1 Center for Molecular Medicine, Maine Medical Center Research Institute,
Scarborough, ME 04074, USA
2 Department of Oncology, Catholic University of Rome, School of Medicine, Rome
00168, Italy
3 Department of Geriatric Medicine, University of Florence, School of Medicine,
Florence 50139, Italy
* Author for correspondence (e-mail: maciat{at}mmc.org)
Accepted 11 March 2003
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Summary |
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Key words: Copper, Fibroblast growth factor, Interleukin 1, S100A13, Tetrathiomolybdate
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Introduction |
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A wide variety of cell types have been shown to synthesize both IL-1
and FGF1; however, their two main sources in mammals are the peripheral blood
monocytes and tissue macrophages, which use a yet unidentified mechanism for
the export of both cytokines. In NIH 3T3 cells the release of FGF1 and
IL-1
is regulated by convergent yet distinct pathways that use cellular
stress to mediate their release into the extracellular compartment
(Tarantini et al., 1995
;
Tarantini et al., 2001
). It is
known that FGF1 is released in response to stress as a latent homodimer that
requires intracellular oxidation of a conserved cysteine residue at position
30 (Tarantini et al., 1995
).
This event enables FGF1 to interact with the extravesicular p40 domain of
synaptotagmin 1 (Syt1) and S100A13
(Carreira et al., 1998
;
Landriscina et al., 2001b
;
LaVallee et al., 1998
;
Tarantini et al., 1998
), and
these interactions facilitate the release of FGF1 as a multiprotein aggregate
containing p40 Syt1 and S100A13
(Landriscina et al., 2001a
).
Interestingly, whereas temperature stress induces the release of the mature
but not the precursor form of IL-1
from NIH 3T3 cells, the expression
of precursor IL-1
represses the release of FGF1 in response to stress
(Tarantini et al., 2001
).
Because (1) FGF1, S100A13 and Syt1 are Cu2+-binding proteins
(Engleka and Maciag, 1992;
Landriscina et al., 2001a
;
Shing, 1988
), (2)
Cu2+-induced oxidation facilitates the cell-free self assembly of
FGF1, p40 Syt1 and S100A13 as a multiprotein complex
(Landriscina et al., 2001a
),
(3) S100A13 expression facilitates the release of FGF1 independently of
transcription (Landriscina et al.,
2001b
), and (4) the Cu2+ chelator, tetrathiomolybdate
(TTM) inhibits the release of FGF1 in response to stress
(Landriscina et al., 2001a
),
it is likely that intracellular Cu2+ metabolism may be responsible
for the stress-induced oxidative event that facilitates the assembly and
release of the multiprotein FGF1 complex. Since FGF1 and IL-1
exhibit
remarkably similar crystallographic structures
(Graves et al., 1990
;
Zhu et al., 1991
), and both
FGF1 and IL-1
are released in response to stress from NIH 3T3 cells
(Tarantini et al., 2001
), we
questioned whether (1) intracellular Cu2+ is involved in the
release of IL-1
in response to stress, (2) the release of IL-1 from
human blood monocytes is also induced by cellular stress, (3) Cu2+
affinity is able to adsorb IL-1
from media conditioned by temperature
stress, (4) the release of IL-1
could be modified by the expression of
S100A13 and (5) whether IL-1
, like FGF1
(Mach and Middaugh, 1995
), is
endowed with molten globule character.
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Materials and Methods |
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Adenoviral transduction of NIH 3T3 cells
An adenoviral vector expressing Myc-S100A13 was prepared as described
(Hardy et al., 1997) at a
concentration of approximately 1013 viral particles per ml. NIH 3T3
cells were transduced by incubation in serum-free DMEM with approximately
103 viral particles and poly-D-lysine (Sigma) for two hours at
37°C. After infection, the adenovirus-containing media was removed and
replaced with serum-containing media (10% FBS) for 24 hours. The transduced
cells were removed by trypsin digestion and seeded for either heat shock or
immunoprecipitation experiments.
Ultracentrifugation and immunofluorescence analysis
Recombinant forms of IL-1 and either S100A13 or S100A13
BR
were incubated at molar ratios (IL-1
:S100A13) of 1:1, 1:5 and 1:10 in
phosphate-buffered saline (PBS) either in the presence or absence of 1 mM
CuCl2 for 30 minutes at 42°C followed by centrifugation at
280,000 g for 18 hours at 4°C and resolution of the pellet
fractions by S100A13 immunoblot analysis as described
(Landriscina et al., 2001b
).
Analysis of IL-1
-DsRed1 and EGFP-S100A13 intracellular trafficking was
performed using transient IL-1
-DsRed1 and EGFP-S100A13 NIH 3T3 cell
cotransfectants subjected to heat shock (42°C, 2h), followed by 4%
formaldehyde fixation and examination using the LTCS-SP confocal system
(Leica) equipped with an inverted DMIRBE microscope using an 100x
objective and the 237 µM confocal pinhole.
Analysis of protein interactions in heat-shock-conditioned media
The heat shock of human U937 and murine NIH 3T3 cells was performed as
previously described (Tarantini et al.,
1995) in serum-free cell medium for 2 hours at 42°C and
control cultures were incubated at 37°C in serum-free cell medium for also
2 hours. Two independent clones from each transfection have been evaluated
with similar results. For the analysis of the release of Myc-S100A13 and
IL-1
-ßGal, DTT-treated media conditioned by heat shock and cell
lysates from the appropriate NIH 3T3 cell transfectants were prepared and
divided into two portions, one of which was processed as described for S100A13
immunoblot analysis of the Myc reporter sequence
(Landriscina et al., 2001a
)
and the other for IL-1
-ßGal immunoblot analysis
(Tarantini et al., 2001
).
Briefly, one portion was concentrated and immunoprecipitated with an
anti-IL-1
antibody for the evaluation of mature IL-1
-ßGal
release, and the second portion was adsorbed to heparin-Sepharose and eluted
at 1.5 M NaCl for the evaluation of Myc-S100A13 release. IL-1
, S100A13
and FGF1 from human U937 cells were resolved by Cu2+-chelator
affinity chromatography (Hi Trap Chelation; Amersham Pharmacia Biotech) and
eluted with 60 mM imidazole. Immunoprecipitated and eluted proteins were
resolved by 8% and 12% acrylamide SDS-PAGE, respectively, and evaluated by
either IL-1
(Tarantini et al.,
2001
) or Myc (Landriscina et
al., 2001b
) immunoblot analysis. The activity of lactate
dehydrogenase in conditioned media was used as an assessment of cell lysis in
all experiments as previously reported
(Tarantini et al., 2001
). The
effects of actinomycin D (Sigma), cycloheximide (Sigma), tetrathiomolybdate
(Sigma-Aldrich) and ZLL (Biomol, USA) on IL-1
release were evaluated as
previously reported (LaVallee et al.,
1998
).
Analysis of the molten globule character of IL-1
Dioleylphosphoglycerol (DOPG) was purchased from Avanti Polar Lipids and
carboxyfluorescein (CF) was purchased from Molecular Probes. For liposome
preparation, DOPG was dried under a nitrogen stream and resuspended in an
aquous 100 mM CF solution at pH 7.0 by vortexing and the lipid suspension
sonicated for 30 minutes, extruded through an Avanti Polar Lipids Miniextruder
and passed on a 10 ml dextran desalting column equilibrated in 10 mM HEPES
containing 150 mM NaCl at pH 7.0 (Pierce, USA). The fluorescence of the
liposomes was monitored in each experiment for 10 minutes by a Fluorolog 3
fluorescent spectroscope (Jobin Yvon, Edison, NJ) at an excitation wavelength
470 nm and emission wavelength 520 nm. The temperature was maintained at
50°C by a Peltier system. -Chymotrypsin served as a negative
control and 0.1% Triton X-100 served as a positive control for micelle lysis.
Human recombinant IL-1
or
-chymotrypsin at different
concentrations were added to the quartz cuvette at the 2 minute time point of
each experiment.
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Results |
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|
Human U937 cells release IL-1 and S100A13 in response to
temperature stress in a Cu2+-dependent manner
While the NIH 3T3 cell has proven to be a useful cell culture system to
study the release of FGF1 and IL-1, we sought to determine whether this
pathway was also used by more physiologic cell types. Since mononuclear cells
are a rich source of both FGF1 and IL-1
, we evaluated the ability of
heat shock to induce the release of FGF1 and IL-1
from PMA-induced
human U937 cells. As shown in Fig.
1B, IL-1
immunoblot analysis of
Cu2+-affinity-adsorbed cell culture media conditioned by heat shock
but not at 37°C from U937 cells, exhibited the presence of both precursor
(
33 kDa) and mature (
19 kDa) forms of IL-1
as
Cu2+-binding proteins. In this experiment, we identified the 33 kDa
protein as the precursor form of IL-1
and not as a reduction-resistant
dimer of mature IL-1
since (1) the Cu2+-induced dimer of the
mature form of IL-1
does not exhibit any affinity for immobilized
Cu2+ and (2) the recombinant form of pIL-1
exhibited similar
Cu2+ affinity to the mature form of IL-1
(data not shown).
Temperature stress also induced the release of FGF1 from human U937 cells
stably transfected with FGF1 (Fig.
1B).
Since these data suggest that both the precursor and the mature forms of
IL-1 are Cu2+-binding proteins, and Cu2+ is able
to regulate the stress-induced release of FGF1
(Tarantini et al., 1995
), we
assessed the ability of the Cu2+ chelator, tetrathiomolybdate (TTM)
to repress the release of IL-1
from both U937 and NIH 3T3 cells in
response to heat shock by the treatment of PMA-activated human U937 cells and
murine NIH 3T3 cells for 18 hours with TTM prior to temperature stress. As
shown in Fig. 1B,C, TTM was
able to inhibit the release of IL-1
at 250 nM from both U937 and NIH
3T3 cells and this is consistent with the concentration of TTM used in
preclinical and clinical studies for the management of solid tumor growth
(Brewer et al., 2000
;
Cox et al., 2001
;
Pan et al., 2002
).
Interestingly, as shown in Fig.
1B, heat shock of PMA-induced U937 cells also enabled the
Cu2+-dependent export of S100A13, a component of the FGF1
multiprotein release complex (Landriscina
et al., 2001b
).
It has been extensively demonstrated that the release of the mature form of
IL-1 from monocytes is highly dependent on its processing from its
precursor form by calpain (Kobayashi et
al., 1990
). In contrast, fibroblasts including NIH 3T3 cells are
not able to process the precursor forms of the IL-1 proteins
(Siders et al., 1993
;
Tarantini et al., 2001
).
Because we have previously demonstrated that NIH 3T3 cells are not able to
export the precursor form of IL-1
in response to temperature stress
(Tarantini et al., 2001
), we
used the lack of precursor IL-1
processing and export in NIH 3T3 cells
as a resource to further study the release of mature IL-1
-independent
of precursor IL-1
processing. As shown in
Fig. 1C, the stress-induced
release of the mature form of IL-1
from NIH 3T3 cells does not involve
intracellular calpain activity and has the same Cu2+-dependent
characteristics for IL-1
export as reported by human U937 cells
(Fig. 1B). These observations
reinforce the premise that the stress-induced pathways responsible for the
regulation of IL-1
by murine NIH 3T3 and human U937 cells may indeed be
similar.
IL-1 uses S100A13 for stress-induced release
Because FGF1 uses the function of the S100A13 gene product to facilitate
its release in response to stress
(Landriscina et al., 2001b)
and S100A13 is released from activated human U937 cells in response to heat
shock in a Cu2+-dependent manner, we questioned whether IL-1
could also use S100A13 for its export into the extracellular compartment. To
address this premise, we examined, by using a cell-free system, the ability of
the recombinant forms of IL-1
and S100A13 to interact and form a
Cu2+- and molar ratio-dependent multiprotein aggregate that would
be susceptible to ultracentrifugation. As shown in
Fig. 2A (right panel), S100A13,
by itself, was not susceptible to precipitation following centrifugation at
280,000 g for 18 hours either in the presence or absence of
Cu2+. Indeed, S100A13 was only present in the pellet fraction when
incubated with the mature form of IL-1
in the presence of
Cu2+ (Fig. 2A, left
panel). In addition, the level of S100A13 present in the pellet fraction
increased as a function of the IL-1
to S100A13 molar ratio with a
maximum between a molar ratio of 1:5 to 1:10
(Fig. 2A), which suggests that
IL-1
and S100A13 may be able to interact in a Cu2+-dependent
manner (Fig. 2A).
|
Since expression of signal peptide-less S100A13 protein in NIH 3T3 cells
leads to its constitutive release at 37°C
(Landriscina et al., 2001b),
and IL-1
and S100A13 are able to interact in a cell-free system in a
Cu2+-dependent manner, we questioned whether the expression of the
mature form of IL-1
in the NIH 3T3 cell could also repress the
constitutive release of intracellular S100A13 at 37°C. Thus, S100A13
containing an N-terminal Myc-epitope tag was transfected into mature
IL-1
-ßGal NIH 3T3 transfectants
(Tarantini et al., 2001
) and
the stable cotransfectants were either maintained at 37°C for 2 hours or
subjected to heat shock. Insert-less vector and mature IL-1
-ßGal
cotransfectants served as controls. As shown in
Fig. 2B, we observed that the
expression of the mature form of IL-1
was able to repress the
constitutive release of Myc-S100A13 at 37°C.
Because these results suggest that IL-1 and S100A13 may be able to
associate with each other in the intracellular compartment in response to
temperature stress, we performed immunoprecipitation and immunoblot analysis
of cell lysates obtained from stable NIH 3T3 cell mature IL-1
transfectants, adenovirally transduced with Myc epitope-tagged S100A13. As
shown in Fig. 2C,
immunoprecipitation of cell lysates with an anti-Myc antibody followed by
IL-1
immunoblot analysis resolves the presence of the mature form of
IL-1
in the Myc immunoprecipitant fraction. However, this interaction
was not observed in cell lysates obtained from the Myc-S100A13-transduced
control NIH 3T3 cells (data not shown).
We also questioned whether the expression of S100A13 as a
Cu2+-binding protein could overcome the requirement for heat
shock-induced transcription by examining the ability of actinomycin D to
repress the export of an IL-1:ß-Gal chimera into the extracellular
compartment when expressed in a stable S100A13 background. As shown in
Fig. 2D, whereas actinomycin D
was able to repress the release of IL-1
-ßGal from insert-less
vector and IL-1
-ßGal NIH 3T3 cell cotransfectants, actinomycin D
was unable to repress the export of IL-1
-ßGal from Myc-S100A13 and
IL-1
-ßGal NIH 3T3 cell cotransfectants in response to heat shock.
In addition, TTM was able to repress the release of IL-1
-ßGal in
response to temperature stress with data similar to those shown in
Fig. 1A using human U937 cells,
and similar results were obtained when cycloheximide was used to inhibit
translation in the stable S100A13 background (data not shown).
Temperature stress induces the cytosolic redistribution of both
IL-1 and S100A13
In order to confirm the biochemical data suggesting an interaction between
IL-1 and S100A13, we obtained transient NIH 3T3 cell cotransfectants
expressing IL-1
-DsRed1 and EGFP-S100A13 chimeric constructs. These
cells were subjected to heat shock and examined for the localization of the
reporter genes using confocal fluorescence microscopy. As shown in
Fig. 3, both IL-1
and
S100A13 exhibited a diffuse cytosol pattern of intracellular distribution at
37°C and this contrasted with the peripheral distribution of both
polypeptides in response to heat shock. This redistribution was observed in
approximately 15% of the transient NIH 3T3 cell cotransfectants and the
fluorescent reporter proteins, EGFP and DsRed1, did not exhibit this
redistribution in response to temperature stress (data not shown). Overlay of
the DsRed1 and EGFP signals demonstrated a significant level of colocalization
(Fig. 3), which suggests that
IL-1
and S100A13 can associate with each other near the inner surface
of the cell periphery in response to heat shock.
|
A S100A13 mutant lacking the basic residue-rich domain is a
dominant-negative regulator of stress-induced IL-1 release
Because our data argue that intracellular IL-1 and S100A13 may be
able to associate in response to temperature stress, we sought to define the
domain in S100A13 responsible for this association. We examined the basic
residue (BR)-rich domain at the C-terminus of S100A13, since this domain is
novel among the various members of the S100 gene family
(Wicki et al., 1996b
). Thus we
deleted the last eleven residues in S100A13 and assessed the ability of the
recombinant form of S100A13
BR to associate in a
Cu2+-dependent manner with IL-1
in a cell-free system. As
shown in Fig. 4A, the
S100A13
BR failed to precipitate the mature form of IL-1
in the
presence of Cu2+. Furthermore, like S100A13
(Landriscina et al., 2001b
),
the recombinant form of S100A13
BR eluted from immobilized
Cu2+ at 35 mM imidazole (data not shown) suggesting that the basic
residue-rich domain is not involved in Cu2+-binding. In addition,
we prepared a S100A13
BR mutant chimera containing a multiple Myc
epitope tag (Myc-S100A13
BR), obtained stable Myc-S100A13
BR and
IL-1
-ßGal NIH 3T3 cell cotransfectants, and evaluated the ability
of the IL-1
-ßGal and Myc-S100A13
BR NIH 3T3 cell
cotransfectants to release mIL-1
-ßGal in response to temperature
stress. As shown in Fig. 4B,
IL-1
-ßGal was not detected in media conditioned by heat shock from
IL-1
-ßGal and Myc-S100A13
BR NIH 3T3 cell cotransfectants.
We also examined whether the expression of Myc-S100A13
BR could function
as a repressor of S100A13 release in response to heat shock and as shown in
Fig. 4C, media conditioned by
temperature stress from the S100A13
BR and IL-1
-ßGal NIH 3T3
cell cotransfectants adenovirally transduced with wild type MycS100A13,
exhibit significantly reduced levels of wild type Myc-S100A13.
|
Recombinant mature IL-1 exhibits molten globule character
Since IL-1 and FGF1 (Prudovsky
et al., 2002
) translocate near the inner surface of the plasma
membrane prior to stress-induced release and FGF1 assumes molten globule
character near 42°C (Mach and
Middaugh, 1995
), a novel feature that may enable it to associate
with and traverse lipid bilayers (Chi et
al., 2001
; Samuel et al.,
2000
; Srimathi et al.,
2002
) at elevated temperatures, we questioned whether the mature
form of IL-1
is also able to exibit molten globule character. We
examined the ability of IL-1
to induce liposome leakage, which would
reflect its solubility within the lipid bilayer. We used fluorescence
spectroscopy to detect the leakage of the fluorescent probe, carboxyfluorescin
(CF), from dioleyl-phosphoglycerol (DOPG) liposomes at 50°C, a temperature
used to exhibit molten globule character in cell-free lipid micelles
(Mach and Middaugh, 1995
). In
this situation, CF trapped within the vesicles is self-quenching at 100 mM but
becomes highly fluorescent when released into the environment. As shown in
Fig. 5, we observed a
significant increase of the fluorescence signal after addition of recombinant
mature IL-1
. This increase in fluorescence was dependent on the
concentration of IL-1
and the addition of 0.5 µM IL-1
was as
efficient as 0.1% Triton X-100 (positive control) for the release of CF from
the DOPG liposomes. We used
-Chymotrypsin as a negative control for
these experiments and its addition (0.5 µM) to the liposomes did not
exhibit increase in fluorescence (Fig.
5).
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Discussion |
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It is well established that the prototype members of the IL-1 gene family
are potent inducers of the latent transcription factor, NF-B
(Baldwin, 1996
;
Beg et al., 1993
). Indeed,
human mononuclear cells are known to be: (1) a rich source of FGF1
(Wahl, 1984
); (2) involved in
the delivery of FGF1 to sites of inflammation
(Sano et al., 1990
;
Remmers et al., 1991
;
Sano et al., 1992
), and (3)
are a target for extracellular IL-1
as a chemoattractant
(Torisu et al., 2000
).
Further, IL-1
is also able to induce the expression of vascular
endothelial growth factor (VEGF) and FGF2 in human endothelial cells
(Ko et al., 1999
;
Torisu et al., 2000
). Thus it
is possible that the response of the tumor microvasculature to stress involves
the release of IL-1
, which may be responsible for the recruitment of
mononuclear cells into the tumor microenvironment in order to deliver and
release FGF1. Since TTM therapy in humans significantly reduces the serum
levels of the FGF prototypes as well as reducing the level of the IL-1
prototypes in media conditioned by breast tumor cells
(Pan et al., 2002
), it is
possible that TTM administration may repress the export of IL-1
within
the tumor microvasculature in vivo. This may further limit the recruitment of
mononuclear cells to sites within the tumor microenvironment where the release
of FGF1 may foster the angiogenic potential of VEGF within the
microvasculature and aid in the promotion of tumor cell proliferation and
survival. This premise is consistent with the recent observation that TTM
treatment is able to suppress the transcriptional activity of NF-
B in
tumor cells in vivo (Pan et al.,
2002
), since the repression of the stress-induced release of
IL-1
by TTM could significantly contribute to the downregulation of
microvascular cell and tumor-derived NF-
B expression. Likewise, recent
studies suggest that Cu2+-chelation may represent an alternative
approach to the management of ß-amyloid deposition in a transgenic mouse
model of Alzheimer disease (Cherny et al.,
2001
). Since IL-1
is significantly increased in the brains
of Alzheimer disease patients (Griffin et
al., 1989
) and is able to induce the expression of the
ß-amyloid precursor gene in human endothelial cells
(Goldgaber et al., 1989
), it
may be likely that the therapeutic effect of Cu2+ chelators in
Alzheimer disease may be due to their ability to attenuate the release of
IL-1
.
It is also well established that mononuclear cells can release both the
precursor and the mature form of IL-1 as biologically active cytokines
in vitro (Siders et al.,
1993
). However, Dinarello has suggested that, since the precursor
form of IL-1
remains bound to the cell surface following its release,
whereas the mature form is predominantly present in biological fluids in vivo,
this may indeed represent evidence for the preferential release of the mature
form of IL-1
(Dinarello,
1994
). Indeed, the lack of pIL-1
release from NIH 3T3 cells
in response to cellular stress (Tarantini
et al., 2001
) is consistent with the data of others
(Siders et al., 1993
) showing
that a variety of different cell types including keratinocytes and
fibroblasts, prefer to release the mature forms of the IL-1 prototypes.
Moreover, the release of the mature forms of IL-1
as a result of
pIL-1
processing may also be dependent upon the activity of
intracellular calpain. Therefore the observation that pIL-1
NIH 3T3
cell transfectants do not contain the mature form of IL-1
in their
cytosol (Tarantini et al.,
2001
) may be due to either their deficiency in the expression of
the appropriate calpain gene product or the presence of high levels of the
calpain inhibitor, calpastatin.
Because S100A13 and IL-1 share similar characteristics of
stress-induced release from human U937 and murine NIH 3T3 cells and S100A13 is
an important component of the multiprotein complex involved in the
stress-induced release of FGF1, we suggest that intracellular S100A13 may be
involved in the formation of a Cu2+-dependant IL-1
:S100A13
heterotetramer that facilitates the export of both polypeptides. Indeed, this
suggestion is reinforced by the observations that IL-1
and S100A13 are
able to form a multiprotein Cu2+-dependent complex in vitro as well
as in a cell-free system, and IL-1
may be able to access intracellular
S100A13 near the inner surface of the plasma membrane. Interestingly,
experimental evidence has established a similar putative model of interaction
between S100 dimers and their target proteins, the annexin gene family members
(Rety et al., 1999
). Thus,
upon divalent ion binding, each S100A11 monomer opens up to accommodate a
target annexin II monomer. In this way, an S100 dimer functionally
crossbridges two homologous target molecules and forms a tight
heterotetrameric complex, which is able to associate with the cytoskeleton and
the lipid bilayer of the plasma membrane
(Schafer and Heizmann, 1996
).
Interestingly, the S100:annexin 2 heterodimer complex has been reported to
associate with phosphatidylserine and function as a key mediator of the
extrinsic coagulation and fibrinolytic systems on the surface of endothelial
cells in response to temperature stress
(Kim and Hajjar, 2002
).
Further, phosphatidylserine is able to flip from the inner to the outer
leaflet of the plasma membrane in response to cellular stress and this feature
is known to be a regulator of stress-mediated vascular endothelial cell
activation in vivo (Ishii et al.,
2001
).
Previous observations from our laboratory suggested that unlike the
stress-induced FGF1 release pathway, the IL-1 export pathway does not
use the function of Syt1 since a Syt1 mutant lacking the
Ca2+-binding C2A domain, which is able to function as a
dominant-negative effector of FGF1 release
(La Vallee et al., 1998
), does
not affect the stress-induced release of IL-1
from NIH 3T3 cells
(Tarantini et al., 2001
).
However, the use of intracellular Cu2+ to facilitate the
stress-induced interaction between IL-1
and S100A13 is a feature that
is conserved between IL-1
and FGF1 release pathways. Although our
observations further suggest that the assembly of the stress-induced
IL-1
:S100A13 complex occurs prior to release, it is not yet known how
this complex is able to traverse the plasma membrane. However, the ability of
IL-1
to associate with and displace CF from an intra-DOPG micelle
locale suggests that, like FGF1 (Mach et
al., 1993
), IL-1
may be endowed with molten globule
character: a biophysical characteristic in which partially unfolded protein
conformations formed as a result of the transition from high tertiary to low
tertiary structure are able to use their secondary structure to achieve low
solubility in aqueous environments, resulting in their association with and
transversion through acidic phospholipid bilayers.
Our data also suggest that the C-terminal basic-rich (BR) domain of S100A13
may be responsible for its interaction with the mature form of IL-1 but
not with Cu2+. Indeed, the C-terminus of other S100 gene family
members have been implicated in mediating their ability to interact with
proteins (Kilby et al., 1996
;
Pozdnyakov et al., 1998
;
Rety et al., 1999
;
Schafer and Heizmann, 1996
).
Interestingly, unlike other S100 gene family members, S100A13 contains a nine
amino acid basic residue-rich domain, which is absent in other S100 gene
family members (Schafer and Heizmann,
1996
; Wicki et al.,
1996a
) with the exception of the recently identified S100A14,
which contains a basic residue-rich domain at its C terminus
(Pietas et al., 2002
). Because
members of the S100 gene family lacking a basic residue-rich domain at their
C-terminus are also exported to the extracellular compartment
(Schafer and Heizmann, 1996
),
we suggest that this domain may be responsible for the association of S100A13
with IL-1
. In addition, we also suggest that the remainder of the
S100A13 structure may be involved in the mechanism to facilitate the
traversion of the S100A13:IL-1
complex through the lipid bilayer and
enable the release of the multiprotein complex. Thus we anticipate that like
IL-1
and FGF1, S00A13 may also exhibit molten globule character.
We also suggest that the stress-induced interaction between the mature form
of IL-1 and the BR domain of S100A13 promotes the release of both
proteins as a Cu2+-dependent complex in which this heterotetramer
is formed by the ability of both proteins to bind Cu2+. It is also
noteworthy that the expression of S100A13 in an IL-1
background results
in an attenuation of the sensitivity of the IL-1
release pathway to the
transcription inhibitor actinomycin D. Since the transcription of the S100A13
gene is not regulated by heat shock (data not shown), it is likely that the
role of cellular stress in the export of the mature form of IL-1
may
not be due to the induction of a classical stress-mediated transcriptional
response; rather, the stress response may involve the regulation of a
post-translational activity that modifies S100A13. Although we do not know the
nature of this putative post-translational activity, our data suggest that the
oxidative character of intracellular Cu2+ is involved in the
regulation of this feature and this may indeed be the responsibility of the
intracellular transport of Cu2+ to both IL-1
and S100A13
that is susceptible to chelation by TTM.
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Acknowledgments |
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