13 translocation in ECV.
|
Effects of the inhibitory chemicals
To confirm the effects of the chemicals we used to block various
translocation pathways, we used anti-ß-COP antibody to confirm the
disruption of the ER-Golgi compartments induced by brefeldin A. Brefeldin A
induces fusion of Golgi to ER and inhibits protein transport to the post Golgi
compartment (Lippincott-Schwartz et al.,
1990). The vesicle coat protein ß-COP is a cis-Golgi
membrane-resident protein and was found in a different location following
brefeldin A treatment in HUVECs (Humphries
et al., 1997
). A scattered distribution of ß-COP indicates
dissociation of the protein from cis-Golgi membranes and fusion between Golgi
and ER compartments (Fig.
7A,B). Similarly, labeled phalloidin, which binds to actin fibers,
was used to monitor actin depolymerization in cells treated with amlexanox
(Fig. 7C,D). The destruction of
tubulin filaments in cells treated with demecolcine was visualized by using a
monoclonal anti-tubulin antibody and a Cy3-conjugated secondary antibody
(Fig. 7E,F).
|
Distinct S100A13 and S100A6 translocation
In order to confirm that the translocating vesicles containing either
S100A13 or S100A6 go through distinct pathways, double staining with two
antibodies obtained from different species was performed. Translocation of
S100A13 (green) was observed by following Cy2-conjugated antibody after
angiotensin II stimulation in Fig.
9A. As described earlier, the protein is mainly observed in the
cell nucleus and in the vesicles within the cytoplasm after angiotensin II
stimulation. With the same cell, S100A6 was also localized in vesicles and in
the cytoplasm (Fig. 9B).
However, a superimposed image of the two pictures
(Fig. 9A,B) reveals that
vesicles containing S100A13 (green) are distinct from the vesicles containing
S100A6 (red) after angiotensin II stimulation
(Fig. 9C). These data confirm
that S100A6 and S100A13 are localized in distinct vesicles as implied by their
use of different translocation pathways.
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Discussion |
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Translocation of S100 proteins may play an important role in assembly of
signaling complexes to activate specific signaling pathways
(Mueller et al., 1999;
Davey et al., 2000
;
Davey et al., 2001
). We
observed relocation of S100A13 in response to either angiotensin II or
thapsigargin (data not shown), both of which are known to elevate calcium
intracellularly. It is tempting to associate the translocation process of
S100A13 with alternation of the intracellular calcium levels even though the
biochemical data indicated that calcium has no effect on the hydrophobic
interaction of the protein. Thus, we hypothesize that an increase in
intracellular calcium levels might not have a direct effect on the
translocation of S100A13. Instead, the translocation process might depend on
the interaction with unknown target proteins, which are activated in the cells
saturated with a high concentration of calcium. A similar observation was
reported in the case of S100A10, which does not bind to calcium either, but
forms a tight complex with another calcium-binding protein, annexin II, and is
thought to be involved in endo- and exocytosis
(Harder and Gerke, 1993
).
Alternatively, the homeostasis of other ions present in the cells might be
influenced by the sudden increase of calcium and then participate in
translocation of S100A13. One potential candidate is copper since it has been
reported lately that it induces the assembly of S100A13, FGF-1 and
synaptotagmin-1 multiprotein aggregate
(Landriscina et al.,
2001
).
Interestingly, translocation after angiotensin II stimulation was also
observed with S100A6. S100A6 is another unique member in the S100
calcium-binding family, as the interaction of S100A6 to its target proteins is
calcium-dependent even though addition of calcium only induces very modest
conformational changes of the protein
(Sastry et al., 1998).
Although no hydrophobic exposure for target protein interaction was observed
in either S100A13 or S100A6 proteins in response to calcium binding, both
proteins translocated in response to an increase of intracellular calcium
levels in ECV cells. This implies that both S100A13 and S100A6 have different
modes for transducing calcium signals and interacting with their target
proteins from other members of the S100 family.
The so-called alternative secretion pathway, which is independent of the
classic ER-Golgi route, seems to be the most favorable pathway for relocation
of S100 proteins since S100 proteins lack the signal sequence to anchor the
plasma membrane for secretion. Several S100 proteins such as S100B, S100A8 and
S100A9 utilize the tubulin pathway for secretion
(Rammes et al., 1997;
Davey et al., 2000
). However,
the translocation of S100A13 in response to angiotensin II was inhibited by
addition of brefeldin A, which impairs the protein transport to the post-Golgi
compartment, and no inhibition of translocation was observed with addition of
demecolcine and amlexanox. Our results
(Fig. 5,
Fig. 8A) imply that the
translocation of S100A13 in ECV is associated with the classic ER-Golgi
pathway, which is novel in the S100 protein family. By contrast, the
translocation of S100A6 is independent of the ER-Golgi and the tubulin
pathway, but actin stress fibers might play an important role instead
(Fig. 6,
Fig. 8B).
In order to identify the type of vesicle we observed in translocation of
S100 proteins, we used the H+-ATPase inhibitor bafilomycin A to
inhibit acidification, which affects a variety of intracellular pathways,
including transport from early to late endosomes and recycling from the plasma
membrane to the trans-Golgi (Palokangas et
al., 1998; Gustafson et al.,
2000
). Apart from inhibiting the H+-ATPase activity in
Golgi cisternae, bafilomycin A also disrupts Golgi stack morphology and might
block the coat formation of the secreting vesicles
(Gustafson et al., 2000
). As
we have observed that S100A13 translocates through the ER-Golgi pathway in
small vesicles, the translocation process might be inhibited by bafilomycin A.
Indeed, translocation of S100A13 but not of S100A6 was inhibited by
bafilomycin A (Fig. 8). The
translocating vesicles carrying S100A13 in response to angiotensin II
stimulation seem to be different from the ones that translocate S100A6 as
shown in Fig. 9C. This confirms
the distinct translocation routes of S100A13 and S100A6 in response to the
increase of intracellular calcium by angiotensin II in the same cellular
environment.
In contrast to other members of the S100 family, we report here a classic translocation pathway of S100A13 in ECV cells in response to intracellular calcium increase by angiotensin II. The vesicles responsible for S100A13 translocation are possibly early endosomes as the translocation process can be inhibited by bafilomycin A. We also identified actin-stress-fiber-dependent translocation of S100A6 in this study. The distinct translocation pathways of the two S100 proteins in the same cell imply the existence of different signal transduction mechanisms of S100 proteins in response to intracellular calcium changes. Co-staining with other Golgi-ER early endosomal markers and searching for the possible target proteins that interact with S100A13 will be our future goals to understand the physiological role of this unique protein in the S100 family.
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Acknowledgments |
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References |
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