From the Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04074
Received for publication, October 20, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Interleukin (IL)1 The interleukin(IL)1-1
gene family is currently comprised of eight members, and these include
the IL1 prototypes, IL1 Amino acid sequence (10) and x-ray crystallographic analysis (11) of
the IL1 and FGF prototypes have revealed rather striking structural
similarities between members of the two gene families. Like the
majority of the IL1 gene family members (1, 2), the FGF gene family
prototypes, FGF1 and FGF2 also lack a classical signal peptide sequence
to direct secretion through the ER-Golgi apparatus (12). Whereas the
release of FGF1 is regulated by a variety of stress conditions
(13-18), the release of FGF2 is not regulated by stress (19), and FGF2
contains structural features that repress the stress-induced release of
FGF1 (19). Because (i) the release pathways utilized by the FGF
prototypes have diverged, and (ii) it is unlikely that the pathway
responsible for the regulation of FGF1 would have evolved independent
of the mechanisms utilized by other signal peptide-deficient gene
products to gain access to the extracellular compartment, we sought to
determine whether other signal peptide-deficient cytokines are
also able to utilize the FGF1 release pathway for export. We focused
our effort on IL1 Plasmids, Transfections, and Cell Culture--
Human
pIL1
Transfectants were grown to 70-80% confluency and prior to the
temperature stress, the cells were washed with DMEM. The heat shock was
performed in DMEM at 42 °C for 2 h, as previously described (12). When FGF1 transfectants were subjected to temperature stress, the
heat shock medium was supplemented with 4 units/ml heparin (Upjohn
Co.). The effects of Brefeldin A (Epicenter Technologies), 2-deoxyglucose (Sigma), and amlexanox (Takeda) on mIL1 Processing of Cell Lysates and Conditioned Medium and Immunoblot
Analysis--
Prior to and after temperature stress, total cell
lysates were obtained by sonication of cell pellets collected in
Germino buffer DT (10 mM Tris, pH 7.6, containing 250 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10 mM 2-mercaptoethanol, and 0.1%
(v/v) Triton X-100) as described previously (26). Conditioned medium
was collected, filtered through a 0.22-µm filter, and then treated
with either dithiothreitol (DTT, Sigma) and
(NH4)2SO4 (Sigma) or were left untreated. DTT treatment was performed at a concentration of 0.1% (w/v) DTT at 37 °C for 2 h, and
(NH4)2SO4 fractionation was
performed at 95% (w/v) saturation as described previously (18). Cell
lysates and DTT- or
(NH4)2SO4-treated medium from
mIl1
Prior to immunoprecipitation, the cells were washed and scraped in cold
phosphate-buffered saline, and cell pellets were obtained by
centrifugation as described previously (26). The pellets were
resuspended in 1 ml of cold NP 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, 2 µg/ml aprotinin, 2 µg/ml leupeptin).
Conditioned medium with or without DTT treatment were filtered through
a 0.22-µm filter and concentrated using a centrifugal filter device
(Ultrafree-15, Millipore) to a volume of ~500 µl. 2 µg/ml
aprotinin, 2 µg/ml leupeptin, and 5 µl of goat anti-human IL1
Analysis of the pIL1 We evaluated the ability of human IL1 mediates proinflammatory
events through its extracellular interaction with the IL1 type I
receptor. However, IL1
does not contain a conventional signal
peptide sequence that provides access to the endoplasmic
reticulum-Golgi apparatus for secretion. Thus, we have studied the
release of the precursor (p) and mature (m) forms of IL1
from NIH
3T3 cells. We have demonstrated that mIL1
but not pIL1
was
released in response to heat shock with biochemical and pharmacological
properties similar to those reported for the stress-mediated release
pathway utilized by fibroblast growth factor (FGF)1. However, unlike
the FGF1 release pathway, the IL1
release pathway appears to
function independently of synaptotagmin (Syt)1 because the expression
of a dominant-negative form of Syt1, which represses the release of
FGF1, did not inhibit the release of mIL1
in response to temperature
stress. Interestingly, whereas the expression of both mIL1
and FGF1
in NIH 3T3 cells did not impair the stress-induced release of either
polypeptide, the expression of both pIL1
and FGF1 repressed the
release of FGF1 in response to temperature stress. These data suggest
that the release of mIL1
requires proteolytic processing of its
precursor form and that mIL1
and FGF1 may utilize similar but
distinct mechanisms for export.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and IL1
, as well as the IL1 receptor
antagonist RA (1), IL-18, and the recently identified members, FIL1
,
FIL1
, FIL1
, and FIL1
(2). The IL1 prototypes are translated as
31-34-kDa precursor proteins that are cleaved by two distinct specific
proteases to produce mature 17-kDa forms of the IL1 prototypes from the
C-terminal end of the precursor (3). Whereas precursor (p) IL1
is
biologically inactive until it is processed into the mature (m) form by
the IL1
-converting enzyme (ICE) (4, 5), pIL1
is biologically active (5). Precursor IL1
is recognized by a
calcium-dependent protease of the calpain family, and this
cleavage results in the formation of the mature counterpart (6).
Interestingly, the N-terminal fragment derived from pIL1
proteolytic
processing contains a functional nuclear localization signal (7) and
can be translocated to the nucleus (8). The ability of pIL1
to bind
the IL1 type I receptors with high affinity, with subsequent activation
of the signal transduction pathway, and the presence of the nuclear
localization sequence anticipates the existence of a biological role
for pIL1
, independent from the activity of mIL1
. Indeed,
comparative studies using the pIL1
and mIL1
forms have suggested
that pIL1
, but not mIL1
, is a negative regulator of cell
migration (9).
because its precursor form is biologically
functional (5, 20), and extracellular IL1
is well described as an
antagonist of FGF-dependent biological activities (21-24).
We report that the release pathway utilized by IL1
exhibits similar
biochemical, pharmacological, and biological properties to the FGF1
release pathway. In contrast, unlike the FGF1 release pathway (16, 17), IL1
does not require the function of synaptotagmin (Syt)1 but does
require the function of an intracellular protease to convert pIL1
to
mIL1
, because only mIL1
is released in response to heat shock.
Lastly, the stress-induced IL1
and FGF1 release pathways may be
convergent, because the expression of pIL1
acts as a
dominant-negative repressor of FGF1 release.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-(1
271) and mIL1
-(113-271) cDNAs (7), as well
as pIL1
·
-gal and mIL1
·
-gal fusion constructs, were
inserted into the expression vector, pMEXneo (25), and stable NIH 3T3 cell transfectants were obtained as previously described (7). The
transfectants were grown on cell culture dishes coated with 10-µg/cm2 fibronectin in Dulbecco's modified Eagle's
medium (DMEM, Cellgro) supplemented with 10% (v/v) bovine calf serum
(BCS, HyClone), 1× antibiotic/antimycotic (Life Technologies, Inc.),
and 400 µg/ml geneticin (G418, Life Technologies, Inc.). The Syt1
constructs, full-length rat p65 Syt1 and its deletion mutant
Syt1
(120-214) in the expression vector pMEXneo/hygro, were obtained
as described (17). The Syt1 constructs were cotransfected into
mIL1
·
-gal NIH 3T3 cell transfectants using 5 µg of DNA mixed
with the multicomponent lipid-based transfection reagent FuGENE6 (Roche
Molecular Biochemicals) following the manufacturer's instructions.
Stable cotransfectants were grown on fibronectin-coated (10 µg/cm2) cell culture dishes in DMEM, supplemented with
10% (v/v) bovine calf serum, 1× antibiotic/antimycotic, 400 µg/ml G418, and 200 µg/ml hygromycin (Roche Molecular
Biochemicals). The human FGF1 construct in the expression vector,
pcDNA3.1/Hygro (Invitrogen) was obtained by digesting the pXZ38
plasmid (12) with EcoRV and HpaI (New England
BioLabs Inc.), isolating the 836-base pair fragment containing FGF1 by
electroelution, ligating into the pcDNA3.1/Hygro expression vector
previously digested with EcoRV, and purifying by
electroelution. The FGF1 pcDNA3.1/Hygro construct was cotransfected
into mIL1
·
-gal and pIL1
·
-gal NIH 3T3 cell transfectants
using 5 µg of DNA mixed with the multicomponent lipid-based
transfection reagent, FuGENE6, following the manufacturer's instructions. Stable cotranfectants were selected and grown as described above. Whereas multiple clones were obtained in the mIL1
,
FGF1, and mIL1
·FGF1 backgrounds, we were limited to studying a
single clone in the pIL1
and pIL1
·FGF1 backgrounds.
release were
evaluated as previously described (17-18).
·
-gal and pIL1
·
gal NIH 3T3 cell transfectants were
further processed by affinity chromatography using a 0.5-ml
p-aminobenzyl 1-thio-
-D-galactopyranoside column (Sigma) previously equilibrated with Germino buffer DT. The
column was washed with Germino buffer DT, followed by a wash with
Germino buffer DT. The proteins were eluted with 2 ml of 0.1 M sodium borate, pH 10 and were concentrated by
centrifugation using Centricon 30 concentrators (Amicon, Inc.).
polyclonal antibodies (1.21 mg/ml, a generous gift from Dr. R. Chizzonite, Hoffmann-La Roche Inc.) were added to either cell lysates
or conditioned medium, and the samples were rotated at 4 °C for
18 h. Protein A-Sepharose (Amersham Pharmacia Biotech) was added,
and the samples were rotated at 4 °C for an additional 2 h. All
IL1
samples either eluted from the p-aminobenzyl 1-thio-
-D-galactopyranoside column or purified by
immunoprecipitation were resolved by either 15% (w/v) SDS-PAGE (native
forms of pIL1
and mIL1
) or 8% (w/v) SDS-PAGE (pIL1
·
-gal
and IL1
·
-gal fusion proteins) and subjected to immunoblot
analysis as previously described (9). Briefly, proteins were
transferred to a nitrocellulose membrane (Hybond C, Amersham Pharmacia
Biotech) and probed with goat anti-human IL-1
polyclonal antibody at
a 1:1200 dilution. IL-1
specific bands were visualized by
chemiluminescence (ECL, Amersham Pharmacia Biotech) following the
manufacturer's instructions.
·
-gal/FGF1 and
mIL1
·
-gal/FGF1 NIH 3T3 cell cotransfectants was accomplished by
dividing individual cell lysates and conditioned medium in half for the
detection of FGF1 and IL1
immunoreactive bands. For FGF1 analysis,
total cell lysates were obtained by sonication in cold lysis buffer containing 1% (v/v) Triton X-100 (Sigma) and adsorbed to a 1-ml heparin-Sepharose CL-6B column (Amersham Pharmacia Biotech), previously equilibrated with 50 mM Tris pH 7.4 containing 10 mM EDTA (TEB). The column was washed with TEB, and the
proteins were eluted with 2 ml of TEB containing 1.5 M
NaCl. The volume of the eluates was reduced using Centricon 10 concentrators (Amicon), and the eluates were analyzed by 15% (w/v)
SDS-PAGE followed by immunoblot analysis using anti-human FGF1
polyclonal antibodies (27) as described previously (16). IL1
was
analyzed by immunoprecipitation using anti-IL1
antibody and resolved
by 8% (w/v) SDS-PAGE followed by immunoblot analysis, as described
above. Cell lysates from mIL1
·
-gal, mIL1
·
-gal/p65 Syt1,
and mIL1
·
-gal/Syt1
(120-214) mutant NIH 3T3 cell
cotransfectants were obtained by sonication in NP lysis buffer and
processed as described below. Conditioned medium from these
cotransfectants were treated with 0.1% (w/v) DTT and divided into two
equal samples. The first sample was adsorbed to heparin-Sepharose CL-6B
(1 ml) and washed with TEB, and samples were eluted with 1.5 M NaCl, as reported above. Cell lysates and conditioned
medium purified by heparin affinity were resolved by 10% (w/v)
SDS-PAGE and subjected to immunoblot analysis using a rabbit anti-rat
Syt1 polyclonal antibody as described previously (17). The second
sample was processed by IL1
immunoprecipitation as described above.
As a negative control, cell lysates were also immunoprecipitated with a
control antibody (goat IgG purified immunoglobulin from pooled normal
goat serum, Sigma). Cell lysates and conditioned medium purified by
IL1
immunoprecipitation were resolved by 8% (w/v) SDS-PAGE,
followed by IL1
immunoblot analysis, as described above. IL1
and
Syt1 specific bands were visualized by chemiluminescence (ECL)
following the manufacturer's instructions. The activity of lactate
dehydrogenase in conditioned medium was utilized as an assessment of
cell lysis in all experiments and was measured by a colorimetric assay
using pyruvate as a substrate (Sigma). Each experiment reported was
repeated at least three times with similar results in all cases, and
representative data are shown in each figure.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
to enter the
extracellular compartment using NIH 3T3 cells because these cells (i) are refractory to the activity of endogenous and exogenous IL1
(data
not shown) and (ii) have proven to be valuable for the study of the
FGF1 release pathway (16-18). Thus, stable NIH 3T3 cell transfectants
expressing either pIL1
or mIL1
with or without the
-gal reporter gene were obtained and were subjected to
temperature stress at 42 °C for 2 h. Cell lysates and
conditioned medium treated with 0.1% (w/v) DTT were processed for
IL1
immunoblot analysis. As shown in Fig.
1, A and B, mIL1
and mIL1
·
-gal were readily visible in medium conditioned by
heat shock. In contrast, neither pIL1
nor pIL1
·
-gal was
detected in heat shock-conditioned medium, but both forms of the
polypeptide were present in cell lysates (Fig. 1, A and
B). These data suggest the following: (i) the mature (residues 113 to 271) but not the precursor (residues 1 to 271) form of
IL1
was able to enter the extracellular compartment in response to
temperature stress, (ii) the release of IL1
does not restrict export
of the reporter gene product,
-gal, and (iii) cell lysis does not
account for the release of mIL1
because the absence of pIL1
in
medium conditioned by heat shock served as a negative control.
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Fig. 1.
Mature IL1 but not
precursor IL1
is released in response to
temperature stress. The mIL1
and pIL1
(A) and
mIL1
·
-gal and pIL1
·
-gal (B) NIH 3T3 cell
transfectants were subject to heat shock (42 °C, 2 h), and CM
was treated with DTT, immunoprecipitated with anti-IL1
, and analyzed
by IL1
immunoblot analysis. A, lane 1, total CL from
mIL1
transfectants; lanes 2 and 3, 37 and
42 °C CM from mIL1
transfectants, respectively; lane
4, total CL from pIL1
transfectants; lanes 5 and
6, 37 and 42 °C CM from pIL1
transfectants,
respectively. B, lane 1, total CL from mIL1
·
-gal
transfectants; lanes 2 and 3, 37 and 42 °C CM
from mIL1
·
-gal transfectants, respectively; lane 4,
total CL from pIL1
·
-gal transfectants; lanes 5 and
6, 37 and 42 °C CM from pIL1
·
-gal transfectants,
respectively. C and D,
mIL1
·
-gal and pIL1
·
-gal NIH 3T3 cell transfectants were
subject to heat shock (42 °C, 2 h), CM was treated with DTT
(0.1%, w/v), (NH4)2SO4 (95%, w/v)
or left untreated. The IL1
·
-gal fusion proteins were isolated
by PATG affinity chromatography (C) or by IL1 IP
(D) followed by IL1 immunoblot analysis. C, lanes
1 and 2, total CL from mIL1
·
-gal transfectants
at 37 and 42 °C, respectively, purified by PATG affinity;
lanes 3-5, 37 °C CM from mIL1
·
-gal
transfectants, after no treatment,
(NH4)2SO4, or DTT treatment,
respectively; lanes 6-8, 42 °C CM from mIL1
·
-gal
transfectants after no treatment,
(NH4)2SO4, or DTT treatment,
respectively. D, lanes 1 and 2, total CL from
mIL1
·
-gal transfectants, purified by IL1
IP with anti-IL1
(lane 1) or control (lane 2) antibody;
lanes 3 and 4, 37 °C CM from mIL1
·
-gal
transfectants after no treatment or DTT treatment, respectively;
lanes 5 and 6, 42 °C CM from mIL1
·
-gal
transfectants, after no treatment or DTT treatment,
respectively.
Because FGF1 (14) but not FGF2 (19) was released from NIH 3T3 cells in
response to heat shock as a DTT-sensitive latent homodimer (14), and
both reducing agents and (NH4)2SO4
were able to activate the heparin affinity of latent FGF1(16), we
questioned whether these reagents would also affect the ability of
IL1 to be recognized by affinity reagents. Because mIL1
·
-gal
does not exhibit heparin-binding affinity (data not shown), we utilized
-gal affinity and IL1
immunoprecipitation to assess this issue. As shown in Fig. 1C, IL1
immunoblot analysis of medium
conditioned by temperature stress was performed using
p-aminobenzyl-1-thio-
-D-galactopyranoside (PATG) affinity and failed to detect the presence of the
IL1
·
-gal fusion product. However, treatment of heat
shocked-conditioned medium with either 0.1% (w/v) DTT or 95% (w/v)
(NH4)2SO4 did resolve the presence
of mIL1
·
-gal in medium conditioned by heat shock from
mIL1
·
-gal NIH 3T3 cell transfectants. In contrast, the use of
IL1
immunoprecipitation detected the presence of IL1
·
-gal in
medium conditioned by heat shock, and the addition of either DTT (Fig.
1D) or (NH4)2SO4 (data
not shown) to the medium did not alter the ability of the anti-IL1
antibody to recognize the fusion protein (Fig. 1D).
These data suggest that whereas mIL1
·
-gal is present in the
extracellular compartment in a conformation that does not efficiently
recognize PATG (unlike FGF1 that utilizes Cys oxidation for export,
Ref. 15), this conformation may not represent mIL1
but rather the
conformation of
-gal. Thus, it is unlikely that mIL1
utilizes Cys
oxidation for export.
We also examined the kinetics and pharmacologic properties of
mIL1·
-gal release in response to temperature stress, because the FGF1 release pathway exhibits relatively slow export kinetics (14).
Moreover, FGF1 is sensitive to agents that interfere with translation
(12), transcription (12), ATP biosynthesis (17), and assembly of the
F-actin cytoskeleton (28) but is not sensitive to Brefeldin A, an agent
that interferes with the function of the ER-Golgi apparatus (14). As
previously observed with FGF1 release (14), the release of
mIL1
·
-gal also required at least 90 min of temperature stress
before IL1
immunoblot analysis was able to resolve the presence of
the fusion protein in medium conditioned by heat shock (Fig.
2A). In addition, IL1
immunoblot analysis revealed that the release of mIL1
·
-gal was
not sensitive to treatment of the mIL1
·
-gal NIH 3T3 cell
transfectants with Brefeldin A (Fig. 2B) but was sensitive
to the presence of 2-deoxyglucose (Fig. 2C) as well as
cycloheximide and actinomycin D (data not shown). Further, the release
of mIL1
·
-gal was also sensitive to treatment with amlexanox
(Fig. 2D), an agent which induces the
Src-dependent disassembly of F-actin stress fibers (28) and
exhibits a dose-response to amlexanox similar to that reported for the
inhibition of FGF1 release in vitro (18). These data suggest
that the pathway utilized by mIL1
·
-gal for entering the
extracellular compartment is dependent upon transcription, translation,
ATP biosynthesis, and the actin cytoskeleton but is independent of the
function of the ER-Golgi apparatus.
|
Because the kinetic and pharmacologic properties of mIL1·
-gal
release were remarkably similar to that previously reported for FGF1,
we examined the potential role of p65 Syt1 in the IL1
release
pathway. Syt1 is the prototype member of a gene family of vesicular
transmembrane Ca2+- and acidic phospholipid-binding
proteins (29) involved in the regulation of conventional exocytosis
(30) and endocytosis (31). Because the extravesicular p40 domain of
Syt1 is released as an aggregate with FGF1 in response to heat shock
(16) and is associated with FGF1 in vivo (18), we utilized a
p65 Syt1 mutant (p65 Syt1
(120-214)) lacking the C2A domain, which
acts as a dominant negative effector of FGF1 release in response to heat shock (17). To evaluate the role of p65 Syt1 in the release of
mIL1
·
-gal, we obtained stable NIH 3T3 cell cotransfectants expressing either p65 Syt1/mIL1
·
-gal or p65
Syt1
(120-214)/mIL1
·
-gal and examined medium conditioned by
heat shock for the presence of p40 Syt1 and IL1
·
-gal using
immunoblot analysis. As shown in Fig.
3A, Syt1 immunoblot analysis
revealed the presence of p40 Syt1 in medium conditioned by heat shock
from the p65 Syt1/mIL1
·
-gal NIH 3T3 cell cotransfectants but
did not report the presence of p40 Syt1 in heat shocked-conditioned
medium from either mIL1
·
-gal NIH 3T3 cell transfectants or p65
Syt1
(120-214)/mIL1
·
-gal NIH 3T3 cell cotransfectants. In
contrast, however, IL1
immunoblot analysis did reveal the presence
of mIL1
·
-gal in medium conditioned by temperature stress
independent of the expression of either p65 Syt1 or p65
Syt1
(120-214) (Fig. 3B). These data suggest that unlike
the stress-induced FGF1 release pathway (17), the IL1
release
pathway may not utilize the function of p65 Syt1. However, it is not
possible to eliminate the function of another member of the Syt gene
family in the regulation of mIL1
release.
|
Although the data with p65 Syt1(120-214) expression suggest that
the IL1
and FGF1 release pathways may have diverged, we questioned
whether the coexpression of IL1
and FGF1 in the NIH 3T3 cell could
confirm this premise. Thus we obtained stable NIH 3T3 cell
cotransfectants in which pIL1
·
-gal and mIL1
·
-gal NIH
3T3 transfectants were cotransfected with FGF1 and examined their
ability to release mIL1
·
-gal, pIL1
·
-gal, and FGF1 in response to heat shock. As shown in Fig.
4, IL1
and FGF1 immunoblot analysis
revealed the presence of both mIL1
·
-gal and FGF1 in medium
conditioned by heat shock from the FGF1/mIL1
·
-gal NIH 3T3 cell
cotransfectants. However, IL1
and FGF1 immunoblot analysis failed to
detect the presence of either pIL1
or FGF1 from the FGF1/pIL1
·
-gal NIH 3T3 cell cotransfectants in response to
temperature stress (Fig. 4). Because the precursor form but not the
mature form of IL1
was able to repress the release of FGF1 in
response to heat shock, the precursor domain of IL1
may contain a
structural feature that may function as a dominant-negative effector of
FGF1 release. Although we do not know the element within the precursor domain of IL1
responsible for this event, these data do suggest that
the IL1
and FGF1 release pathways may indeed be convergent.
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It is well established that the precursor forms of the IL1 prototypes
are cell-associated polypeptides whereas the mature forms of the IL1
prototypes are present in either biological fluids or cell culture
medium (32-35); an observation which suggests that the mature forms of
the IL1 prototypes may be preferred for release. Indeed, in transgenic
mice manipulated to overexpress mIL1 in basal keratinocytes, high
levels of mIL1
were released into the circulation (36). In contrast,
however, mechanical deformation of human keratinocytes was required to
induce a rapid release of pIL1
(37). Whereas both precursor and
mature forms of the IL1 prototypes have also been detected in cell
culture medium conditioned by mononuclear cells treated with
lipopolysaccharides (34, 38), the appearance of the precursor forms
correlated with the release of LDH, a cytosolic protein used to monitor
the release of cytosol because of compromised plasma membrane integrity (39). It should be noted that our experimental conditions also employed
LDH release, and we did not observe any significant levels of
extracellular LDH in response to temperature stress. However, because
the pIL1
·
-gal release studies were limited to a single clone,
we cannot eliminate the possibility that a critical ratio of
pIL1
·
-gal expression may enable pIL1
·
-gal to be
released in response to heat shock.
Our data are consistent with the observation that human bladder
carcinoma cells selectively release mIL1 but not pIL1
in vitro (40). Moreover, Siders, et al. (41) using a
variety of different cell types transfected with either the precursor or mature forms of the IL1 prototypes observed that the mature forms of
IL1
and IL1
were also preferred for release and that the release
of mIL1
from pIL1
was dependent upon the presence of a
calpain-like protease activity (41). Interestingly, whereas macrophages
from the ICE-null mouse were able to release pIL1
but not
mIL1
in response to lipopolysaccharide stimulation, these cells were
also deficient in IL1
release suggesting a possible role for ICE in
the release of IL1
(42). Because we were unable to observe the
presence of mIL1
in cytosol derived from the pIL1
NIH 3T3 cell
transfectants, even under conditions of apoptosis where ICE is
functional (data not shown), it is unlikely that ICE expression is
involved in the formation of mIL1
in the NIH 3T3 cell. Rather, the
NIH 3T3 cells may be deficient in the expression of the appropriate
calpain and as a result are not capable of processing intracellular
pIL1
. Alternatively, this may also be because of the presence of a
calpain inhibitor (43) or because of the absence of calpain activation
in response to heat shock. However, our data do suggest that the
proteolytic conversion of pIL1
to mIL1
may be prerequisite for
the appearance of IL1
in the extracellular compartment.
Although the ability of pIL1 to repress the release of FGF1 in
response to heat shock suggests a connection between the IL1
and
FGF1 release pathways, we do not know whether mIL1
requires the
function of helper genes to gain access to the extracellular compartment. However, it is interesting to note that like FGF1 (18,
44), early studies with IL1
using gel exclusion chromatography also
noted the presence of IL1
in high molecular weight complexes (34,
45). Whether these represent the IL1
equivalent of the multiprotein
aggregate of FGF1(18) is not known. It is intriguing to speculate that
like the FGF1 release pathway, the release of mIL1
in response to
temperature stress may require the function of an unknown set of helper genes.
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ACKNOWLEDGEMENTS |
---|
We thank the officers of Takeda
Pharmaceuticals, Ltd. and Hoffman-LaRoche, Inc, for their generous
supply of amlexanox and IL1 antibody, respectively and Julia
Frothingham and Norma Albrecht for expert administrative assistance.
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FOOTNOTES |
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* 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.
Present address: Dept. of Geriatric Medicine, University of
Florence, School of Medicine, Florence, Italy 50100.
§ Supported by a fellowship from the Catholic University of Rome.
¶ Present address: Div. of Lung Diseases, NHLBI, National Institutes of Health, Bethesda, MD 20892.
To whom correspondence should be addressed: Center for
Molecular Medicine, Maine Medical Center Research Inst., 81 Research Drive, Scarborough, ME 04074. Tel.: 207-885-8200; Fax: 207-885-8179; E-mail: maciat@mail.mmc.org.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.C000714200
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ABBREVIATIONS |
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The abbreviations used are:
IL, interleukin;
CL, cell lysate;
CM, conditioned medium;
ER, endoplasmic reticulum;
FGF, fibroblast growth factor;
-gal,
-galactosidase;
ICE, IL1
converting enzyme;
PATG, p-aminobenzyl-1-thio-
-D-galactopyranoside;
Syt, Synaptotagmin;
p, precursor;
m, mature;
DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis;
DMEM, Dulbecco's
modified Eagle's medium;
2DG, 2-deoxyglucose;
IP, immunoprecipitation,
LDH, lactate dehydrogenase.
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