Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04074
Received for publication, January 19, 2001, and in revised form, April 3, 2001
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ABSTRACT |
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S100A13, a member of the S100 gene family of
Ca2+-binding proteins has been previously
characterized as a component of a brain-derived heparin-binding
multiprotein aggregate/complex containing fibroblast growth factor 1 (FGF1). We report that while expression of S100A13 in NIH 3T3 cells
results in the constitutive release of S100A13 into the extracellular
compartment at 37 °C, co-expression of S100A13 with FGF1 represses
the constitutive release of S100A13 and enables NIH 3T3 cells to
release S100A13 in response to temperature stress. S100A13 release in
response to stress occurs with kinetics similar to that observed for
the stress-induced release of FGF1, but S100A13 expression is able to
reverse the sensitivity of FGF1 release to inhibitors of transcription
and translation. The release of FGF1 and S100A13 in response to heat
shock results in the solubility of FGF1 at 100% (w/v) ammonium sulfate
saturation, and the expression of a S100A13 deletion mutant lacking its
novel basic residue-rich domain acts as a dominant negative effector of
FGF1 release in vitro. Surprisingly, the expression of
S100A13 also results in the stress-induced release of a Cys-free FGF1
mutant, which is normally not released from NIH 3T3 cells in response
to heat shock. These data suggest that S100A13 may be a component of
the pathway for the release of the signal peptide-less polypeptide,
FGF1, and may involve a role for S100A13 in the formation of a
noncovalent FGF1 homodimer.
FGF11 and FGF2 are the
prototype members of a large family of heparin-binding growth factor
genes that regulate numerous biological processes such as neurogenesis,
mesoderm formation, and angiogenesis (1, 2). FGF1 and FGF2 lack a
classical signal peptide sequence that provides access to the
conventional endoplasmic reticulum (ER)-Golgi secretion pathway, a
characteristic that led to the hypothesis that the release of these
polypeptides may proceed through novel release/export pathways (2). Our
laboratory previously demonstrated that FGF1, but not FGF2, is released
as a latent homodimer by a transcription- and
translation-dependent mechanism in response to a variety of
cellular stresses including heat shock (3), hypoxia (4), and serum
starvation (5). Conversely, the disruption of communication between the
ER and Golgi apparatus by brefeldin A does not prevent the release of
FGF1 from NIH 3T3 cells, confirming that FGF1 release may occur through
a nonconventional pathway (6).
FGF1 is released in vitro as a reducing agent- and
denaturant-sensitive complex, which contains the p40 extravesicular
domain of the Ca2+-binding protein, p65 synaptotagmin
(Syt)1 (7). The release of FGF1 in response to stress is dependent on
Syt1 expression, since the expression of either a deletion mutant
lacking 95 amino acids from the extravesicular portion of Syt1 or
an antisense-Syt1 gene is able to repress FGF1 release in NIH
3T3 cells (7, 8). In addition, FGF1 purified from ovine brain as a high
molecular weight aggregate exists as a component of a noncovalent
heparin-binding complex with p40 Syt1 and S100A13 (9).
The interleukin (IL)-1 gene family of proinflammatory polypeptides also
contains functional cytokines that act as extracellular, receptor-dependent mediators of cellular function (10, 11), yet the majority of the members of the IL-1 gene family lack a classical signal peptide sequence for conventional secretion (10, 11).
Interestingly, crystallographic analysis of the FGF and IL-1 prototypes
demonstrates a remarkable level of structural similarity between these
seemingly disparate polypeptides (12, 13). Recent studies have
suggested that the pathway utilized for the release of IL-1 Recently, we have reported that amlexanox, an anti-allergic drug that
binds S100A13 (15), is able to inhibit the release of FGF1 and p40 Syt1
in response to temperature stress (9, 16). Because amlexanox also
induces a Src-dependent and reversible disassembly of actin
stress fibers (16), these data have suggested a role for the actin
cytoskeleton in the regulation of FGF1 release (16).
S100A13 is a member of a large gene family of Ca2+-binding
proteins characterized by the absence of a classical signal peptide sequence and the presence of two Ca2+-binding EF-hand
domains (17, 18). S100A13 is a novel member of the S100 gene family
that encodes a protein containing a positively charged
carboxyl-terminal domain that may be involved in specific protein
interactions (19). While members of the S100 gene family, including
S100A13 (20), are expressed in a variety of tissues and organs (17,
18), the expression of a few members of the S100 gene family have been
implicated in the regulation of human pathology including
neurodegenerative diseases, cardiomyopathies, cancer, and chronic
inflammation (17, 18). Interestingly, members of the S100 gene family
have been shown to be constitutively released into the extracellular
compartment (17, 18, 21), where they have been characterized as
leukocyte chemoattractants and regulators of macrophage activation (17,
18, 22).
Since S100A13 was purified from the ovine brain as a part of the
multiprotein aggregate containing FGF1 and p40 Syt1, we sought to
determine whether S100A13 is involved in the release of FGF1. We report
that S100A13 expression facilitates the release of FGF1 into the
extracellular compartment in response to temperature stress in
vitro and that S100A13 expression is able to revert the dependence
for both transcription and translation in the release of FGF1 in
response to heat shock. We further describe the unanticipated observation that S100A13 expression is able to export a Cys-free FGF1
mutant, suggesting that S100A13 may be involved in the formation of the
FGF1 homodimer, a prerequisite for export.
Plasmid Constructs--
The plasmid pT7T3-S100A13 containing the
cDNA encoding murine S100A13 was a generous gift from Dr. Beat W. Schafer (University of Zurich, Switzerland), and all restriction
enzymes were obtained from New England Biolabs. The S100A13 cDNA
was obtained by digesting the pT7T3-S100A13 plasmid with
NotI and BstZ17I. The fragment was cloned
into the pCS2-6-Myc tag vector digested with EcoRI, filled-in by the Klenow reaction (New England Biolabs Inc.), and digested with NotI. The Myc tag was ligated at the amino
terminus of the S100A13 protein and consisted of a 13-amino acid
epitope (MEQKLISEEDLNE) repeated six times and recognized by the 9E10 monoclonal anti-Myc antibody (Oncogene Research Products). For expression in mammalian cells, the Myc-S100A13 DNA was digested using
HindIII and ligated into the pcDNA3.1-Hygro (+) vector
(Invitrogen) also excised with HindIII. For expression in
prokaryotic cells, S100A13 cDNA was digested from pT7T3-S100A13
using HindIII and BstZ17I. The fragment
was ligated into the prokaryotic expression vector, pGEX-KG, digested
with XbaI, filled-in by the Klenow reaction, and further
digested with HindIII. The pGEX-KG vector contains the
sequence encoding for the glutathione S-transferase (GST) that was used to purify the recombinant S100A13. For the preparation of
the S100A13 Cell Culture--
Murine NIH 3T3 cells were obtained from the
American Type Culture Collection (ATCC) and grown as previously
described (3). NIH 3T3 cells, FGF1 NIH 3T3 (3), and Cys-free FGF1 NIH
3T3 cell transfectants (6) were cotransfected with the gene encoding murine Myc-S100A13 cloned into the pcDNA3.1-Hygro (+) vector or with the insertless expression vector. FGF1 NIH 3T3 cells were also
transfected with the same eukaryotic expression vector containing the
DNA sequence encoding for the Myc-S100A13 Temperature Stress, Processing of Conditioned Media, and
Immunoblot Analysis--
NIH 3T3 cell transfectants were grown to
70-80% confluency, and prior to temperature stress, the cells were
washed with Dulbecco's modified Eagle's medium containing 5 units/ml
heparin (Upjohn). The heat shock was performed as previously described
(3, 7, 8) in Dulbecco's modified Eagle's medium containing 5 units/ml heparin for 110 min at 42 °C. Control cultures were incubated at
37 °C in Dulbecco's modified Eagle's medium containing 5 units/ml heparin. Two independent clones from each transfection have been evaluated with similar results. The effects of 2-deoxyglucose (Sigma),
brefeldin A (Epicenter Technologies), amlexanox (Takeda), latrunculin
(Molecular Probes, Inc., Eugene, OR), actinomycin D (Sigma), and
cycloheximide (Sigma) on FGF1 and Myc-S100A13 release were evaluated as
previously reported (3, 6, 8, 9, 16). Following heat shock, the
conditioned media were collected, filtered, and treated with either
0.1% (w/v) DTT (Sigma) for 2 h at 37 °C or fractionated with
ammonium sulfate (Sigma) at 100% (w/v) saturation as previously
described (3, 6). After ammonium sulfate fractionation, conditioned
media were centrifuged at 10,000 × g for 50 min, and
the pellets were resuspended in 20 ml of 50 mM Tris, pH
7.4, containing 10 mM EDTA (TEB). Pellets and supernatants were dialyzed against 50 mM Tris, pH 7.4, for 18 h and
treated with 0.1% (w/v) DTT for 2 h at 37 °C. DTT- and
ammonium sulfate-treated conditioned media were adsorbed to a
heparin-Sepharose CL-6B (1-ml) column (Amersham Pharmacia Biotech),
equilibrated with TEB, and the adsorbed proteins were washed with TEB
and eluted with 2.5 ml of TEB containing NaCl concentrations specified
under "Results." Eluted proteins were concentrated using Centricon
10 (Amicon, Inc.), resolved by 15% (w/v) SDS-PAGE, transferred to a
nitrocellulose membrane (Hybond C; Amersham Pharmacia Biotech), and
immunoblotted with an anti-Myc monoclonal antibody (Oncogene Research
Products) or an anti-FGF1 polyclonal antibody (23), and bands were
visualized by chemiluminescence (ECL; Amersham Pharmacia Biotech).
Total cell lysates were obtained from cells incubated at 37 °C by
resuspending cell pellets 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) and analyzed by immunoblot analysis.
For the analysis of the release of FGF1 and Myc-S100A13 Purification of Recombinant S100A13, Preparation of S100A13
Antiserum, and Analysis of S100A13 Heparin Affinity--
The
GST-S100A13 fusion protein was generated as described above (25), with
the exception that the BL21 E. coli were lysed in 50 mM Tris buffer, pH 8.8, containing 10 mM EDTA,
10 mM glucose, and 10 µg/ml lysozyme, and the
thrombin-cleaved S100A13 protein was further purified by C8
(PerkinElmer Life Sciences) reverse-phase high pressure liquid
chromatography as previously described for FGF1 (26). Female
6-month-old New Zealand White rabbits (Hazelton Research Animals) were
injected intradermally with 1 mg of recombinant S100A13 suspended in
Freund's complete adjuvant (Calbiochem) and boosted with 200 µg of
protein suspended in Freund's incomplete adjuvant (Calbiochem). The
antibody was purified by incubating the serum with immobilized
recombinant S100A13 on a Problot polyvinylidene difluoride membrane
(Applied Biosystem Inc.) blocked at 42 °C with 24 mM
Tris, pH 7.4, containing 136 mM NaCl, 2 mM KCl,
0.1% (v/v) Tween 20, and 5% (w/v) bovine serum albumin. The membrane was washed three times with 0.05% (v/v) Triton X-100 in 50 mM Tris, pH 7.4, containing 150 mM NaCl, and
the antibody was eluted with 0.2 M glycine, pH 2.8, and
neutralized with 1 M K2HPO4, pH 7.4. For the evaluation of S100A13 heparin affinity, 5 µg of
recombinant S100A13 were adsorbed to heparin-Sepharose (1 ml),
previously equilibrated with TEB. The adsorbed proteins were washed
with TEB and eluted with 2.5 ml of TEB containing increasing
concentrations of NaCl. Eluted proteins were concentrated using
Centricon 3 (Amicon, Inc.), resolved by 15% (w/v) SDS-PAGE, and
immunoblotted with the rabbit anti-S100A13 antibody.
S100A13 Exhibits Low Heparin Affinity and Is Constitutively
Released in Vitro--
Because we have previously characterized ovine
brain-derived S100A13 as a component of a high affinity heparin-binding
multiprotein aggregate/complex containing FGF1 and the p40
extravesicular domain of p65 Syt1 (9), we sought to characterize the
heparin-binding character of S100A13. As shown in Fig.
1A, while recombinant murine S100A13 was able to bind heparin-Sepharose, its affinity for heparin was low, since S100A13 immunoblot analysis reported its presence in the
0.5 M NaCl elution fraction. Thus, like p40 Syt1 (7), brain-derived S100A13 (9) may gain heparin affinity by its ability to
associate either directly or indirectly with p40 Syt1 and/or FGF1.
Because the level of endogenous S100A13 expression in NIH 3T3 cells is
low (data not shown), we constructed a chimera in which six copies of
the Myc epitope were fused at the 5'-end of the S100A13 cDNA and
obtained stable Myc-S100A13 NIH 3T3 cell transfectants and FGF1 and
Myc-S100A13 NIH 3T3 cell cotransfectants. As a control, we also
obtained FGF1 NIH 3T3 cells cotransfected with the insert-less vector
used to transfect the Myc-S100A13 cDNA. Myc immunoblot analysis of
cell lysates from Myc-S100A13 NIH 3T3 cell transfectants, Myc-S100A13
and FGF1 NIH 3T3 cell cotransfectants, and insertless vector and FGF1
NIH 3T3 cell cotransfectants confirmed the expression of Myc-S100A13 as
a polypeptide with an apparent molecular mass of 27 kDa in the
Myc-S100A13 NIH 3T3 cell transfectants (Fig. 2B) and Myc-S100A13 and FGF1
NIH 3T3 cell cotransfectants (Fig. 2A). This band was not
present in cell lysates derived from the insert-less vector and FGF1
NIH 3T3 cell cotransfectants (Fig. 2A). Myc epitope
immunoblot analysis demonstrated a slight retardation in the
electrophoretic mobility of Myc-S100A13 from the expected molecular
mass of 21 kDa, an alteration that may be due to a change in the
conformation of the protein upon binding to metal ions. Indeed, this
observation is consistent with similar alterations established for a
variety of other S100 proteins (27-29). As shown in Fig.
1B, the Myc-S100A13 chimera obtained from lysates of
Myc-S100A13 NIH 3T3 cell transfectants exhibited similar heparin
affinity as the S100A13 recombinant protein purified from the
prokaryotic expression system. Because temperature stress of the
Myc-S100A13 NIH 3T3 cell transfectants did not alter the heparin
affinity of the Myc-S100A13 chimera (Fig. 1B), we suggest
that the Myc-S100A13 protein may not be subjected to
temperature-mediated post-translational modifications, which may affect
its heparin affinity.
We have previously reported that NIH 3T3 cells transfected with FGF1
and p65 Syt1 are able to release FGF1 and the p40 extravesicular domain
of Syt1 in response to cellular stress (7, 8) and that FGF1 and the p40
extravesicular domain of p65 Syt1 are present in the extracellular
compartment as a reducing agent- and denaturant-sensitive complex (7).
Because the expression of p40 Syt1 in NIH 3T3 cells results in the
stress-independent, constitutive release of p40 Syt1 (8), we questioned
whether Myc-S100A13 may also be able to access the extracellular
compartment either spontaneously or under conditions of temperature
stress. Myc-S100A13 NIH 3T3 cell transfectants were subjected to heat
shock (110 min at 42 °C) or maintained for 110 min at 37 °C, and
conditioned media were treated with 0.1% (w/v) DTT, adsorbed to
heparin-Sepharose, eluted at 1.5 M NaCl, and analyzed for
Myc-S100A13 release by immunoblot analysis. As shown in Fig.
2B, we observed that Myc-S100A13 was present in media
conditioned independently of temperature stress and that stress
conditions did not significantly increase the level of Myc-S100A13 in
the extracellular compartment. Kinetic analysis of the
temperature-independent Myc-S100A13 release from Myc-S100A13 NIH 3T3
cell transfectants demonstrated that Myc-S100A13 was detectable in
media conditioned at 37 °C after 30 min with an increase in the
level of extracellular Myc-S100A13 observed after prolonged time
periods (hours) at 37 °C (data not shown).
FGF1 Is Able to Repress the Constitutive Release of
S100A13--
The Myc-S100A13 and FGF1 NIH 3T3 cell cotransfectants
were also subjected to heat shock in order to study the simultaneous release of Myc-S100A13 and FGF1. Conditioned media were obtained from
both Myc-S100A13 and FGF1 and insert-less vector and FGF1 NIH 3T3 cell
cotransfectants following heat shock and processed for FGF1 and Myc
immunoblot analysis. We observed that both cotransfectants released
similar levels of FGF1 in response to stress, and the Myc-S100A13 and
FGF1 NIH 3T3 cell cotransfectants also released the Myc-S100A13 chimera
in response to temperature stress (Fig. 2A). The Myc-S100A13
and FGF1 NIH 3T3 cell cotransfectants were further evaluated for the
capacity to constitutively release Myc-S100A13 independent of stress,
and Myc immunoblot analysis was able to detect the presence of
Myc-S100A13 only in media conditioned at 37 °C for time periods
greater than 8 h (data not shown). Further, FGF1 immunoblot
analysis revealed that FGF1 was not present in media conditioned at
37 °C for this prolonged time period (data not shown). These data
suggest that (i) the constitutive release of Myc-S100A13 at 37 °C is
significantly impaired but is up-regulated in response to stress in a
FGF1-rich background represented by the Myc-S100A13 and FGF1 NIH 3T3
cell cotransfectants and (ii) the constitutive release of Myc-S100A13
occurs unattenuated in a FGF1-poor background represented by the
Myc-S100A13 NIH 3T3 cell transfectants.
Because FGF1 and p40 Syt1 are known to be released in response to heat
shock with rather slow kinetics (8), we evaluated whether the
stress-induced release of Myc-S100A13 also proceeds with similar
kinetics. The Myc-S100A13 and FGF1 NIH 3T3 cell cotransfectants were
subjected to heat shock for 10, 20, 30, 60, and 90 min, and the
conditioned media were processed for FGF1 and Myc immunoblot analysis.
As shown in Fig. 2C, both FGF1 and Myc-S100A13 were released
with similar kinetics, and both required at least 90 min of exposure to
temperature stress to gain access to the extracellular compartment.
These results suggest that FGF1 (6), p40 Syt1 (8), and Myc-S100A13 also
exhibit similar kinetics of release from NIH 3T3 cells in response to
stress when NIH 3T3 cell Myc-S100A13 transfectants are cotransfected
with FGF1.
The Expression of Myc-S100A13 Overcomes the Transcriptional and
Translational Requirements for FGF1 Release in Response to Heat
Shock--
Since we have previously reported that the release of FGF1
and p40 Syt1 is dependent on transcription and translation (3, 8), we
evaluated the effect of actinomycin D and cycloheximide on the heat
shock-induced release of Myc-S100A13 and FGF1. Myc-S100A13 and FGF1 NIH
3T3 cell cotransfectants and insert-less vector and FGF1 NIH 3T3 cell
cotransfectants were subjected to heat shock in the presence and
absence of actinomycin D (10 µg/ml) or cycloheximide (10 µg/ml),
and conditioned media were analyzed for FGF1 and Myc-S100A13 release by
immunoblot analysis. We observed that FGF1 NIH 3T3 cells cotransfected
with Myc-S100A13 were able to release FGF1 and Myc-S100A13 in response
to heat shock in the presence of either actinomycin D or
cycloheximide (Fig. 3B). In contrast, the
treatment of insert-less vector and FGF1 NIH 3T3 cell cotransfectants
with actinomycin D and cycloheximide completely inhibited FGF1 release (Fig. 3A). Because these data demonstrated that Myc-S100A13
expression was able to revert the sensitivity of FGF1 release to
actinomycin D and cycloheximide, we evaluated whether S100A13 gene
expression was induced during heat shock. However, analysis of the
steady-state levels of the transcripts for S100A13 at 37 and 42 °C
demonstrated that S100A13 is not a heat shock gene (data not shown).
Thus, we suggest that the ability of Myc-S100A13 expression to revert the actinomycin D- and cycloheximide-sensitive component of the FGF1
release pathway may be due to a heat shock-induced post-translational event mediated by S100A13.
Because we have previously reported that the stress-mediated release of
FGF1 and p40 Syt1 requires the biosynthesis of ATP (8), is sensitive to
reagents that affect the integrity of the actin cytoskeleton (9, 16),
and is not affected by brefeldin A (8), a reagent that disrupts
communication between the ER-Golgi apparatus (30), we sought to
determine whether the stress-induced release of Myc-S100A13 exhibited
similar behavior. Thus, Myc-S100A13 and FGF1 NIH 3T3 cell
cotransfectants were subjected to heat shock in the presence and
absence of the appropriate biochemical reagent, and their conditioned
media were subjected to Myc and FGF1 immunoblot analysis. As shown in
Fig. 3C, the release of FGF1 and Myc-S100A13 was inhibited
by treatment with (i) 2-deoxyglucose, a reagent that inhibits ATP
biosynthesis (31), and (ii) amlexanox and latrunculin, reagents that
induce the collapse of actin cytoskeleton through different
mechanisms (16, 32). In addition, the release of Myc-S100A13 and FGF1
was insensitive to treatment with brefeldin A, a reagent that disrupts
ER-Golgi communication (30).
Since Myc-S100A13 is constitutively released from Myc-S100A13 NIH 3T3
cell transfectants and its release is significantly inhibited by the
expression of FGF1, we evaluated the pharmacological properties of the
constitutive release of S100A13. We observed that, like the
constitutive release of p40 Syt1 (8), the constitutive release of
Myc-S100A13 is insensitive to amlexanox, 2-deoxyglucose, brefeldin A, actinomycin D, and cycloheximide (data not shown). In
contrast, amlexanox and 2-deoxyglucose are able to repress the release
of both FGF1 and Myc-S100A13 in response to heat shock from FGF1 and
Myc-S100A13 NIH 3T3 cell cotransfectants. Furthermore, while FGF1 is
able to repress the release of S100A13, it is not able to repress the
constitutive release of p40 Syt1 (8). These data suggest that the
constitutive release of both Myc-S100A13 and p40 Syt1 occurs using
pathways divergent from the stress-dependent pathway of
FGF1 release and also argues that the constitutive release pathways
exhibited by Myc-S100A13 and p40 Syt1 (8) may be different.
Myc-S100A13 May Be Released as a Complex with FGF1 in Response to
Heat Shock--
The term S100 refers to the ability of S100 gene
translation products to be resistant to precipitation at 100% (w/v)
ammonium sulfate saturation (18). In contrast, FGF1 is precipitated by ammonium sulfate fractionation at 85% (w/v) saturation (33), and
ammonium sulfate fractionation has been utilized to fractionate FGF1
from numerous organs and tissues (9, 34). To evaluate the ability of
S100A13 to be precipitated by ammonium sulfate fractionation, we
performed 100% (w/v) ammonium sulfate fractionation using media
conditioned by heat shock from Myc-S100A13 NIH 3T3 cell transfectants.
Following dialysis, pellets and supernatants from the 100% (w/v)
ammonium sulfate fractionated media were adsorbed to heparin-Sepharose
and eluted with 1.5 M NaCl. As anticipated, Myc immunoblot
analysis revealed that Myc-S100A13 released by the Myc-S100A13 NIH 3T3
cell transfectants in response to heat shock was present only in the
supernatant fraction (data not shown).
Because these data suggested that Myc-S100A13 may be resistant to
precipitation following 100% (w/v) ammonium sulfate fractionation, we
hypothesized that if Myc-S100A13 is complexed with FGF1 as a result of
stress-induced release, the Myc-S100A13- and FGF1-containing complex
should be characterized by enhanced solubility of FGF1 in the
supernatant fraction following 100% (w/v) ammonium sulfate saturation.
To assess this premise, media conditioned by heat shock from
Myc-S100A13 and FGF1 NIH 3T3 cell cotransfectants and from insert-less
vector and FGF1 NIH 3T3 cell cotransfectants were subjected to 100%
(w/v) ammonium sulfate fractionation and pellet and supernatant
fractions obtained by centrifugation. After DTT treatment, pellet and
supernatant fractions were adsorbed to heparin-Sepharose, eluted at 1.5 M NaCl, and the presence of FGF1 and Myc-S100A13 assessed
by FGF1 and Myc immunoblot analysis. As shown in Fig.
4A, higher levels of FGF1 were
resolved in the pellet fraction from media obtained from
temperature-stressed insert-less vector and FGF1 NIH 3T3 cell
cotransfectants, whereas media obtained from temperature-stressed
Myc-S100A13 and FGF1 NIH 3T3 cell cotransfectants exhibited increased
levels of FGF1 and Myc-S100A13 in the supernatant fraction. These data
suggest that Myc-S100A13 may be able to alter the solubility of FGF1 in media conditioned by heat shock, and the solubility of FGF1 under conditions of 100% (w/v) ammonium sulfate saturation may be a consequence of an association between FGF1 and Myc-S100A13.
A S100A13 Mutant Lacking the Basic Residue-rich Domain Functions as
a Dominant Negative Regulator of FGF1 Release--
The
carboxyl-terminal domains of several S100 proteins have been described
as the structural regions responsible for mediating the interaction
between S100 polypeptides and specific target proteins (17, 18).
Because the carboxyl terminus of S100A13 (last 11 amino acid residues)
is characterized by the presence of 6 basic amino acid residues (Fig.
5A), a feature absent in the
other members the S100 protein family (19), we evaluated whether the
carboxyl-terminal basic residue-rich domain present in S100A13 is
involved in the release of FGF1 and Myc-S100A13. We produced a deletion
mutant of Myc-S100A13 (Myc-S100A13
We also evaluated the ability of the FGF1 and Myc-S100A13 The Expression of Myc-S100A13 Induces the Release of Cys-free FGF1
in Response to Temperature Stress--
We have previously reported
that (i) FGF1 is released as a homodimer in response to heat shock (3,
6), (ii) a Cys-free mutant of FGF1 is not able to access the
extracellular compartment in response to stress (6), and (iii) residue
Cys30 in FGF1 is responsible for dimer formation and export
into the extracellular compartment (35). Since the solubility of
extracellular FGF1 under conditions of 100% (w/v) ammonium sulfate
saturation suggests that S100A13 and FGF1 may be able to associate, we
evaluated whether S100A13 was able to affect the release of the
Cys-free mutant of FGF1. As a result, the Cys-free FGF1 NIH 3T3 cell
transfectants were cotransfected with either Myc-S100A13 or insertless
vector, and media conditioned by temperature stress from both
cotransfectants were evaluated for the presence of FGF1 and
Myc-S100A13. FGF1 and Myc immunoblot analysis revealed that while the
Cys-free FGF1 mutant was not released in response to heat shock from
the insertless vector control and FGF1 cotransfectants, the Cys-free
FGF1 mutant was released in response to temperature stress in cells
expressing both Cys-free FGF1 and the Myc-S100A13 chimera (Fig.
4C). In addition, the Myc-S100A13 and Cys-free FGF1 NIH 3T3
cell cotransfectants were also able to release Myc-S100A13 in response
to heat shock (Fig. 4C). Moreover, as previously described
for the Myc-S100A13 and FGF1 NIH 3T3 cell cotransfectants, Cys-free
FGF1 was also able to suppress the constitutive release of Myc-S100A13
(Fig. 4C). The heat shock-induced release of Cys-free FGF1
and Myc-S100A13 from Cys-free FGF1 and Myc-S100A13 NIH 3T3 cell
cotransfectants (i) occurred with kinetics similar to that observed for
the release of FGF1, (ii) was sensitive to amlexanox and 2-deoxyglucose
treatment, two reagents that are able to inhibit the stress-induced
release of FGF1 (6, 7), (iii) was insensitive to brefeldin A, a feature
also exhibited by the release of FGF1, and (iv) was insensitive to
treatment with actinomycin D and cycloheximide (data not shown). These
data suggest that the expression of Myc-S100A13 may be able to affect
the release of FGF1 either as a monodimer or as a noncovalent dimer.
Since our results have suggested that Myc-S100A13 is able to (i)
associate with FGF1 in response to temperature stress and (ii) induce
the temperature-dependent release of Cys-free FGF1, we
evaluated the capacity of Myc-S100A13 to be released in association with Cys-free FGF1. Myc-S100A13 and Cys-free FGF1 NIH 3T3 cell cotransfectants were subjected to heat shock, conditioned media were
collected and fractionated at 100% (w/v) ammonium sulfate, and pellet
and supernatant fractions were obtained. As shown in Fig.
4B, FGF1 and Myc immunoblot analysis revealed the presence of significant levels of Cys-free FGF1 and Myc-S100A13 in the supernatant fraction, suggesting that Myc-S100A13 may also be able to
associate with the monomeric form of FGF1 under conditions of
temperature stress.
Our data suggest that S100A13 is involved in the regulation of the
stress-induced FGF1 release pathways. Thus, like the stress-induced release of FGF1 (3), p40 Syt1 (7, 8), and murine IL-1 The finding that Myc-S100A13 expression is able to overcome the
requirement for transcription and translation in the release of FGF1 is
consistent with the observation that some members of the S100 gene
family have been characterized not only as stress-induced genes
(36-39) but also as Cu2+-binding proteins (17, 18, 40,
41). It is interesting to note that S100B was independently
characterized from neural tissue as an inhibitor of
Cu2+-mediated L-ascorbate oxidation (40).
Moreover, S100A4, S100A6, S100A7, and S100B are up-regulated in several
human tumor cells, where they are associated with increased
invasiveness of transformed cells and acquisition of metastatic
phenotype (17). The murine analog of S100A8, the CP-10 protein, has
been also associated with inflammation, since it is expressed and
released in macrophages and endothelial cells only after their
activation by interleukin-1 and lipopolysaccharide (17, 42). However,
the observation that the S100A13 gene is not induced in response to
heat shock in NIH 3T3 cells does not eliminate the possibility that
other members of the S100 gene family may participate in the release of
FGF1 and that the expression of S100A13 may compensate for their function.
While it is difficult to anticipate how S100A13 participates in these
release pathways, we suggest that S100A13 may be able to orient FGF1
and Cys-free FGF1 in a manner that enables these polypeptides to
form noncovalent homodimers. Indeed, Cu2+ oxidation is able
to induce FGF1 homodimer (26) and FGF1 IL-1 It is interesting that latrunculin and amlexanox, which are known to
attenuate the F-actin cytoskeleton (16, 32), also inhibit the heat
shock-induced release of FGF1 and Myc-S100A13. Indeed, this suggests
that the cytoskeleton may play an important role in the FGF1 release
pathway. The premise is further supported by the association of the
members of the S100 gene family with actin stress fibers, which is
dependent upon the physiological status of the cell (48). As a result,
it is possible that S100A13 may act as an adaptor between FGF1 and
F-actin structures. The F-actin cytoskeleton may be involved in at
least two stages of the stress-induced release of FGF1 and Myc-S100A13:
(i) the transport of the complex to the cell membrane proceeding along
F-actin stress fibers with the potential participation of myosin
molecular motors (49) and (ii) exocytosis, which has been demonstrated
to depend on the submembrane actin cortex during classical protein
secretion (50). Because both actin-dependent transport and
exocytosis are energy-dependent processes (50), this
suggestion is consistent with the ability of 2-deoxyglucose to repress
the stress-induced release of FGF1 and Myc-S100A13. Interestingly,
unlike latrunculin (32), amlexanox does not interfere with the
stability of submembrane F-actin (16), and this further implies that
the F-actin stress fibers may be involved in the regulation of the
transport of FGF1, p40 Syt1, and Myc-S100A13 in the nonclassical
release of these polypeptides.
The observation that S100A13 is constitutively released in
vitro is also noteworthy, since a similar observation has been made with the intracellular p40 fragment of p65 Syt1 (8).
Interestingly, the function of the C2A domain in p40 Syt1 has been
implicated in lipid bilayer penetration (51). However, unlike p40 Syt1, which exhibits constitutive release in FGF1 NIH 3T3 cell transfectants (8), the expression of FGF1 in Myc-S100A13 NIH 3T3 cell transfectants represses the constitutive release of Myc-S100A13. Although it is
difficult to interpret the significance of these data, it is possible
that the ability of FGF1 to attenuate constitutive release of
Myc-S100A13 but not the constitutive release of p40 Syt1 may reflect
the ability of intracellular FGF1 to prefer an association with S100A13
rather than with p40 Syt1. Indeed, the observation that FGF1, S100A13,
and p40 Syt1 are present as a noncovalent aggregate/complex in neural
tissue suggests that the self-aggregration properties attributed to
both the extravesicular p40 domain of p65 Syt1 (52) and S100 gene
family members (17, 18) may be involved in the arrangement of a
conformation-sensitive aggregate complex that may facilitate the
release of FGF1. The observations that FGF1 (35), p40 Syt1 (53), and
S100A13 (54) are able to associate with acidic phospholipids further
suggest an additional complexity to the stoichiometric interactions
between these polypeptides.
Although we do not know how this multiprotein aggregate/complex gains
access to the extracellular compartment, it is likely that the basic
residue-rich domain at the carboxyl terminus of S100A13 may be involved
in regulating this function. Several studies have reported that the
carboxyl-terminal domain of S100 proteins is involved in mediating the
interaction of S100 polypeptides in their dimeric state with their
target proteins (17, 18). Because deletion of the basic residue-rich
domain at the carboxyl terminus of Myc-S100A13 results in the
generation of a dominant-negative effector of FGF1 release in response
to temperature stress, it is likely that this domain may be involved in
the regulation of FGF1 export under heat shock conditions. However, it
is unlikely that the S100A13 basic residue-rich domain is involved in
mediating the ability of Myc-S100A13 to traverse the plasma membrane,
since the Myc-S100A13
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
is quite
similar yet distinct from the pathway utilized by FGF1 export, since
both proteins are released in response to temperature stress with
similar biochemical and pharmacologic properties (14). In contrast,
however, the IL-1
release pathway requires the proteolytic
processing of the 31-kDa precursor form of IL-1
to the 17-kDa mature
form for IL-1
release and does not appear to utilize Syt1 for
export, since a dominant negative Syt1 mutant blocks FGF1 but not
IL-1
release (14). Although we have not been able to eliminate the
function of other Syt gene family members in the stress-induced release
of IL-1
, the observation that the precursor form of IL-1
is able
to block the release of FGF1 in response to temperature stress suggests that the FGF1 and IL-1
release pathways may be mechanistically linked (14).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
88-98 mutant, the plasmid pT7T3-S100A13 was digested with BsaBI and PpuMI, and the S100A13 fragment
was purified by gel electrophoresis. Two oligonucleotides lacking the
sequence encoding for the last 11 amino acids of S100A13 were ligated
in the digested plasmid. The two oligonucleotides and their
complementary chains were designed with a BsaBI site at the
5'-end and a PpuMI site at the 3'-end and with a
complementary overlapping sequence in the middle. The sequences
of the oligonucleotides used are as follows:
5'-GAATCAGGACTCAGAGCTGAGGTTCAGTGAATACTGGAGACTGAT-3'; 5'-AGTATTCACTGAACCTCAGCTCTGAGTCCTGATTC-3';
5'-TGGAGAGCTGGCAAAGGAAGTCTAAAGCTTGCTGTCCAGCAG-3'; 5'-GTCCTGCTGGACAGCAAGCTTTAGACTTCCTTTGCCAGCTCTCCAATCAGTCTCC-3'. The
S100A13
88-98 construct was ligated into the pCS2-6Myc tag vector
with the six-Myc tag repeat sequence fused at the amino terminus of the
protein and the Myc-S100A13
88-98 sequence cloned into the
pcDNA3.1-Hygro (+) expression vector as described above.
88-98 mutant. All transfections were performed using a multicomponent lipid-based reagent
(FuGENE 6; Roche), and stable clones of the NIH 3T3 cell cotransfectants were obtained, expanded, and grown in Dulbecco's modified Eagle's medium (Cellgro) supplemented with 10% (v/v) bovine
calf serum (HyClone), 1× antibiotic/antimycotic (Life Technologies, Inc.), 0.15 g/liter hygromicin B (Life Technologies), and 0.4 g/liter
Geneticin (G418; Life Technologies) on human fibronectin-coated dishes
(10 µg/cm2).
88-98 from
NIH 3T3 cell cotransfectants, conditioned media and cell lysates were
divided into two portions, one of which was processed by
heparin-Sepharose adsorption for FGF1 immunoblot analysis and the other
processed by immunoprecipitation with anti-Myc antibody for the
detection of Myc-S100A13
88-98 and Myc-S100A13. Briefly, conditioned
media were filtered, treated with 0.1% (w/v) DTT for 2 h at
37 °C, and concentrated using a centrifugal filter device (Ultrafree-15; Millipore Corp.) to a volume of ~500 µl, and
aprotinin and leupeptin were added to each sample to yield a final
concentration of 2 µg/ml. Cell lysates and concentrated conditioned
media were rotated for 18 h at 4 °C in the presence of 1 µg
of mouse anti-Myc antibody. Protein A-Sepharose (Amersham Pharmacia
Biotech) was added, and the samples were rotated for an additional
2 h at 4 °C. Immunoprecipitated proteins were eluted with
sample buffer, boiled, resolved by 12% (w/v) SDS-PAGE, and
immunoblotted with anti-Myc antibody. The activity of lactate
dehydrogenase (Sigma) in conditioned media was utilized as an
assessment of cell lysis in all experiments as previously reported
(24).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The heparin affinity of S100A13.
A, recombinant S100A13 was obtained as described under
"Materials and Methods," and 5 µg of recombinant S100A13 was
adsorbed to heparin-Sepharose, eluted with 0.5, 1.0, and 1.5 M NaCl as described (7); the fractions were resolved by
15% (w/v) SDS-PAGE and subjected to S100A13 immunoblot analysis using
a rabbit anti-S100A13 antibody. Lane 1, 5 µg of
recombinant S100A13; lane 2, 0.5 M
NaCl fraction; lane 3, 1.0 M NaCl
fraction; lane 4, 1.5 M NaCl
fraction; lane 5, flow-through fraction.
B, Myc-S100A13 NIH 3T3 cell transfectants were subjected to
heat shock (110 min at 42 °C), and cell lysates were obtained from
control (37 °C) and stressed cultures as described under
"Materials and Methods." Proteins were adsorbed to
heparin-Sepharose, eluted at 0.5, 1.0, and 1.5 M NaCl, and
resolved by 15% (w/v) SDS-PAGE. Myc-S100A13 was evaluated by Myc
immunoblot analysis. Lane 1, 0.5 M
NaCl fraction from control cells; lane 2, 1.0 M NaCl fraction from control cells; lane
3, 1.5 M NaCl fraction from control cells;
lane 4, 0.5 M NaCl fraction from
heat-shocked cells; lane 5, 1.0 M
NaCl fraction from heat-shocked cells; lane 6,
1.5 M NaCl fraction from heat-shocked cells.
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Fig. 2.
The release of Myc-S100A13 in response to
temperature stress. A and B, S100A13 is
released with a Myc reporter in response to heat shock. Myc-S100A13 and
FGF1 NIH 3T3 cell cotransfectants and insert-less vector and FGF1 NIH
3T3 cell cotransfectants (A) and Myc-S100A13 NIH 3T3 cell
transfectants (B) were either subjected to heat shock (110 min at 42 °C) or incubated for 110 min at 37 °C. Conditioned
media were treated with 0.1% DTT, adsorbed to heparin-Sepharose, and
eluted at 1.5 M NaCl. Eluted proteins were concentrated,
resolved by 15% (w/v) SDS-PAGE, and analyzed by FGF1 and Myc
immunoblot analysis. A, upper panel, FGF1
immunoblot analysis; lower panel, Myc immunoblot analysis.
Lane 1, total cell lysate from Myc-S100A13 and
FGF1 NIH 3T3 cotransfectants; lane 2, total cell
lysate from insert-less vector and FGF1 NIH 3T3 cotransfectants;
lanes 3 and 4, media conditioned at 37 and 42 °C from Myc-S100A13 and FGF1 NIH 3T3 cotransfectants;
lanes 5 and 6, media conditioned at 37 and 42 °C from insert-less vector and FGF1 NIH 3T3 cotransfectants.
B, lane 1, total cell lysate from
Myc-S100A13 NIH 3T3 transfectants; lanes 2 and
3, media conditioned at 37 and 42 °C from Myc-S100A13 NIH
3T3 transfectants. C, the kinetics of FGF1 and Myc-S100A13
release in response to temperature stress. Myc-S100A13 and FGF1 NIH 3T3
cell cotransfectants were subjected to heat shock for 10 (lane 2), 20 (lane 3), 30 (lane 4), 60 (lane 5), and
90 (lane 6) min or incubated for 90 min at
37 °C (lane 1). Conditioned media were
processed as described above and analyzed by FGF1 (upper
panel) and Myc (lower panel)
immunoblot analysis.
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Fig. 3.
Pharmacology of FGF1 and Myc-S100A13 release
in response to temperature stress. A and B,
the ability of Myc-S100A13 to overcome the requirement for
transcription and translation for FGF1 release in response to
temperature stress. Myc-S100A13 and FGF1 NIH 3T3 cell cotransfectants
(B) and insert-less vector and FGF1 NIH 3T3 cell
cotransfectants (A) were incubated for 2 h at 37 °C
in the presence and absence of 10 µg/ml cycloheximide or 10 µg/ml
actinomycin D and then either subjected to heat shock (110 min at
42 °C) or incubated for 110 min at 37 °C in the presence and
absence of the same concentration of the two reagents. Conditioned
media were treated with 0.1% (w/v) DTT, adsorbed to heparin-Sepharose,
and eluted at 1.5 M NaCl. Eluted proteins were
concentrated, resolved by 15% (w/v) SDS-PAGE, and analyzed by FGF1 and
Myc immunoblot analysis. A, FGF1 immunoblot analysis.
B, upper panel, FGF1 immunoblot
analysis; lower panel, Myc immunoblot analysis.
Lanes 1 and 2, media conditioned at 37 and 42 °C from control cells; lanes 3 and
4, media conditioned at 37 and 42 °C from cells incubated
with cycloheximide; lanes 5 and 6,
media conditioned at 37 and 42 °C from cells incubated with
actinomycin D. C, effect of amlexanox, latrunculin,
2-deoxyglucose, and brefeldin A on FGF1 and Myc-S100A13 release in
response to temperature stress. Myc-S100A13 and FGF1 NIH 3T3 cell
cotransfectants were subjected to heat shock in the presence and
absence of 0.375 mM amlexanox, 400 nM
latrunculin, 50 mM 2-deoxyglucose, and 0.5 µg/ml
brefeldin A. For the evaluation of brefeldin A and 2-deoxyglucose
activity, the cells were pretreated prior to the heat shock with the
same concentrations of the reagents for 30 and 60 min, respectively,
and conditioned media were collected and processed as described above.
Upper panel, FGF1 immunoblot analysis; lower
panel, Myc immunoblot analysis: lanes 1 and
2, media conditioned at 37 and 42 °C from control cells;
lanes 3 and 4, media conditioned at 37 and 42 °C from amlexanox-treated cells; lanes
5 and 6, media conditioned at 37 and 42 °C
from latrunculin-treated cells; lanes 7 and
8, media conditioned at 37 and 42 °C from
2-deoxyglucose-treated cells; lanes 9 and
10, media conditioned at 37 and 42 °C from brefeldin
A-treated cells. The figure is a composite of several
experiments.
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Fig. 4.
Ammonium sulfate fractionation and the
release of FGF1 and Myc-S100A13 in response to heat shock.
A, solubility of Myc-S100A13 and FGF1 at ammonium sulfate
saturation. Myc-S100A13 and FGF1 NIH 3T3 cell cotransfectants and
insertless vector and FGF1 NIH 3T3 cell cotransfectants were subjected
to heat shock; conditioned media were collected and subjected to 100%
(w/v) ammonium sulfate fractionation; and pellet and supernatant
fractions were obtained by centrifugation. After DTT treatment, the
pellet and supernatant fractions were adsorbed to heparin-Sepharose,
eluted at 1.5 M NaCl, resolved by 15% (w/v) SDS-PAGE, and
evaluated by FGF1 and Myc immunoblot analysis. Upper panel,
FGF1 immunoblot analysis; lower panel, Myc immunoblot
analysis. Lanes 1 and 2, 37 and
42 °C supernatant fractions from Myc-S100A13 and FGF1 NIH 3T3 cell
cotransfectants; lanes 3 and 4, 37 and
42 °C pellet fractions from Myc-S100A13 and FGF1 NIH 3T3 cell
cotransfectants; lanes 5 and 6, 37 and
42 °C supernatant fractions from insert-less vector and FGF1 NIH 3T3
cell cotransfectants; lanes 7 and 8,
37 and 42 °C pellet fractions from insertless vector and FGF1 NIH
3T3 cell cotransfectants. B, solubility of Myc-S100A13 and
Cys-free FGF1 at ammonium sulfate saturation. Myc-S100A13 and Cys-free
FGF1 NIH 3T3 cell cotransfectants were subjected to heat shock;
conditioned media were collected and subjected to 100% (w/v) ammonium
sulfate fractionation; and pellet and supernatant fractions were
obtained as described above and analyzed by FGF1 and Myc immunoblot
analysis. Upper panel, FGF1 immunoblot analysis; lower
panel, Myc immunoblot analysis. Lanes 1 and
2, 37 and 42 °C pellet fractions from Myc-S100A13 and
Cys-free FGF1 NIH 3T3 cell cotransfectants; lanes
3 and 4, 37 and 42 °C supernatant fractions
from Myc-S100A13 and Cys-free FGF1 NIH 3T3 cell cotransfectants.
C, ability of Myc-S100A13 to export Cys-free FGF1 in
response to temperature stress. Myc-S100A13 and FGF1, Myc-S100A13 and
Cys-free FGF1, and insertless vector and Cys-free FGF1 NIH 3T3 cell
cotransfectants were subjected to heat shock, and conditioned media
were collected, treated with 0.1% (w/v) DTT, adsorbed to
heparin-Sepharose, and eluted with 1.5 M NaCl. The eluted
fractions were resolved by 15% (w/v) SDS-PAGE and analyzed by FGF1 and
Myc immunoblot analysis. Upper panel, FGF1 immunoblot
analysis; lower panel, Myc immunoblot analysis.
Lane 1, total cell lysate from Myc-S100A13 and
FGF1 NIH 3T3 cell cotransfectants; lane 2, total
cell lysate from insert-less vector and Cys-free FGF1 NIH 3T3 cell
cotransfectants; lane 3, total cell lysate from
Myc-S100A13 and Cys-free FGF1 NIH 3T3 cell cotransfectants;
lanes 4 and 5, media conditioned at 37 and 42 °C from Myc-S100A13 and FGF1 NIH 3T3 cell cotransfectants;
lanes 6 and 7, media conditioned at 37 and 42 °C from Myc-S100A13 and Cys-free FGF1 NIH 3T3 cell
cotransfectants; lanes 8 and 9, media
conditioned at 37 and 42 °C from insert-less vector and Cys-free
FGF1 NIH 3T3 cell cotransfectants.
88-98) lacking the last 11 residues at the carboxyl terminus of the protein and cotransfected FGF1
NIH 3T3 cell transfectants with this deletion mutant. Analysis of the
heparin affinity of the Myc-S100A13
88-98 mutant obtained from cell
lysates of the FGF1 and Myc-S100A13
88-98 NIH 3T3 cell
cotransfectants demonstrated that the Myc-S100A13
88-98 protein is
devoid of heparin affinity (data not shown), suggesting that the basic
residue-rich region of Myc-S100A13 may be responsible for its ability
to associate with heparin.
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Fig. 5.
Ability of
Myc-S100A13 88-98 to inhibit the release of
FGF1 in response to temperature stress. A, schematic
illustration of the domain structure of S100A13 (top) and
the deletion mutant S100A13
88-98 (bottom). B,
Myc-S100A13 and FGF1 and S100A13
88-98 and FGF1 NIH 3T3 cell
cotransfectants were subjected to heat shock, and, following DTT
treatment, conditioned media were either adsorbed to heparin-Sepharose
for evaluation of FGF1 release or concentrated and immunoprecipitated
with anti-Myc antibody for the evaluation of Myc-S100A13
88-98 and
Myc-S100A13 release. Eluted and immunoprecipitated proteins were
resolved by 15% (w/v) and 12% (w/v) SDS-PAGE, respectively, and
evaluated by FGF1 and Myc immunoblot analysis. Upper panel,
FGF1 immunoblot analysis; lower panel, Myc immunoblot
analysis. Lane 1, cell lysate from Myc-S100A13
and FGF1 NIH 3T3 cell cotransfectants; lane 2,
cell lysate from Myc-S100A13
88-98 and FGF1 NIH 3T3 cell
cotransfectants; lanes 3 and 4, media
conditioned at 37 and 42 °C from Myc-S100A13 and FGF1 NIH 3T3 cell
cotransfectants; lanes 5 and 6, media
conditioned at 37 and 42 °C from Myc-S100A13
88-98 and FGF1 NIH
3T3 cell cotransfectants.
88-98 NIH
3T3 cell cotransfectants to release FGF1 and the Myc-S100A13
88-98 deletion mutant in response to temperature stress. The FGF1 and Myc-S100A13 NIH 3T3 cell cotransfectants were used as a positive control. The cotransfectants were subjected to heat shock and, following DTT treatment, the conditioned media were either adsorbed to
heparin-Sepharose for the evaluation of FGF1 release or concentrated and immunoprecipitated with an anti-Myc antibody for an evaluation of
Myc-S100A13
88-98 release. FGF1 immunoblot analysis demonstrated that while FGF1 was present in media conditioned by temperature stress
from FGF1 and Myc-S100A13 NIH 3T3 cell cotransfectants, FGF1 was not
detected in media conditioned by heat shock from FGF1 and
Myc-S100A13
88-98 NIH 3T3 cell cotransfectants (Fig. 5B).
In contrast, however, Myc-S100A13
88-98 was present in media conditioned by heat shock from the FGF1 and Myc-S100A13
88-98 NIH
3T3 cell cotransfectants (Fig. 5C). These data suggest that the basic residue-rich domain in Myc-S100A13 may not be required for
the heat shock-induced export of Myc-S100A13, but this domain may be
necessary for the export of FGF1 into the extracellular compartment in
response to temperature stress.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(14), the
stress-induced release of Myc-S100A13 is sensitive to agents that
interfere with ATP biosynthesis and organization of the F-actin
cytoskeleton but is insensitive to disruption of intracellular
communication between the ER-Golgi apparatus. However, several
observations suggest that S100A13, unlike p40 Syt1, is able to
facilitate the release of FGF1. Indeed, expression of Myc-S100A13 was
able to overcome the inhibitory activity of actinomycin and
cycloheximide on FGF1 release and was able to induce the release of
Cys-free FGF1 in response to stress, suggesting a functional role for
S100A13 in the FGF1 release pathway as a potential modifier of a
stress-induced post-translational event.
heterodimer
formation (26). Additional evidence from S100 crystallographic studies
demonstrates that members of the S100 gene family are able to form
stable Ca2+-independent homodimers and through an
alteration in their conformation enable the two EF-hands in S100 to
hold polypeptides, such as annexin 2 (Anx2) (43), enabling the
carboxyl-terminal domain of the polypeptide to interact with other
proteins (17, 18). In studies utilizing the interaction between S100A11
and Anx1 (44, 45) as well as between S100A10 and Anx2 (43), the
orientation established by these conformational changes yields the
formation of stable heterotetramers
(S100A112·Anx12 and
S100A102·Anx22). In the situations with FGF1,
it is intriguing to speculate that S100A13 may be able to form similar
heterotetramers with these polypeptides. Indeed, this may explain how
S100A13 is able to facilitate the release of Cys-free FGF1, since it is
possible that when interacting with native FGF1, S100A13 may be able to orient its conformation to expose Cys30, which is not
exposed to solvent in its native conformation (46), for FGF1 homodimer
oxidation. In the absence of Cys30, S100A13 may enable
Cys-free FGF1 to form the noncovalent equivalent of the FGF1
Cys30 homodimer as a component of a
FGF2·S100A132 heterotetramer so that it can
be released in response to heat shock. This would be consistent with
the observation that the stress-induced release of Cys-free FGF1 and
Myc-S100A13 exhibits similar kinetics and responsiveness to
pharmacological agents including amlexanox, 2-deoxyglucose, brefeldin
A, cycloheximide, and actinomycin D (data not shown). In
addition, the S100A13-dependent increase in the solubility
of Cys-free FGF1 under conditions of 100% (w/v) ammonium sulfate
saturation also supports the premise that S100A13 may be able to
associate with the monomeric form of FGF1 in response to temperature
stress, and this association may involve the formation of a noncovalent
heterotetramer complex that may enable Cys-free FGF1 to access the
release pathway. Interestingly, the ability of S100A13 to force FGF1
into the supernatant fraction under conditions of saturated ammonium
sulfate fractionation would be consistent with this suggestion, since
solubility in ammonium sulfate is well described as being sensitive to
alterations in protein conformation (47).
88-98 mutant is released in response to heat
shock. This is consistent with the observations that members of the
S100 gene family lacking a basic residue-rich domain at their carboxyl terminus are released into the extracellular compartment following expression in mammalian cells (17, 18, 21) and may imply that the
carboxyl-terminal basic residue-rich domain in S100A13 functions to
associate with target proteins, while the remainder of the S100A13 may
be involved in penetration through the lipid bilayer. Indeed, S100B and
S100A10 do not contain a basic residue-rich domain, yet S100B is
released by glial cells (55, 56), and S100A10 has been reported not
only to associate with plasminogen in the extracellular compartment but
is able to stimulate tissue-dependent plasminogen
activation either alone or as a complex with Anx2 (57). These and other
studies (58, 59) have implicated the function of S100, Anx2, and
phosphatidylserine as key mediators of the extrinsic coagulation and
fibrinolytic systems on the surface of the endothelial cell (57-59).
Further, phosphatidylserine flipping from the inner leaflet to the
outer leaflet of the plasma membrane is known to be a regulator of
vascular hemostasis (60), and phosphatidylserine is also able to flip
to the outer leaflet of the plasma membrane in response to heat shock
(60). Because all of the known components of the stress-induced FGF1
release pathway have been characterized as phosphatidylserine-binding proteins (35, 53, 54) and S100A13 has been characterized as an
Anx2-binding protein (61), we anticipate that Anx2 and phosphatidylserine flipping may be involved in mediating the export of
these polypeptides.
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ACKNOWLEDGEMENTS |
---|
We thank the officers of Takeda
Pharmaceuticals Ltd. and Hoffman-LaRoche, Inc. for the generous supply
of amlexanox and IL-1 antibody, respectively, B. W. Schafer for
the murine S100A13 cDNA, and N. Albrecht for expert administrative assistance.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL32348 and AG98503 (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.
Supported in part by a fellowship from the Catholic University of Rome.
§ Present address: Dept. of Geriatric Medicine, University of Florence, School of Medicine, Florence, Italy 50139.
¶ To whom correspondence should be addressed: Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Dr., Scarborough, ME 04074. Tel.: 207-885-8200; Fax: 207-885-8179; E-mail: maciat@mmc.org.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M100546200
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ABBREVIATIONS |
---|
The abbreviations used are: FGF, fibroblast growth factor; DTT, dithiothreitol; ER, endoplasmic reticulum; IL, interleukin; PAGE, polyacrylamide gel electrophoresis; Syt, synaptotagmin.
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REFERENCES |
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